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Maria Paola COSTI

Professore Ordinario presso: Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103


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Pubblicazioni

- Aprepitant as anticancer drug [Brevetto]
Michael, Khun; Peer, Bork; Costi, Maria Paola; Luciani, Rosaria
abstract

The present invention relates to the use of a heterocyclic compound according to generalstructural formula (I) for the treatment of cancer. In a preferred aspect the compound according 5to formula (I) is aprepitant.BACKGROUND OF THE INVENTIONCancer is a disorder in which a population of cells has become, in varying degrees, unresponsive 10to the control mechanisms that normally govern proliferation and differentiation. Therapeuticagents used in clinical cancer therapy can be categorized into several groups, including,alkylating agents, antibiotic agents, antimetabolic agents, biologic agents, hormonal agents, andplant-derived agents.15Cancerous cells display uncontrolled growth (growth and division beyond the normal limits),invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread toother locations in the body via lymph or blood). Much effort has been spent to find novel anticanceragents. Cancer therapy is also being attempted by modulating the enzymatic activity oftarget proteins in cancer cells. For example, compounds were found which inhibit enzymes used 20in purine and pyrimidine synthesis such as thymidylate synthase (TS), dihydrofolate reductase(DHFR), and glycinamide ribonucleotide formyl transferase (GARFT). By inhibiting theformation of precursor purine and pyrimidine nucleotides, the formation of DNA and RNA iscomprimised, which are necessary for the growth and survival of both normal cells and cancercells. As cancer cells are rapidly dividing they heavily rely on a sufficient supply of nucleotides 25to afford such rapid divisions. However, therapeutic target proteins, such as the aforementionedenzymes respond differently to treatment depending on the type of cancer and the the geneticpredisposition of the patient.Due to the multiple different forms of cancer there is a need for additional pharmaceutically 30active substances which can be used to treat cancer.SUMMARY OF THE INVENTION2The inventors have identified a compound which unexpectedly is useful to effectively treatcancer.


- Phenylboronic acid derivative inhibitors of beta-lactamases, their preparation, pharmaceutical compositions, and therapeutic use. [Brevetto]
Shoichet, ; Brian, K.; Costi, Maria Paola; Tondi, Donatella
abstract

PCT Int. Appl. (2000), 41 pp. CODEN: PIXXD2 WO 0035904 A1 20000622 CAN 133:53675 AN 2000:421128


- QUINOXALINE DERIVATIVES AS ANTITUBERCULOSIS AGENTS [Brevetto]
Costi, Maria Paola; Ferrari, Stefania; Saxena, Puneet; Venturelli, Alberto; Farina, Davide Salvatore Francesco; Luciani, Rosaria; Gadupudi, Ramakrishna
abstract

The invention refers to a new class of quinoxaline derivatives and pharmaceutical acceptable salts thereof, useful as anti tuberculosis agents, in particular in the treatment of pathologies caused by Mycobacterium tuberculosis.


- SPECIFIC INHIBITORS OF PTERIDINE REDUCTASE WITH ANTIPARASITIC ACTION. [Brevetto]
Costi, Maria Paola; G., Paglietti
abstract

The processes for the preparation of alkyl derivatives of N-[4-(2,4-diaminopteridin-6- ylmethylamino)benzoyl]-N-piperidin-2-carboxylic acid and N-[4-(2,4-diaminopteridin-6- ylmethylamino)benzoyl] -N-piperidin-4-carboxylic acid and of N-[4-(2,4-diaminopteridin-6- ylmethylamino)benzoyl]-N-pyrrolidin-2-carboxylic acid and N-[4-(2,4-diaminopteridin-6- ylmethylamino)benzoyl]-N-pyrrolidin-4-carboxylic acid. Their specific properties of inhibition versus Pteridine reductase of parasitic species. The processes for the preparation of alkyl derivatives of 1-[4-(quinoxalin-2- ylamino)benzoyl]piperidin-mono and di-carboxylic acid and alkyl derivatives of 1-[4- (quinoxalin-2-ylamino)benzoyl]pyrrolidin-mono and di-carboxylic acid. The specific properties of inhibition versus Pteridine reductase of parasitic species. The processes for the preparation of alkyl derivatives of 4-(6,7-dimethoxy-quinoxalin-2- ylmethoxy)benzoic acid and alkyl derivatives of 2-(phenylsulfanil)-quinoxalin-5,7-diamine and their specific properties of inhibition versus Pteridine reductase of bacterial species. The pharmaceutical composition and their use in the treatment and prevention of parasitic infections caused by Leishmania and Tripanosoma and their antiparasitic action in combination with Pyrimetamine.


- STRUTTURA DEL CRISTALLO DEL COMPLESSO DI TIMIDILATO SINTETASI (TS) CON UN LIGANDO [Brevetto]
Stefano, Mangani; Cecilia, Pozzi; Ferrari, Stefania; Costi, Maria Paola
abstract

Gli inventori hanno scoperto che alcuni "piccoli" peptidi con una sequenza comprendente da 3 a 10 amminoacidi inibiscono la proteina Timidilato sintasi attraverso il legame ad un nuovo sito localizzato all'interfaccia di dimerizzazione della proteina, riuscendo a stabilizzare la conformazione inattiva della proteina. La scoperta di questo nuovo sito di legame e di peptidi in grado di legarsi ad esso ha permesso di verificare l'esistenza di un nuovo meccanismo di inibizione di cellule tumorali (in particolare di cellule di cancro ovarico) in cui la funzione catalitica della Timidilato sintasi è inibita (mediante stabilizzazione della sua forma inattiva) senza che, allo stesso tempo, venga bloccata la sua funzione di repressione della traduzione genica. Questo nuovo meccanismo, consente di evitare l'accumulo di alte concentrazioni di Timidilato sintasi nelle cellule cancerogene, responsabile dell'insorgere di resistenze ai farmaci tumorali classici, per esempio ai farmaci a base di platino che si usano nel trattamento dei tumori ovarici.Di conseguenza, questa scoperta consente di fornire un nuovo strumento per disegnare nuovi inibitori della Timidilato sintasi utili nel trattamento del cancro e particolarmente vantaggiosi per invertire la resistenza delle cellule tumorali ai classici farmaci antitumorali. Dal punto di vista pratico, la domanda di brevetto relativa ai peptidi troverà utilità nella commercializzazione dei specifici inibitori per le indicazioni terapeutiche sopra descritte. La domanda di brevetto diretta alla struttura cristallina del complesso proteina-inibitore, potrà essere sfruttata producendo e vendendo il cristallo della proteina-inibitore per screening di nuovi potenziali farmaci e per il "drug design" di nuovi inibitori della Timidilato sintasi in grado di interagire con la proteina attraverso il nuovo meccanismo di inibizione individuato dagli inventori. Tali nuovi farmaci avrebbero il vantaggio di trattare il cancro senza provocare l'insorgenza di resistenze; oppure potrebbero essere somministrati in combinazione con gli antitumorali classici per potenziarne l'attività terapeutica.Si potrebbe pensare, ad esempio, di stipulare un contratto di licenza e cessione del know how (oppure un contratto di cessione della domanda di brevetto+know how) con un'azienda che si occupa della produzione e vendita di cristalli proteici o complessi proteici per scopi di ricerca. Le società clienti di tali produttori sono normalmente aziende farmaceutiche interessate all'acquisto di strutture cristalline di target farmacologici per svolgere screening e "drug design" di nuovi potenziali farmaci.


- USO DI INIBITORI DELLA PTERIDINA REDUTTASI PER LA PREVENZIONE E/O IL TRATTAMENTO DI INFEZIONI PARASSITARIE [Brevetto]
Costi, Maria Paola; Wade, R; Henrich, S; D, Montejenues; Ferrari, Stefania; Venturelli, Alberto; Lazzari, Sandra; Guerrieri, Davide; Nerini, Erika
abstract

La presente invenzione riguarda l’uso di composti inibitori dellapteridina reduttasi per la prevenzione e/o il trattamento di infezioni parassitarie.


2021 - Evidence of Pyrimethamine and Cycloguanil Analogues as Dual Inhibitors of Trypanosoma brucei Pteridine Reductase and Dihydrofolate Reductase [Articolo su rivista]
Tassone, Giusy; Landi, Giacomo; Linciano, Pasquale; Francesconi, Valeria; Tonelli, Michele; Tagliazucchi, Lorenzo; Costi, Maria Paola; Mangani, Stefano; Pozzi, Cecilia
abstract

Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine-nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.


2021 - Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting [Articolo su rivista]
Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; D'Arca, Domenico; Pacifico, Salvatore; Guerrini, Remo; Spyrakis, Francesca; Gozzi, Gaia; Lauriola, Angela; Santucci, Matteo; Cannazza, Giuseppe; Tagliazucchi, Lorenzo; Cazzato, Addolorata Stefania; Losi, Lorena; Ferrari, Stefania; Ponterini, Glauco; Costi, Maria P
abstract

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor alpha (FRalpha) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRalpha selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.


2021 - Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase [Articolo su rivista]
Vitiello, Simone; Caselli, Monica; Pavesi, Giorgia; Santucci, Matteo; Ferrari, Stefania; Costi, Maria Paola; Ponterini, Glauco
abstract

The observables associated with protein intrinsic fluorescence – spectra, time decays, anisotropies – offer opportunities to monitor in real time and non-invasively a protein‘s functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.


2021 - Structural bases for the synergistic inhibition of human thymidylate synthase and ovarian cancer cell growth by drug combinations [Articolo su rivista]
Pozzi, C.; Santucci, M.; Marverti, G.; D'arca, D.; Tagliazucchi, L.; Ferrari, S.; Gozzi, G.; Losi, L.; Tassone, G.; Mangani, S.; Ponterini, G.; Costi, M. P.
abstract


2021 - VIrtual screening identifies cross-class inhibitors of clinically relevant serine- and metallo-beta lactamases [Abstract in Atti di Convegno]
Gianquinto, E.; Santucci, M.; Maso, L.; Cross, S.; Sannio, F.; Verdirosa, F.; De Luca, F.; Docquier, Jd; Cendron, L.; Tondi, D.; Venturelli, A.; Cruciani, G.; Costi, Mp; Spyrakis, F.
abstract


2020 - A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells [Articolo su rivista]
Marverti, Gaetano; Gozzi, Gaia; Maretti, Eleonora; Lauriola, Angela; Severi, Leda; Sacchetti, Francesca; Losi, Lorena; Pacifico, Salvatore; Ferrari, Stefania; Ponterini, Glauco; Leo, Eliana Grazia; Costi, Maria Paola; D'Arca, Domenico
abstract

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.


2020 - Designing Chimeric Molecules for Drug Discovery by Leveraging Chemical Biology [Articolo su rivista]
Borsari, C.; Trader, D. J.; Tait, A.; Costi, M. P.
abstract

After the first seed concept introduced in the 18th century, different disciplines have attributed different names to dual-functional molecules depending on their application, including bioconjugates, bifunctional compounds, multitargeting molecules, chimeras, hybrids, engineered compounds. However, these engineered constructs share a general structure: a first component that targets a specific cell and a second component that exerts the pharmacological activity. A stable or cleavable linker connects the two modules of a chimera. Herein, we discuss the recent advances in the rapidly expanding field of chimeric molecules leveraging chemical biology concepts. This Perspective is focused on bifunctional compounds in which one component is a lead compound or a drug. In detail, we discuss chemical features of chimeric molecules and their use for targeted delivery and for target engagement studies.


2020 - High-resolution crystal structure of Trypanosoma brucei pteridine reductase 1 in complex with an innovative tricyclic-based inhibitor [Articolo su rivista]
Landi, G.; Linciano, P.; Tassone, G.; Costi, M. P.; Mangani, S.; Pozzi, C.
abstract

The protozoan parasite Trypanosoma brucei is the etiological agent of human African trypanosomiasis (HAT). HAT, together with other neglected tropical diseases, causes serious health and economic issues, especially in tropical and subtropical areas. The classical antifolates targeting dihydrofolate reductase (DHFR) are ineffective towards trypanosomatid parasites owing to a metabolic bypass by the expression of pteridine reductase 1 (PTR1). The combined inhibition of PTR1 and DHFR activities in Trypanosoma parasites represents a promising strategy for the development of new effective treatments for HAT. To date, only monocyclic and bicyclic aromatic systems have been proposed as inhibitors of T. brucei PTR1 (TbPTR1); nevertheless, the size of the catalytic cavity allows the accommodation of expanded molecular cores. Here, an innovative tricyclic-based compound has been explored as a TbPTR1-targeting molecule and its potential application for the development of a new class of PTR1 inhibitors has been evaluated. 2,4-Diaminopyrimido[4,5-b]indol-6-ol (1) was designed and synthesized, and was found to be effective in blocking TbPTR1 activity, with a K i in the low-micromolar range. The binding mode of 1 was clarified through the structural characterization of its ternary complex with TbPTR1 and the cofactor NADP(H), which was determined to 1.30 Å resolution. The compound adopts a substrate-like orientation inside the cavity that maximizes the binding contributions of hydrophobic and hydrogen-bond interactions. The binding mode of 1 was compared with those of previously reported bicyclic inhibitors, providing new insights for the design of innovative tricyclic-based molecules targeting TbPTR1.


2020 - Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign [Articolo su rivista]
Linciano, P.; Cullia, G.; Borsari, C.; Santucci, M.; Ferrari, S.; Witt, G.; Gul, S.; Kuzikov, M.; Ellinger, B.; Santarem, N.; Cordeiro da Silva, A.; Conti, P.; Bolognesi, M. L.; Roberti, M.; Prati, F.; Bartoccini, F.; Retini, M.; Piersanti, G.; Cavalli, A.; Goldoni, L.; Bertozzi, S. M.; Bertozzi, F.; Brambilla, E.; Rizzo, V.; Piomelli, D.; Pinto, A.; Bandiera, T.; Costi, M. P.
abstract

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives.


2020 - New insights into bioactive compounds from the medicinal plant Spathodea campanulata P. Beauv. and their activity against Helicobacter pylori [Articolo su rivista]
Ngnameko, C. R.; Marchetti, L.; Zambelli, B.; Quotadamo, A.; Roncarati, D.; Bertelli, D.; Njayou, F. N.; Smith, S. I.; Moundipa, P. F.; Costi, M. P.; Pellati, F.
abstract

The medicinal plant Spathodea campanulata P. Beauv. (Bignoniaceae) has been traditionally applied for the prevention and treatment of diseases of the kidney and urinary system, the skin, the gastrointestinal tract, and inflammation in general. The present work shows for the first time how chemical components from this plant inhibit Helicobacter pylori growth by urease inhibition and modulation of virulence factors. The crude extract and the main fractions of S. campanulata bark were tested on H. pylori isolated strains and the active ones were further fractionated. Fractions and sub-fractions of the plant crude extract were characterized by ultra-high-performance liquid chromatographic tandem high resolution-mass spectrometry detection (UHPLC-HRMS). Several phenolics and triterpenoids were identified. Among the sub-fractions obtained, SB2 showed the capacity to inhibit H. pylori urease in a heterologous bacterial model. One additional sub-fraction (SE3) was able to simultaneously modulate the expression of two adhesins (HopZ and BabA) and one cytotoxin (CagA). The flavonol kaempferol was identified as the most interesting compound that deserves further investigation as a new hit for its capacity to modulate H. pylori virulence factors.


2020 - Virtual screening identifies broad-spectrum β-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases [Articolo su rivista]
Spyrakis, F.; Santucci, M.; Maso, L.; Cross, S.; Gianquinto, E.; Sannio, F.; Verdirosa, F.; De Luca, F.; Docquier, J. -D.; Cendron, L.; Tondi, D.; Venturelli, A.; Cruciani, G.; Costi, M. P.
abstract

Bacteria are known to evade β-lactam antibiotic action by producing β-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available β-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors.


2019 - Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform [Articolo su rivista]
Moraes, Carolina B.; Witt, Gesa; Kuzikov, Maria; Ellinger, Bernhard; Calogeropoulou, Theodora; Prousis, Kyriakos C.; Mangani, Stefano; Di Pisa, Flavio; Landi, Giacomo; Iacono, Lucia Dello; Pozzi, Cecilia; Freitas-Junior, Lucio H.; dos Santos Pascoalino, Bruno; Bertolacini, Claudia P.; Behrens, Birte; Keminer, Oliver; Leu, Jennifer; Wolf, Markus; Reinshagen, Jeanette; Cordeiro-da-Silva, Anabela; Santarem, Nuno; Venturelli, Alberto; Wrigley, Stephen; Karunakaran, Deepa; Kebede, Bethlehem; Pöhner, Ina; Müller, Wolfgang; Panecka-Hofman, Joanna; Wade, Rebecca C.; Fenske, Martina; Clos, Joachim; Alunda, José María; Corral, María Jesús; Uliassi, Elisa; Bolognesi, Maria Laura; Linciano, Pasquale; Quotadamo, Antonio; Ferrari, Stefania; Santucci, Matteo; Borsari, Chiara; Costi, Maria Paola; Gul, Sheraz
abstract

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion–toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.


2019 - COMPOSTI AD ATTIVITÀ ANTIPARASSITARIA [Brevetto]
Costi, Maria Paola; Costantino, Luca; Linciano, Pasquale; Ferrari, Stefania; Cordeiro da Silva, Anabela
abstract

La presente invenzione si riferisce a composti a struttura 5-(2-nitroetil)-2,4,6-triaminopirimidinica derivatizzata in posizione 1 sulla catena etilenica con anelli eteroaromatici o fenil-benzileteri. L’invenzione, inoltre, riguarda l’uso di detti composti come agenti antiparassitari contro le infezioni da Trypanosoma brucei e Trypanosoma cruzi.


2019 - Cyclic Peptides Acting as Allosteric Inhibitors of Human Thymidylate Synthase and Cancer Cell Growth [Articolo su rivista]
Pacifico, Salvatore; Santucci, Matteo; Luciani, Rosaria; Saxena, Puneet; Linciano, Pasquale; Ponterini, Glauco; Lauriola, Angela; D'Arca, Domenico; Marverti, Gaetano; Guerrini, Remo; Costi, Maria Paola
abstract

Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness.


2019 - Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics [Articolo su rivista]
Borsari, Chiara; Dolores Jimenez-Anton, María; Eick, Julia; Bifeld, Eugenia; Jose Torrado, Juan; Isabel Olías-Molero, Ana; Jesús Corral, María; Santarem, Nuno; Baptista, Catarina; Severi, Leda; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Reinshagen, Jeanette; Witt, Gesa; Linciano, Pasquale; Tait, Annalisa; Costantino, Luca; Luciani, Rosaria; Tejera Nevado, Paloma; Zander-Dinse, Dorothea; Franco, Caio H.; Ferrari, Stefania; Moraes, Carolina B.; Cordeiro-da-Silva, Anabela; Ponterini, Glauco; Clos, Joachim; María Alunda, Jose; Costi, Maria Paola
abstract

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)- 3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 mM for Leishmania infantum, 3.4 mM for L. donovani, 6.7 mM for L. major), Trypanosoma cruzi (EC50 7.5 mM) and T. brucei (EC50 0.8 mM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.


2019 - Enhancement of Benzothiazoles as Pteridine Reductase-1 Inhibitors for the Treatment of Trypanosomatidic Infections [Articolo su rivista]
Linciano, Pasquale; Pozzi, Cecilia; Iacono, Lucia Dello; di Pisa, Flavio; Landi, Giacomo; Bonucci, Alessio; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Santarem, Nuno; Baptista, Catarina; Franco, Caio; Moraes, Carolina B; Müller, Wolfgang; Wittig, Ulrike; Luciani, Rosaria; Sesenna, Antony; Quotadamo, Antonio; Ferrari, Stefania; Pöhner, Ina; Cordeiro-da-Silva, Anabela; Mangani, Stefano; Costantino, Luca; Costi, Maria Paola
abstract

2-Amino-benzo[ d]thiazole was identified as a new scaffold for the development of improved pteridine reductase-1 (PTR1) inhibitors and anti-trypanosomatidic agents. Molecular docking and crystallography guided the design and synthesis of 42 new benzothiazoles. The compounds were assessed for Trypanosoma brucei and Leishmania major PTR1 inhibition and in vitro activity against T. brucei and amastigote Leishmania infantum. We identified several 2-amino-benzo[ d]thiazoles with improved enzymatic activity ( TbPTR1 IC50 = 0.35 μM; LmPTR1 IC50 = 1.9 μM) and low μM antiparasitic activity against T. brucei. The ten most active compounds against TbPTR1 were able to potentiate the antiparasitic activity of methotrexate when evaluated in combination against T. brucei, with a potentiating index between 1.2 and 2.7. The compound library was profiled for early ADME toxicity, and 2-amino- N-benzylbenzo[ d]thiazole-6-carboxamide (4c) was finally identified as a novel potent, safe, and selective anti-trypanocydal agent (EC50 = 7.0 μM). Formulation of 4c with hydroxypropyl-β-cyclodextrin yielded good oral bioavailability, encouraging progression to in vivo studies.


2019 - Evidence of destabilization of the human thymidylate synthase (hTS) dimeric structure induced by the interface mutation Q62R [Articolo su rivista]
Pozzi, C.; Lopresti, L.; Santucci, M.; Costi, M. P.; Mangani, S.
abstract

In human cells, thymidylate synthase (TS) provides the only source of 2’-deoxythymidyne-5’-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.


2019 - Excited-state intramolecular proton transfer in a bioactive flavonoid provides fluorescence observables for recognizing its engagement with target proteins [Articolo su rivista]
Vanossi, D.; Caselli, M.; Pavesi, G.; Borsari, C.; Linciano, P.; Costi, M. P.; Ponterini, G.
abstract

A benzothiophene-substituted chromenone with promising activity against Leishmania and Trypanosoma species exhibits peculiar fluorescence properties useful for identifying its complexes with target proteins in the microorganism proteomes. The emission spectra, anisotropy and time profiles of this flavonoid strongly change when moving from the free to the protein-bound forms. The same two types of emission are observed in organic solvents and their mixtures with water, with the relative band intensities depending on the solvent ability to establish hydrogen bonds with the solute. The regular emission prevails in protic solvents, while in aprotic solvents the anomalously red-shifted emission occurs from a zwitterionic tautomeric form, produced in the excited state by proton transfer within the intramolecularly H-bonded form. This interpretation finds support from an experimental and theoretical investigation of the conformational preferences of this compound in the ground and lowest excited state, with a focus on the relative twisting about the chromenone-benzothiophene interconnecting bond. An analysis of the absorption and emission spectra and of the photophysical properties of the two emitting tautomers highlights the relevance of the local microenvironment, particularly of the intra- and intermolecular hydrogen bonds in which this bioactive compound is involved, in determining both its steady-state and time-resolved fluorescence behaviour.


2019 - Exploring acyclic boronic derivatives as potent beta-lactamases inhibitors with broad spectrum activity [Relazione in Atti di Convegno]
Maso, L.; Quotadadamo, A.; Bellio, P.; Montanari, Martina; Celenza, G.; Costi, Mp:; Tondi, D.; Cendron, L.
abstract

Background: Bacterial resistance has become a worldwide concern after the emergence of pan-resistant clinical isolates. beta-lactamases enzymes (BLs) represent one of the major mechanisms of bacterial resistance. Among them, the metallo depedent subclass (MBLs) are particularly worrisome, given their capability of hydrolysing beta-lactam antibiotics as well as any inhibitors currently in use. In particular, New Delhi metallo-BL-1 (NDM-1), the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. While for serine-beta-lactamases (SBLs) inhibitors are available in therapy, for MBLs no inhibitor have been at the present approved, compromising the efficacy of treatments of bacterial infections. Objectives: Our study aimed to characterize promising β-lactamases inhibitors with broad spectrum activity toward both serine and metallo BLs. We focused our attention on boron based benzothiophene derivatives and used a small library, based on such scaffold, to develop non-beta-lactam-like compounds able to bind BLs active site and inhibit their activity. Our choice has been encouraged by the recently approved serine BLs inhibitor vaborbactam and promising studies on cyclic boronic inhibitors. Methods: We performed an in depth structural and mechanicistic study of a small library of acyclic boronic benzothiophene derivatives with broad spectrum (both SBLs and MBLs) activity. Results: In solution kinetic characterization as well as extensive X-Ray crystallographic analysis of our best candidates have been performed and will be presented here.


2019 - Hits and Lead Discovery in the Identification of New Drugs against the Trypanosomatidic Infections [Capitolo/Saggio]
Calogeropoulou, Theodora; Magoulas, George E.; Pöhner, Ina; Wade, Rebecca C.; Panecka-Hofman, Joanna; Linciano, Pasquale; Ferrari, Stefania; Costi, Maria Paola; Santarem, Nuno; Dolores Jiménez-Antón, Ma; Isabel Olías-Molero, Ana; Cordeiro da Silva and José María Alunda, Anabela
abstract


2019 - IL GIOCO MOLECOLARE TRA DRUG DISCOVERY E CHEMICAL BIOLOGY [Articolo su rivista]
Costi, Maria Paola; Guerrini, Remo; Ponterini, Glauco
abstract

La chemical biology è una disciplina vicina alla chimica farmaceutica strettamente connessa al processo di drug discovery mediante uno scambio di ruolo di inibitori/ligandi e probe molecolari. In tre esempi di applicazione della chemical biology-drug discovery vengono illustrati i linker per la bioconiugazione, le sonde fluorescenti in studi di interazione farmacorecettore cellulare (target engagement) e l’indagine proteomica dei meccanismi biologici d’azione di inibitori specifici.


2019 - Optimization of N-alkylation in the Synthesis of Methotrexate and Pteridine-based Derivatives Under Microwave-Irradiation [Articolo su rivista]
Quotadamo, A.; Linciano, P.; Costi, M. P.; Venturelli, A.
abstract

Methotrexate (MTX) and its pteridine-based derivatives represent an attractive chemotype for the development of bioactive molecules. However, the synthesis of pteridines suffers from several drawbacks. Here we describe a new efficient and improved microwave-assisted lab-scale process for the preparation of MTX and congeners. Starting from the commercially available 2,4-diamino-6-(hydroxymethyl)pteridine (Pt-OH), MTX was obtained with an overall 94% yield through a three steps procedure. The crucial yield-limiting and time-consuming step of S N 2 substitution between halogenated pteridine and nucleophilic aromatic amine was taken. The innovative process, conducted under microwave irradiation, improved yield and purity, and in particular reduced the reaction time from days to 20 minutes. The optimized protocol was successfully applied to the synthesis of diverse pteridine-based derivatives and to the preparation in gram-scale of antiparasitic MTX derivatives for in vivo studies. This new optimized synthetic procedure therefore represents a worthy alternative to the current protocols for the preparation of pteridine-based derivatives.


2019 - SAR Studies and Biological Characterization of a Chromen-4-one Derivative as an Anti-Trypanosoma brucei Agent [Articolo su rivista]
Borsari, Chiara; Santarem, Nuno; Macedo, Sara; Jiménez-Antón, María Dolores; Torrado, Juan J.; Olías-Molero, Ana Isabel; Corral, María J.; Tait, Annalisa; Ferrari, Stefania; Costantino, Luca; Luciani, Rosaria; Ponterini, Glauco; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Behrens, Birte; Reinshagen, Jeanette; Alunda, José María; Cordeiro-da-Silva, Anabela; Costi, Maria Paola
abstract

Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure–activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC50 = 1.1 μM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.


2019 - Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors [Articolo su rivista]
Pozzi, Cecilia; Ferrari, Stefania; Luciani, Rosaria; Costi, Maria; Mangani, Stefano
abstract

Abstract Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.


2019 - Structural Comparison of Enterococcus faecalis and Human Thymidylate Synthase Complexes with the Substrate dUMP and Its Analogue FdUMP Provides Hints about Enzyme Conformational Variabilities [Articolo su rivista]
Pozzi, Cecilia; Ferrari, Stefania; Luciani, Rosaria; Tassone, Giusy; Costi, Maria Paola; Mangani, Stefano
abstract

Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) using N⁵,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.


2019 - Structural Insights into the Development of Cycloguanil Derivatives as Trypanosoma brucei Pteridine-Reductase-1 Inhibitors [Articolo su rivista]
Landi, G.; Linciano, P.; Borsari, C.; Bertolacini, C. P.; Moraes, C. B.; Cordeiro-Da-Silva, A.; Gul, S.; Witt, G.; Kuzikov, M.; Costi, M. P.; Pozzi, C.; Mangani, S.
abstract

Cycloguanil is a known dihydrofolate-reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was developed, resulting in 1 and 2a having IC50 values of 692 and 186 nM, respectively, toward TbPTR1. Structural analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of hydrophobic interactions, H-bonds, and halogen bonds. Moreover, in vitro cell-growth-inhibition tests indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1 activity in T. brucei is a promising new strategy for the treatment of human African trypanosomiasis. For this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.


2019 - The 1,10-phenanthroline ligand enhances the antiproliferative activity of dna-intercalating thiourea-pd(Ii) and-pt(ii) complexes against cisplatin-sensitive and-resistant human ovarian cancer cell lines [Articolo su rivista]
Marverti, G.; Gozzi, G.; Lauriola, A.; Ponterini, G.; Belluti, S.; Imbriano, C.; Costi, M. P.; D’Arca, D.
abstract

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four dierent thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative ecacy. This paralleled both a higher intracellular drug accumulation and a more ecient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.


2019 - X-ray-crystallography deciphers the activity of broad spectrum boronic acid β-Lactamases inhibitors [Articolo su rivista]
Cendron, Laura; Quotadamo, Antonio; Maso, Lorenzo; Bellio, Pierangelo; Montanari, Martina; Celenza, Giuseppe; Venturelli, Alberto; Costi, Maria Paola; Tondi, Donatella
abstract

Recent decades have witnessed a dramatic increase of multidrug resistant (MDR) bacteria, compromising the efficacy of available antibiotics, and a continual decline in the discovery of novel antibacterials. We recently reported the first library of benzo[b]thiophen-2-ylboronic acid inhibitors sharing broad spectrum activity against -Lactamases (BLs). The ability of these compounds to inhibit structurally and mechanistically different types of -Lactamases has been here structurally investigated. An extensive x-ray crystallographic analysis of boronic acids (BAs) binding to proteins representative of serine BLs (SBLs) and metallo -Lactamases (MBLs) have been conducted to depict the role played by the boronic group in driving molecular recogni-tion, especially in the interaction with MBLs. Our derivatives are the first case of non-cyclic boronic acids active against MBLs and represent a productive route toward potent broad-spectrum inhibitors.


2018 - Aryl thiosemicarbazones for the treatment of trypanosomatidic infections [Articolo su rivista]
Linciano, Pasquale; Moraes, Carolina B.; Alcantara, Laura M.; Franco, Caio H.; Pascoalino, Bruno; Freitas-Junior, Lucio H.; Macedo, Sara; Santarem, Nuno; Cordeiro-da-Silva, Anabela; Gul, Sheraz; Witt, Gesa; Kuzikov, Maria; Ellinger, Bernhard; Ferrari, Stefania; Luciani, Rosaria; Quotadamo, Antonio; Costantino, Luca; Costi, Maria Paola
abstract

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50= 2.31 μM, LiEC50= 6.14 μM, TcEC50= 1.31 μM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4′-dimethoxy-[1.1′-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.


2018 - Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth [Articolo su rivista]
Saxena, Puneet; Severi, Leda; Santucci, Matteo; Taddia, Laura; Ferrari, Stefania; Luciani, Rosaria; Marverti, Gaetano; Marraccini, Chiara; Tondi, Donatella; Mor, Marco; Laura, Scalvini; Vitiello, Simone; Losi, Lorena; Fonda, Sergio; Pacifico, Salvatore; Guerrini, Remo; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola
abstract

LR and [D-Gln4]LR peptides bind the mono- mer−monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pron]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.


2018 - Development of a Focused Library of Triazole-Linked Privileged-Structure-Based Conjugates Leading to the Discovery of Novel Phenotypic Hits against Protozoan Parasitic Infections [Articolo su rivista]
Uliassi, Elisa; Piazzi, Lorna; Belluti, Federica; Mazzanti, Andrea; Kaiser, Marcel; Brun, Reto; Moraes, Carolina B; Freitas-Junior, Lucio H; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura
abstract

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50values of 1.8 and 1.9 μg mL-1against T. cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.


2018 - Human Thymidylate Synthase Inhibitors Halting Ovarian Cancer Growth [Articolo su rivista]
Ferrari, Stefania; Severi, Leda; Cecilia, Pozzi; Quotadamo, Antonio; Ponterini, Glauco; Losi, Lorena; Marverti, Gaetano; Costi, Maria Paola
abstract

Human thymidylate synthase (hTS) has an important role in DNA biosynthesis, thus it is essential for cell survival. TS is involved in the folate pathways, specifically in the de novo pyrimidine biosynthesis. Structure and functions are intimately correlated, account for cellular activity and, in a broader view, with in vivo mechanisms. hTS is a target for anticancer agents, some of which are clinical drugs. The understanding of the detailed mechanism of TS inhibition by currently used drugs and of the interaction with the mechanism of action of other anticancer agents can suggest new perspective of TS inhibition able to improve the anticancer effect and to overcome drug resistance. TS-targeting drugs in therapy today are inhibitors that bind at the active site and that mostly resemble the substrates. Nonsubstrate analogs offer an opportunity for allosteric binding and novel mode of inhibition in the cancer cells. This chapter illustrates the relationship among the large number of hTS actions at molecular and clinical levels, its role as a target for ovarian cancer therapy, in particular in cases of overexpression of hTS and other folate proteins such as those induced by platinum drug treatments, and address the potential combination of TS inhibitors with other suitable anticancer agents.


2018 - IN SILICO/IN VITRO APPROACHES FOR THE IDENTIFICATION OF NEW CARBAPENEMASE INHIBITORS [Poster]
Spyrakis, F.; Tondi, D.; Celenza, G.; D’Arrigo, G.; Gianquinto, E.; Santucci, M.; Cross, S.; Bellio, P.; Cendron, L.; Docquier, JD.; Blàzquez, J.; Venturelli, A.; Cruciani, G.; Costi, MP.
abstract

The ongoing emergence of bacterial strains resistant to even third- and fourth-generation β-lactam antibiotics is one of the most pressing and challenging issues in clinical therapy. Resistance is most often mediated by beta-lactamases (BLs), and in particular by carbapenemases (i.e KPC, NDM-1, VIM), which have emerged in both Gram-positive and Gram-negative bacteria (1). Among these, NDM-1 has emerged as a global health threat, capable of easy propagation to other species. No inhibitors are available for NDM-1 so far, despite the number of reports claiming the design and development of novel inhibitors for this enzyme. To this aim we applied a multidisciplinary approach integrating in silico/in vitro analysis for the design and identification of novel non-covalent BL inhibitors. Through a structure-based in silico screening of commercially libraries we identified several promising candidates active against class A and class B carbapenemases. The binding affinities of the high scoring hits were measured in vitro revealing, for some of them, low micromolar affinity towards BLs. Experimental analyses confirmed a 30% and a 50% prediction success rate for KPC-2 and NDM-1, respectively. These results overcome the standard virtual screening success rate which, for random screens, typically range from 0.1 to 0.5%. For NDM-1 the best inhibitors efficacy against resistant bacterial strains overexpressing NDM-1 was also validated, confirming their favorable synergistic effect in combination with the carbapenem meropenem (2). At the same time, we developed a multiligand set of covalent boronic β-lactamase inhibitors using a combination of molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. The analyses confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of its inhibition profile by each compound of the set. Interestingly, this work allowed the identification of potent multi-spectrum KPC-2 and NDM-1 inhibitors able to significantly potentiate the activity of cefotaxime on resistant clinical isolates (MIC reduction, 32-fold) and opened the way to the potential use the designed compounds set as a diagnostic tool for sensitive detection of clinically-relevant β-lactamases (3).


2018 - In This Issue, Volume 9, Issue 5 [Recensione in Rivista]
Costi, Maria Paola
abstract

EFFICIENT SYNTHESES OF COCAINE VACCINES 3 AND THEIR IN VIVO EVALUATION 4 Cocaine addiction and abuse remain major health and societal 5 issues in the United States. Despite this, no therapeutic treat- 6 ment for cocaine addiction is available and current interventions 7 only address withdrawal symptoms interventions. 8 In this issue, Janda and colleagues (DOI: 10.1021/acsmedchem- 9 lett.8b00051) present the development and in vivo evaluation of 10 two cocaine vaccines. Each vaccine consists of one of two hydro- 11 lytically stable haptens developed previously by the researchers, 12 GND or GNE, conjugated to tetanus toxoid (TT) and formulated 13 with alum and cytosine-guanine oligonucleotide (CpG ODN 14 1826) adjuvants. Both vaccines, termed GND-TT and GNE-TT, 15 triggered the production of antibodies with high affinity for 16 cocaine in mice, and these newly generated antibodies success- 17 fully blocked the stimulatory activity of cocaine. Biodistribution 18 studies revealed the ability of GND-TT and GNE-TT to seques- 19 ter free cocaine in the blood and to reduce the drug’s concen- 20 tration in the brain. These results suggest that GND- and GNE-TT g 21 conjugates are suitable for further preclinical development. SYNTHESIS OF PHOSPHATIDYLSERINE AND ITS 23 STEREOISOMERS: THEIR ROLE IN ACTIVATION OF 24 BLOOD COAGULATION 25 Blood coagulation is initiated by the formation of a complex 26 between a transmembrane protein called tissue factor (TF) and 27 circulatory factor VIIa (FVIIa). Phosphatidylserine facilitates this 28 process; however, the mechanism by which this occurs remains 29 enigmatic. Herein, Mallik et al. (DOI: 10.1021/acsmedchem- 30 lett.8b00008) report the synthesis of all stereoisomers of 31 phosphatidylserine (PS) using a novel synthetic method and 32 subsequent studies of each phosphatidylserine isomer with 33 circulatory factor VIIa and tissue factor TF. An assay involving 34 recombinant FVIIa and full length TF present in a reconstituted 35 membrane model revealed that the configuration of both the 36 headgroup and the glycerol backbone of PS play a role in enhanced 37 TF-FVIIa complex activity. The authors also performed molecular 38 dynamics simulations and suggest that changes in chiral centers 39 affect the interactions between the PS head groups and glycerol 40 backbone with TF and FVIIa. 41 This study thus provides new insights into structure−activity 42 relationship and protein−lipid interactions in the blood g 43 coagulation. In This Issue


2018 - New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting [Articolo su rivista]
Rondanin, R.; Lettini, G.; Oliva, P.; Baruchello, R.; Costantini, C.; Trapella, C.; Simoni, D.; Bernardi, T.; Sisinni, L.; Pietrafesa, M.; Ponterini, G.; Costi, M. P.; Vignudelli, T.; Luciani, R.; Matassa, D. S.; Esposito, F.; Landriscina, Matteo
abstract

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.


2018 - Oxaliplatin plus leucovorin and 5-fluorouracil (FOLFOX-4) as a salvage chemotherapy in heavily-pretreated platinum-resistant ovarian cancer [Articolo su rivista]
Conteduca, Vincenza; Gurioli, Giorgia; Rossi, Lorena; Scarpi, Emanuela; Lolli, Cristian; Schepisi, Giuseppe; Farolfi, Alberto; De Lisi, Delia; Gallà, Valentina; Luca Burgio, Salvatore; Menna, Cecilia; Amadori, Andrea; Losi, Lorena; Amadori, Dino; Costi, Maria Paola; De Giorgi, Ugo
abstract

Background: The purpose of this study was to evaluate the clinical impact of oxaliplatin, leucovorin, and 5- fluorouracil (FOLFOX-4) chemotherapy in terms of the response rate, progression-free/overall survival (PFS/OS) and safety profile in patients with heavily pretreated recurrent epithelial ovarian cancer. Methods: Clinical data were reviewed in 29 patients who received FOLFOX-4 as more than third-line chemotherapy, consisting of 85 mg/m2 of oxaliplatin, 200 mg/m2 of leucovorin, and bolus 400 mg/m2 on day 1 of 5-fluorouracil, followed by a 22-h infusion of 600 mg/m2 of 5-fluorouracil for 2 consecutive days every 3 weeks. We also compared the efficacy and toxicity of FOLFOX-4 with that of topotecan, a standard treatment, given at a dosage of 1.5 mg/m2 every three weeks in 26 patients. Results: The median age of enrolled patients was 60 years (range 33 to 85). A median of 4 cycles (range 1–17) of FOLFOX-4 were administered. Complete response and partial response were observed in one (3.5%) and 5 (17.2.2%) patients, respectively, while stable disease was reported in 8 (27.6%) patients. Among all patients, grade 3–4 anemia, neutropenia, and thrombocytopenia were observed in 0 (0%), 5 (17.2%), and 3 (10.3%) cases, respectively. Grade 3–4 fatigue was recorded in one (3.4%) patient and diarrhea in 2 (6.9%). Median PFS and OS were 2.8 months [95% confidence interval (CI) 1.7–4.9] and 6.2months (95% CI 2.4–14.6), respectively. No significant differences in terms of efficacy and toxicity were observed between patients receiving FOLFOX-4 and those treated with topotecan. Conclusions: The FOLFOX-4 regimen would seem to obtain similar survival rates to those of standard therapy with topotecan in platinum-resistant ovarian cancer. Further randomized trials are warranted to confirm our findings.


2018 - Pharmacokinetics and disposition of miltefosine in healthy mice and hamsters experimentally infected with Leishmania infantum [Articolo su rivista]
Jimenez-Anton, M. D.; Garcia-Calvo, E.; Gutierrez, C.; Escribano, M. D.; Kayali, N.; Luque-Garcia, J. L.; Olias-Molero, A. I.; Corral, M. J.; Costi, M. P.; Torrado, J. J.; Alunda, J. M.
abstract

Miltefosine is the only currently available oral drug for treatment of leishmaniasis. However, information on the pharmacokinetics (PK) of miltefosine is relatively scarce in animals. PK parameters and disposition of the molecule was determined in healthy NMRI mice and Syrian hamsters infected and treated with different miltefosine doses and regimens. Long half-life of the molecule was confirmed and differential pattern of accumulation of the drug was observed in analyzed organs in mice and hamster. Long treatment schedules produced miltefosine levels over IC 50 value against L. infantum intracellular amastigotes for at least 24 days in spleen and liver of infected hamsters. The observed differential pattern of organ accumulation of the drug in mice and hamster supports the relevance of both species for translational research on chemotherapy of leishmaniasis.


2018 - pH-Promoted Release of a Novel Anti-Tumour Peptide by “Stealth” Liposomes: Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells [Articolo su rivista]
Sacchetti, Francesca; Marverti, Gaetano; D’Arca, Domenico; Severi, Leda; Maretti, Eleonora; Iannuccelli, Valentina; Pacifico, Salvatore; Ponterini, Glauco; Costi, Maria Paola; Leo, Eliana
abstract

Purpose: To evaluate the potential effects of PEGylated pH-sensitive liposomes on the intracellular activity of a new peptide recently characterized as a novel inhibitor of the human thymidylate synthase (hTS) over-expressed in many drug-resistant human cancer cell lines. Methods: Peptide-loaded pH-sensitive PEGylated (PpHL) and non-PEGylated liposomes (nPpHL) were carefully characterized and delivered to cis-platinum resistant ovarian cancer C13* cells; the influence of the PpHL on the drug intracellular activity was investigated by the Western Blot analysis of proteins involved in the pathway affected by hTS inhibition. Results: Although PpHL and nPpHL showed different sizes, surface hydrophilicities and serum stabilities, both carriers entrapped the drug efficiently and stably demonstrating a pH dependent release; moreover, the different behavior against J774 macrophage cells confirmed the ability of PEGylation in protecting liposomes from the reticuloendothelial system. Comparable effects were instead observed against C13* cells and biochemical data by immunoblot analysis indicated that PEGylated pH-sensitive liposomes do not modify the proteomic profile of the cells, fully preserving the activity of the biomolecule. Conclusion: PpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.


2018 - Proteomic and bioinformatic studies for the characterization of response to pemetrexed in platinum drug resistant ovarian cancer [Articolo su rivista]
Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I.; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P.
abstract

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.


2018 - Repurposing of drugs targeting yap-tead functions [Articolo su rivista]
Elisi, Gian Marco; Santucci, Matteo; D’Arca, Domenico; Lauriola, Angela; Marverti, Gaetano; Losi, Lorena; Scalvini, Laura; Bolognesi, Maria Laura; Mor, Marco; Costi, Maria Paola
abstract

Drug repurposing is a fast and consolidated approach for the research of new active compounds bypassing the long streamline of the drug discovery process. Several drugs in clinical practice have been reported for modulating the major Hippo pathway’s terminal effectors, namely YAP (Yes1-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and TEAD (transcriptional enhanced associate domains), which are directly involved in the regulation of cell growth and tissue homeostasis. Since this pathway is known to have many cross-talking phenomena with cell signaling pathways, many efforts have been made to understand its importance in oncology. Moreover, this could be relevant to obtain new molecular tools and potential therapeutic assets. In this review, we discuss the main mechanisms of action of the best-known compounds, clinically approved or investigational drugs, able to cross-talk and modulate the Hippo pathway, as an attractive strategy for the discovery of new potential lead compounds.


2018 - Scaffolds and Biological Targets Avenue to Fight Against Drug Resistance in Leishmaniasis [Capitolo/Saggio]
Borsari, C.; Quotadamo, A.; Ferrari, S.; Venturelli, A.; Cordeiro-da-Silva, A.; Santarem, N.; Costi, M. P.
abstract

Leishmaniasis is causing a major health problem worldwide and only five to six drugs are available for its control. Among them, antimonials, amphotericin, and miltefosine have been introduced starting in the 1940s and have proved to be effective for many years. However, the appearance of resistance threatens the success of anti-Leishmania treatment and spontaneous resistance to classical first- and second-line anti-Leishmania therapy reveals the major need to develop novel and less toxic drugs. A deep understanding of the mechanisms of drug resistance may help in the design of novel anti-Leishmania agents to be exploited in combination or as second-line therapy. Moreover, the discovery and validation of novel and druggable targets represent a useful tool to tackle drug resistance in leishmaniasis. Some biological processes such as redox metabolism, folate biosynthesis, protein synthesis, and turnover are potentially suitable to develop novel effective agents since they involve proteins showing a unique biological profile with respect to the human metabolism. Phenotypic screening and target-based drug discovery are promising tools to be exploited in drug discovery and development for leishmaniasis. Herein, we outline the most recent efforts in the identification of novel chemical scaffolds using both drug discovery strategies. We finally discuss the breakthroughs in the identification and validation of new biological targets, including Leishmania infantum initiation factor 4A, Leishmania casein kinase 1.2 (LmCK1.2), and methionyl-tRNA synthetase in Leishmania parasites. Different medicinal chemistry teams are currently exploring these targets aiming to pick out compounds displaying an improved profile to reinforce the drug pipeline.


2018 - Synthesis, biological evaluation and molecular modeling of novel azaspiro dihydrotriazines as influenza virus inhibitors targeting the host factor dihydrofolate reductase (DHFR) [Articolo su rivista]
Francesconi, V.; Giovannini, L.; Santucci, M.; Cichero, E.; Costi, M. P.; Naesens, L.; Giordanetto, F.; Tonelli, M.
abstract

Recently we identified cycloguanil-like dihydrotriazine derivatives, which provided host-factor directed antiviral activity against influenza viruses and respiratory syncytial virus (RSV), by targeting the human dihydrofolate reductase (hDHFR) enzyme. In this context we deemed interesting to further investigate the structure activity relationship (SAR) of our first series of cycloguanil-like dihydrotriazines, designing two novel azaspiro dihydrotriazine scaffolds. The present study allowed the exploration of the potential chemical space, around these new scaffolds, that are well tolerated for maintaining the antiviral effect by means of interaction with the hDHFR enzyme. The new derivatives confirmed their inhibitory profile against influenza viruses, especially type B. In particular, the two best compounds shared potent antiviral activity (4: EC50 = 0.29 μM; 6: EC50 = 0.19 μM), which was comparable to that of zanamivir (EC50 = 0.14 μM), and better than that of ribavirin (EC50 = 3.2 μM). In addition, these two compounds proved to be also effective against RSV (4: EC50 = 0.40 μM, SI ≥ 250; 6: EC50 = 1.8 μM, SI ≥ 56), surpassing the potency and selectivity index (SI) of ribavirin (EC50 = 5.8 μM, SI > 43). By a perspective of these results, the above adequately substituted azaspiro dihydrotriazines may represent valuable hit compounds worthy of further structural optimization to develop improved host DHFR-directed antiviral agents.


2017 - Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity [Articolo su rivista]
Di Pisa, F.; Landi, G.; Dello Iacono, L.; Pozzi, C.; Borsari, C.; Ferrari, S.; Santucci, M.; Santarem, N.; Cordeiro-Da-Silva, A.; Moraes, C. B.; Alcantara, L. M.; Fontana, V.; Freitas-Junior, L. H.; Gul, S.; Kuzikov, M.; Behrens, B.; Pohner, I.; Wade, R. C.; Costi, M. P.; Mangani, S.
abstract

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.


2017 - Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors [Articolo su rivista]
Panecka-Hofman, Joanna; Pöhner, Ina; Spyrakis, Francesca; Zeppelin, Talia; Di Pisa, Flavio; Dello Iacono, Lucia; Bonucci, Alessio; Quotadamo, Antonio; Venturelli, Alberto; Mangani, Stefano; Costi, Maria Paola; Wade, Rebecca C.
abstract

Background Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR–TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter. Methods We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR–TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. Results Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures. Conclusions The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents. General significance Apart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.


2017 - Compounds having antiparasitic activity [Brevetto]
Alunda, José; Borsari, Chiara; CORDEIRO DA SILVA, Anabela; Costantino, Luca; Costi, Maria Paola; Ferrari, Stefania
abstract

La presente invenzione si riferisce anuovi composti che presentano l’anello delcromen-4-one legato a diversi anelli eteroaromatici. L’invenzione include una nuova classe di composti di formula generale (B) che presentano l’anello del cromen-4-one legato ad un benzotiofene. L’invenzione, inoltre, riguarda l’uso di detti composti come potenziali agenti antiparassitari verso infezioni da Leishmania spp, Trypanosoma brucei e Trypanosoma cruzi.


2017 - Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-beta-lactamases [Articolo su rivista]
Santucci, Matteo; Spyrakis, Francesca; Cross, Simon; Quotadamo, Antonio; Farina, Davide; Tondi, Donatella; De Luca, Filomena; Docquier, Jean-Denis; Prieto, Ana Isabel; Ibacache, Claudia; Blázquez, Jesús; Venturelli, Alberto; Cruciani, Gabriele; Costi, Maria Paola
abstract

β-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (Ki= 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases.


2017 - Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones? [Articolo su rivista]
Sacchetti, Francesca; D'Arca, Domenico; Genovese, Filippo; Pacifico, Salvatore; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Context: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. Objective: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. Materials and methods For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. Results and discussion Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. Conclusions Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.


2017 - Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity [Articolo su rivista]
Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R. Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J.; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B.; Freitas-Junior, Lucio H.; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura
abstract

Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibitionTbGAPDH= 64% and % inhibitionTcTR= 65%). In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed.


2017 - Design, synthesis and biological evaluation of non-covalent AmpC Beta-Lactamases inhibitors [Articolo su rivista]
Genovese, Filippo; Lazzari, Sandra; Venturi, Ettore; Costantino, Luca; Blazquez, Jesus; Ibacache Quiroga, · Claudia; Costi, Maria Paola; Tondi, Donatella
abstract

Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, beta-lactamases enzymes (BLs), able to inactivate most beta-lactam core antibiotics, represent a key target to block prolonging antibiotics half-life. Several approaches aimed at inhibiting BLs have been so far undertaken, mainly involving beta-lactam like or covalent inhibitors. Applying a structure based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC beta lactamases: it has a Ki of 1 µM, a ligand efficiency of 0.38 kcal ∙ mol–1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate beta-lactamase expression in cell culture. Its features make it a good candidate for chemical optimization: starting from its crystallographic complex with AmpC, eleven analogues were designed to complement additional AmpC sites, then synthesized and tested against clinical resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them exert a higher potency in in vivo tests, showing improved synergic potency with ceftazidime in resistant clinically isolated strains.


2017 - Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To InhibitTrypanosoma bruceiPteridine Reductase in Support of Early-Stage Drug Discovery [Articolo su rivista]
Linciano, Pasquale; Dawson, Alice; Pöhner, Ina; Costa, David M; Sá, Monica S; Cordeiro-da-Silva, Anabela; Luciani, Rosaria; Gul, Sheraz; Witt, Gesa; Ellinger, Bernhard; Kuzikov, Maria; Gribbon, Philip; Reinshagen, Jeanette; Wolf, Markus; Behrens, Birte; Hannaert, Véronique; Michels, Paul A M; Nerini, Erika; Pozzi, Cecilia; di Pisa, Flavio; Landi, Giacomo; Santarem, Nuno; Ferrari, Stefania; Saxena, Puneet; Lazzari, Sandra; Cannazza, Giuseppe; Freitas-Junior, Lucio H; Moraes, Carolina B; Pascoalino, Bruno S; Alcântara, Laura M; Bertolacini, Claudia P; Fontana, Vanessa; Wittig, Ulrike; Müller, Wolfgang; Wade, Rebecca C; Hunter, William N; Mangani, Stefano; Costantino, Luca; Costi, Maria P
abstract

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme-and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 mu M, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.


2017 - Feasibility Study on a Measurement Method and a Portable Measuring System to Estimate the Concentration of Cloxacillin and β -Lactamase in Milk [Articolo su rivista]
Ferrari, Luca; Rovati, Luigi; Costi, Maria Paola; Luciani, Rosaria; Venturelli, Alberto; Cattini, Stefano
abstract

Antibiotics are nowadays used and abused worldwide in common veterinary practice to treat diseases, prevent infections, and promote animal's growth. Drug resistance occurrence is a relevant phenomenon that can inactivate the antibiotic. Moreover, antibiotics used for animals are also found in milk and it poses serious health risks to humans. Another negative effect is related to milk processors since antibiotics cause detrimental effects on cheese and yogurt starter bacteria. Given its consumption as both beverage and derivatives, milk is one of the most regulated products in food industry. Nowadays several commercial tests are available to investigate antibiotics in milk, but they generally provide a qualitative result, require bulky procedure, and are time consuming. In this paper, we investigate the use of a chromogenic cephalosporin to quantify the concentration of cloxacillin - a β-lactam difficult to be detected by using "cowside" screenings which is the drug of choice in the method of mastitis control. The proposed measurement method and prototype have been demonstrated to be able to detect cloxacillin in milk at concentrations lower than the MRL set by the European Commission. Moreover, they are also able to detect the illegal practice of using β-lactamase to degrade β-lactams in milk.


2017 - Host dihydrofolate reductase (DHFR)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus [Articolo su rivista]
Tonelli, Michele; Naesens, Lieve; Gazzarrini, Sabrina; Santucci, Matteo; Cichero, Elena; Tasso, Bruno; Moroni, Anna; Costi, Maria Paola; Loddo, Roberta
abstract

We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC50 = 0.060 μM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC50∼0.008 μM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC50 = 5.8 μM, SI > 43).


2017 - In silico identification and in vitro evaluation of natural inhibitors of leishmania major pteridine reductase I [Articolo su rivista]
Herrmann, Fabian C.; Sivakumar, Nirina; Jose, Joachim; Costi, Maria P.; Pozzi, Cecilia; Schmidt, Thomas J.
abstract

In a continuation of our computational efforts to find new natural inhibitors of a variety of target enzymes from parasites causing neglected tropical diseases (NTDs), we now report on 15 natural products (NPs) that we have identified as inhibitors of Leishmania major pteridine reductase I (LmPTR1) through a combination of in silico and in vitro investigations. Pteridine reductase (PTR1) is an enzyme of the trypanosomatid parasites’ peculiar folate metabolism, and has previously been validated as a drug target. Initially, pharmacophore queries were created based on four 3D structures of LmPTR1 using co-crystallized known inhibitors as templates. Each of the pharmacophore queries was used to virtually screen a database of 1100 commercially available natural products. The resulting hits were submitted to molecular docking analyses in the substrate binding site of the respective protein structures used for the pharmacophore design. This approach led to the in silico identification of a total of 18 NPs with predicted binding affinity to LmPTR1. These compounds were subsequently tested in vitro for inhibitory activity towards recombinant LmPTR1 in a spectrophotometric inhibition assay. Fifteen out of the 18 tested compounds (hit rate = 83%) showed significant inhibitory activity against LmPTR1 when tested at a concentration of 50 µM. The IC50values were determined for the six NPs that inhibited the target enzyme by more than 50% at 50 µM, with sophoraflavanone G being the most active compound tested (IC50= 19.2 µM). The NPs identified and evaluated in the present study may represent promising lead structures for the further rational drug design of more potent inhibitors against LmPTR1.


2017 - Label-free fiber optic optrode for the detection of class C beta-lactamases expressed by drug resistant bacteria [Articolo su rivista]
Zuppolini, S; Quero, G; Consales, M; Diodato, L; Vaiano, P; Venturelli, A; Santucci, M; Spyrakis, F; Costi, Mp; Giordano, M; Cutolo, A; Cusano, A; Borriello, Alessandro
abstract

This paper reports the experimental assessment of an automated optical assay based on label free optical fiber optrodes for the fast detection of class C beta-lactamases (AmpC BLs), actually considered as one of the most important sources of resistance to beta-lactams antibiotics expressed by resistant bacteria. Reflection-type long period fiber gratings (RT-LPG) have been used as highly sensitive label free optrodes, while a higher affine boronic acid based ligand was here selected to enhance the overall assay performances compared to those obtained in our first demonstration. In order to prove the feasibility analysis towards a fully automated optical assay, an engineered system was developed to simultaneously manipulate and interrogate multiple fiber optic optrodes in the different phases of the assay. The automated system tested in AmpC solutions at increasing concentrations demonstrated a limit of detection (LOD) of 6 nM, three times better when compared with the results obtained in our previous work. Moreover, the real effectiveness of the proposed optical assay has been also confirmed in complex matrices as the case of lysates of Escherichia coli overexpressing AmpC. (C) 2017 Optical Society of America


2017 - Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds [Articolo su rivista]
Borsari, Chiara; Santarem, Nuno; Torrado, Juan; Olías, Ana Isabel; Corral, María Jesús; Baptista, Catarina; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Gribbon, Philip; Reinshagen, Jeanette; Linciano, Pasquale; Tait, Annalisa; Costantino, Luca; Freitas-Junior, Lucio H.; Moraes, Carolina B.; Bruno dos Santos, Pascoalino; Alcântara, Laura Maria; Franco, Caio Haddad; Bertolacini, Claudia Danielli; Fontana, Vanessa; Tejera Nevado, Paloma; Clos, Joachim; Alunda, José María; Cordeiro-da-Silva, Anabela; Ferrari, Stefania; Costi, Maria Paola
abstract

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2’-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3–4.2 μM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-β-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.


2017 - Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule [Articolo su rivista]
Bruno, Stefano; Uliassi, Elisa; Zaffagnini, Mirko; Prati, Federica; Bergamini, Christian; Amorati, Riccardo; Paredi, Gianluca; Margiotta, Marilena; Conti, Paola; Costi, Maria Paola; Kaiser, Marcel; Cavalli, Andrea; Fato, Romana; Bolognesi, Maria Laura
abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.


2017 - Multiligand approach to the identification of resistant strains overexpressing beta-lactamase [Relazione in Atti di Convegno]
Costi, Maria Paola; Santucci, Matteo; Spyrakis, Francesca; Cross, Simon; Farina, Davide Salvatore Francesco; Tondi, Donatella; Quotadamo, Antonio; Docquier, Jean Denis; Croce, Filomena; Prieto, Ana Isabel; Ibacache, Claudia; Blázquez, Jesús; Venturelli, Alberto; Cruciani, Gabriele
abstract

β-Lactamases (BLs) are among the main responsible of the threatening spread of Gram- negative pathogens resistant to available antibiotics, including last resort carbapenems. [1-2] The rapid detection of resistant BLs responsible of bacterial infections represents a critical challenge for the choice of the most appropriate therapy. As a consequence approaches aiming at the identification of new antibacterial agents or at the identification of the organisms conferring resistance in bacterial infections are strictly connected. Phenyl-boronic acids have been identified as valuable compounds preventing beta-lactam antibiotics hydrolysis by BLs and therefore prolonging antibiotics antimicrobial action. Diagnostic applications of boronic acids were previously foreseen highlighting the limited efficacy of a single boronic compound in the BLs detection: to reliably characterize each BL in the cellular environment a panel of molecules is needed. [3-5] In the present work we propose the inhibitory activity characterization of a benzen-boronic acids set against a selected panel of therapeutically relevant Extended Spectrum Beta-Lactamases (ESBLs). A multi-ligand approach is applied to BLs detection representing an improvement in the reliable recognition of a specific target enzyme: it relies on the differential profile of a selected inhibitor’s set against each considered BL in a panel of ESBLs. The exploited strategy includes covalent molecular docking, screening, synthetic chemistry, enzyme inhibition kinetic and cellular studies on clinical isolates. The inhibitory profile of the benzene-boronic acids compounds set against resistant bacteria overexpressing serine- and metallo- ESBLs belonging to class A, B, C and D has been obtained. Among the developed boronic acids set, one compound was able to potentiate cefotaxime against KPC-2 carbapenemase with a MIC value reduction of 32 fold, presenting an interesting antibacterial activity against NDM-1 and VIM-2 overexpressing resistant bacteria as well (32-fold improvement). [6] The obtained results open the way to the potential use of our selected compounds set as diagnostic tool for timely BLs detection.


2017 - On the feasibility of a CENTA-based biosensor to measure antibiotics concentration in milk [Relazione in Atti di Convegno]
Ferrari, Luca; Rovati, Luigi; Costi, Maria Paola; Venturelli, Alberto; Luciani, Rosaria; Cattini, Stefano
abstract

In veterinary practice antibiotics are used and abused all over the world both to treat diseases and prevent infections and, to increase feed efficiency thus promoting growth in food producing animals. With more than 30 years history, β-lactam antibiotics are nowadays one of the most important group of antibiotics in veterinary medicine. In this paper we investigate the possibility to realize a measurement method suitable for field testing and we propose a competitive biosensor based on a relatively low-cost chromogenic cephalosporin (CENTA) for the quantitative analysis of β-lactams concentration in milk. In particular, the reported preliminary study has been focused on cloxacillin and the reported results indicates that the method is potentially able to detect cloxacillin at a concentration of about one order of magnitude lower than the maximal residue limits (MRL) set by the European Commission.


2017 - On the feasibility of a CENTA-based biosensor to measure antibiotics concentration in milk [Relazione in Atti di Convegno]
Ferrari, L.; Rovati, L.; Costi, M. P.; Venturelli, A.; Luciani, R.; Cattini, S.
abstract

In veterinary practice antibiotics are used and abused all over the world both to treat diseases and prevent infections and, to increase feed efficiency thus promoting growth in food producing animals. With more than 30 years history, β-lactam antibiotics are nowadays one of the most important group of antibiotics in veterinary medicine. In this paper we investigate the possibility to realize a measurement method suitable for field testing and we propose a competitive biosensor based on a relatively low-cost chromogenic cephalosporin (CENTA) for the quantitative analysis of β-lactams concentration in milk. In particular, the reported preliminary study has been focused on cloxacillin and the reported results indicates that the method is potentially able to detect cloxacillin at a concentration of about one order of magnitude lower than the maximal residue limits (MRL) set by the European Commission.


2017 - Target-based approaches for the discovery of new antimycobacterial drugs [Articolo su rivista]
Borsari, Chiara; Ferrari, Stefania; Venturelli, Alberto; Costi, Maria Paola
abstract

Tuberculosis (TB) is a major global health problem and control of the disease is hampered by the increasing emergence of multidrug resistance (MDR) strains. Novel drugs are urgently needed to overcome drug resistance. Among the most relevant targets of the past 3 years, herein we consider nine enzymes that have been studied in a target-based approach. These targets are involved mainly in the biosynthesis of the cell wall, α-glucan, coenzyme A and acyl carrier protein precursor, and in energy production, DNA metabolism, and pyrimidine synthesis. Some leads and many hits have been discovered using a target-based approach and can be further developed in a drug discovery process.


2017 - The Future of Drug Development for Neglected Tropical Diseases: How the European Commission Can Continue to Make a Difference [Articolo su rivista]
Pierce, Raymond J.; MacDougall, Jane; Leurs, Rob; Costi, Maria Paola
abstract

In this article, the four coordinators of neglected tropical disease (NTD) drug development projects funded under the European Commission (EC) Framework Programme 7 argue that the EC should reassess their funding strategy to cover the steps necessary to translate a lead compound into a drug candidate for testing in clinical trials, and suggest ways in which this might be achieved.


2017 - TRAP1: a viable therapeutic target for future cancer treatments? [Articolo su rivista]
Lettini, Giacomo; Maddalena, Francesca; Sisinni, Lorenza; Condelli, Valentina; Matassa, Danilo Swann; Costi, Maria Paola; Simoni, Daniele; Esposito, Franca; Landriscina, Matteo
abstract

Introduction: HSP90 molecular chaperones (i.e., HSP90α, HSP90β, GRP94 and TRAP1) are potential therapeutic targets to design novel anticancer agents. However, despite numerous designed HSP90 inhibitors, most of them have failed due to unfavorable toxicity profiles and lack of specificity toward different HSP90 paralogs. Indeed, a major limitation in this field is the high structural homology between different HSP90 chaperones, which significantly limits our capacity to design paralog-specific inhibitors. Area covered: This review examines the relevance of TRAP1 in tumor development and progression, with an emphasis on its oncogenic/oncosuppressive role in specific human malignancies and its multifaceted and context-dependent functions in cancer cells. Herein, we discuss the rationale for considering TRAP1 as a potential molecular target and the strategies used to date, to achieve its compartmentalized inhibition directly in mitochondria. Expert opinion: TRAP1 targeting may represent a promising strategy for cancer therapy, based on the increasing and compelling evidence supporting TRAP1 involvement in human carcinogenesis. However, considering the complexity of TRAP1 biology, future strategies of drug discovery need to improve selectivity and specificity toward TRAP1 respect to other HSP90 paralogs. The characterization of specific human malignancies suitable for TRAP1 targeting is also mandatory.


2016 - An Improved Synthesis of CENTA, a Chromogenic Substrate for β-Lactamases [Articolo su rivista]
QUOTADAMO, ANTONIO; LINCIANO, PASQUALE; DAVOLI, Paolo; TONDI, Donatella; COSTI, Maria Paola; VENTURELLI, Alberto
abstract

7-β-Thien-2-yl-acetamido-3-[(4-nitro-3-carboxyphenyl) thiomethyl]-3-cephem-4-carboxylic acid (CENTA) is a yellow chromogenic β-lactamases (BL) substrate. It hydrolyses readily in the presence of all BL and is therefore suitable for kinetic studies, the detection of BL enzymes in crude extracts and chromatographic fractions. CENTA is commercially available at a high price because of the cumbersome synthetic protocol, the only currently available for its preparation. Here we describe a new efficient and improved process for the preparation of CENTA. Starting from the easily available 7-aminocephalosporanic acid (7-ACA) through a three-step synthesis, CENTA was obtained with a 75% overall yield. The newly developed process proceeds through a pivotal intermediate in cephalosporin chemistry, which may be used as starting compound for the development of new cephalosporin derivatives.


2016 - Comparing Drug Images and Repurposing Drugs with BioGPS and FLAPdock: The Thymidylate Synthase Case [Articolo su rivista]
Siragusa, Lydia; Luciani, Rosaria; Borsari, Chiara; Ferrari, Stefania; Costi, Maria Paola; Cruciani, Gabriele; Spyrakis, Francesca
abstract

Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent invitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinaseIIα as a control and tested a series of its inhibitors against the TS template. Ellagic acid and apigenin were identified as TS inhibitors, and various flavonoids were selected and synthesized in a second-round selection. The compounds were demonstrated to be active in the low-micromolar range.


2016 - Decoding the structural basis for carbapenem hydrolysis by class A β- lactamases: fishing for a pharmacophore [Articolo su rivista]
Tondi, Donatella; Cross, Simon; Venturelli, Alberto; Costi, Maria Paola; Cruciani, Gabriele; Spyrakis, Francesca
abstract

Nowadays clinical therapy witnesses a challenging bacterial resistance limiting the available armament of antibiotics. Over decades strains resistant to all antibiotics have been selected while medicinal chemists were not able to develop agents capable of destroying them or to prevent their extension. In particular, carbapenem-resistant Enterobacteriaceae (CRE), representing one of the most common human pathogens, have been reported with increased frequency since their first identification twenty years ago. The enterobacterial carbapenemases differ from the extended spectrum β-lactamases (ESBL) in their ability to hydrolyze β-lactams, cephalosporins and most importantly the last resort monobactams and carbapenems. They are progressively spreading throughout the world therefore leaving no effective β-lactam to cure bacterial infections. Several BL-carbapenemase X-ray structures have been determined making these enzymes attractive targets for structure-based drug design studies. However very little has been done so far to powerfully address the inhibitor design issues for this emerging type of BL. Here we focus on the structural basis for molecular recognition and for broad spectrum activity of class A carbapenemases: based on available 3-dimensional structural information we identify a theoretical pharmacophoric model as a starting point for the development of needed carbapenemases inhibitors. Targetable "hot spots" on the enzyme-binding site were identified using a computational approach.


2016 - DESIGNING NOVEL INHIBITORS FOR CARBAPENEMASES: A MULTIDISCIPLINARY APPROACH [Abstract in Atti di Convegno]
Spyrakis, Francesca; Bellio, Pierangelo; Vicario, Mattia; Cendron, Laura; Marcoccia, Francesca; Perilli, Mariagrazia; Cruciani, Gabriele; Costi, Maria Paola; Celenza, Giuseppe; Tondi, Donatella
abstract

The extensive use of beta-lactam antibiotics has created major resistance problems leading to increased morbidity, mortality and health-care costs. Resistance is most often mediated by eta-lactamases (BLs), which have emerged in both Gram-positive and Gram-negative bacteria [1,2]. Medicinal chemists have introduced eta-lactam-based molecules that inhibit or are stable to their action. These molecules are themselves eta-lactams, making it easier for bacteria to respond by adapting previously evolved mechanisms. Many bacteria are now resistant to these anti-resistance compounds. Thus there is a growing need for new broad-spectrum eta-lactamases inhibitors in general and especially against carbapenemases (i.e KPC, NDM-1, VIM)[3,4]. Their substrate promiscuity, their resistance to available drugs, the easiness of variants appearance and transferability make carbapenemases the most worrisome BLs [2]. Focusing on the de novo, non beta-lactam like derivatives we identified, through a structure-based in silico screening of commercially available library using FLAP, several promising candidates active against class A and class B carbapenemases. The binding affinities of the high scoring hits were measured in vitro revealing, for some of them, low micromolar affinity towards BLs. To investigate the potential of these compounds to reverse antibiotic resistance, we are undertaking antimicrobial activity studies in bacterial cell culture. The ability of novel compounds to synergize antibiotics against pathogenic resistant bacteria, as well as their ability to evade those mechanisms normally involved in resistance to eta-lactam-based inhibitors are now under evaluation. Moreover, since our inhibitors are novel, non eta-lactam based, we expect them to do not up-regulate eta-lactamase expression in cell culture. X-ray crystallography studies are now in progress to confirm our docking prediction and to deeply investigate the structural requirement necessary for proficuous hit to lead generation. Keywords: carbapenemases resistance, SBDD, in silico screening, enzyme inhibition, antimicrobial activity, x-ray crystallography


2016 - Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology [Articolo su rivista]
Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pela', Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D'Arca, Domenico
abstract

Demonstrating a candidate drug' s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.


2016 - Long period fiber grating working in reflection mode as valuable biosensing platform for the detection of drug resistant bacteria [Articolo su rivista]
Quero, G.; Zuppolini, S.; Consales, M.; Diodato, L.; Vaiano, P.; Venturelli, A.; Santucci, M.; Spyrakis, F.; Costi, M. P.; Giordano, M.; Borriello, A.; Cutolo, A.; Cusano, A.
abstract

We report here on a reflection-type long period fiber grating (RT-LPG) biosensor for the fast detection of class C (AmpC) β-lactamases (BLs), actually considered as one of the most important source of resistance to β-lactam antibiotics expressed by resistant bacteria. A standard LPG working in transmission configuration is first transformed in a more practical probe working in reflection mode and successively coated with a primary high refractive index (HRI) overlay of atactic polystyrene (aPS) in order to increase its surrounding refractive index sensitivity (SRI) in biological solutions. The aPS-coated RT-LPG is then coated by a secondary layer of poly(methylmethacrylate)-co-methacrylic acid (PMMA-co-MA) in order to provide the necessary surface functionalities to promote a stable covalent bioreceptors immobilization. The BLs detection has been performed by using the 3-aminophenylboronic acid (3-APBA) as biorecognition element, due to its excellent inhibition properties against class C BLs and specificity. Results here provided demonstrate that the proposed label free biosensor is capable of reliable detection of purified AmpC BLs in phosphate buffer solutions (PBS) with concentrations as low as one hundred nM, with a lowest limit of detection (LOD) of the order of a few tens of nM. The real effectiveness of the proposed biosensor has been also confirmed in lysate samples, which contain Escherichia coli bacteria overexpressing AmpC BLs.


2016 - Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs [Articolo su rivista]
Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Poehner, Ina; Henrich, Stefan; Trande, Matteo; Cordeiro da Silva, Anabela; Santarem, Nuno; Baptista, Catarina; Tait, Annalisa; Di Pisa, Flavio; Dello, Iacono; Lucia, Landi; Giacomo, ; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Reinshagen, Jeanette; Witt, Gesa; Gribbon, Philip; Kohler, Manfred; Keminer, Oliver; Behrens, Birte; Costantino, Luca; Tejera Nevado, Paloma; Bifeld, Eugenia; Eick, Julia; Clos, Joachim; Torrado Santiago, Juan; Jiménez Antón, María D.; Corral, María J.; Alunda, José Ma; Pellati, Federica; Wade, Rebecca C.; Ferrari, Stefania; Mangani, Stefano; Costi, Maria Paola
abstract

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.


2016 - Virtual Screening and X-ray Crystallography Identify Non-Substrate Analog Inhibitors of Flavin-Dependent Thymidylate Synthase [Articolo su rivista]
Luciani, Rosaria; Saxena, Puneet; Surade, Sachin; Santucci, Matteo; Venturelli, Alberto; Borsari, Chiara; Marverti, Gaetano; Ponterini, Glauco; Ferrari, Stefania; Blundell, Tom L; Costi, Maria Paola
abstract

Thymidylate synthase-X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima ThyX in complex with a non-substrate analog inhibitor. Given the active site similarities between Mtb-ThyX and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.


2015 - Alanine Mutants of the Interface Residues of Human Thymidylate Synthase Decode Key Features of the Binding Mode of Allosteric Anticancer Peptides [Articolo su rivista]
Tochowicz, Anna Maria; Santucci, Matteo; Saxena, Puneet; Guaitoli, Giambattista; Costi, Maria Paola
abstract

Allosteric peptide inhibitors of thymidylate synthase (hTS) bind to the dimer interface and stabilize the inactive form of the protein. Four interface residues were mutated to alanine, and interaction studies were employed to decode the key role of these residues in the peptide molecular recognition. This led to the identification of three crucial interface residues F59, L198, and Y202 that impart activity to the peptide inhibitors and suggest the binding area for further inhibitor design.


2015 - ANTICANCER DRUGS [Brevetto]
Costantino, Luca; Costi, Maria Paola; Ponterini, Glauco; Marverti, Gaetano; Franchini, Silvia; Tondi, Donatella; D'Arca, Domenico; Ferrari, Stefania; Luciani, Rosaria; Venturelli, Alberto; Sammak, Susan; Lauriola, Angela; Gozzi, Gaia
abstract

Composti destabilizzanti l’omodimero timidilato sintasi


2015 - Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity [Articolo su rivista]
Eadsforth, T. C.; Pinto, A.; Luciani, R.; Tamborini, L.; Cullia, G.; De Micheli, C.; Marinelli, L.; Cosconati, S.; Novellino, E.; Lo Presti, L.; Cordeiro Da Silva, A.; Conti, P.; Hunter, W. N.; Costi, M. P.
abstract

The bifunctional enzyme N5,N10-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 μM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 μM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP+ and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.


2015 - Corrigendum to "Theoretical analysis of the addition of hydroxylamine to uracil and 5-fluorouracil as a model for the thymidylate synthase reaction" [J. Mol. Struct. (Theochem) 343 (1995) 1-9] [Articolo su rivista]
Rastelli, G.; Costi, M. P.
abstract


2015 - Current and Future Chemotherapy for Chagas Disease [Articolo su rivista]
Gaspar, L.; Moraes, C. B.; Freitas Junior, L. H.; Ferrari, Stefania; Costantino, Luca; Costi, Maria Paola; Coron, R. P.; Smith, T. K.; Siqueira Neto, J. L.; McKerrow, J. H.; Cordeiro da Silva, A.
abstract

Human American trypanosomiasis, commonly called Chagas disease, is one of the most neglected illnesses in the world and remains one of the most prevalent chronic infectious diseases of Latin America with thousands of new cases every year. The only treatments available have been introduced five decades ago. They have serious, undesirable side effects and disputed benefits in the chronic stage of the disease - a characteristic and debilitating cardiomyopathy and/or megavisceras. Several laboratories have therefore focused their efforts in finding better drugs. Although recent years have brought new clinical trials, these are few and lack diversity in terms of drug mechanism of action, thus resulting in a weak drug discovery pipeline. This fragility has been recently exposed by the failure of two candidates; posaconazole and E1224, to sterilely cure patients in phase 2 clinical trials. Such setbacks highlight the need for continuous, novel and high quality drug discovery and development efforts to discover better and safer treatments. In this article we will review past and current findings on drug discovery for Trypanosoma cruzi made by academic research groups, industry and other research organizations over the last half century. We also analyze the current research landscape that is now better placed than ever to deliver alternative treatments for Chagas disease in the near future.


2015 - Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery [Articolo su rivista]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pela', Michela; Pinetti, Diego; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Recently, octapeptide LSCQLYQR (LRp), reducing growth of cis-platinum (cDDP) resistant ovarian carcinoma cells by inhibiting the monomer–monomer interface of the human enzyme thymidylate synthase, has been identified. As the peptide is not able to cross the cell membrane it requires an appropriate delivery system. In this work the application of SLNs, biocompatible and efficient tools for the intracellular drug transport, applied especially for lipophilic drugs, was exploited for the delivery of the hydrophilic peptide LRp. SLNs formulated in the absence/presence of small amount of squalene showed dimensions below 150 nm, negative zeta potential and good stability to the freeze-drying process. Even though the particles formulated with squalene exhibited a less ordered crystal lattice and a lower surface hydrophobicity, a rapid drug release from these nanocarriers occurred as a result of the relevant expulsion of the drug from the lipid core during lipid crystallization. On the contrary, SLNs formulated in the absence of squalene were able to incorporate more stably the peptide showing considerable cytotoxic effect on cDDP resistant C13* ovarian carcinoma cell line at concentration 50 times lower than that used previously with a marketed delivery system. From the cell cycle analysis by the propidium iodide test in SLNs-peptide treated cancer cells an increase of apoptosis percentage was observed, indicating that SLNs were able to carry efficiently the peptide until its enzymatic target.


2015 - Hotspots in an obligate homodimeric anticancer target. structural and functional effects of interfacial mutations in human thymidylate synthase [Articolo su rivista]
Salo Ahen, Outi M. H.; Tochowicz, Anna; Pozzi, Cecilia; Cardinale, Daniela; Ferrari, Stefania; Boum, Yap; Mangani, Stefano; Stroud, Robert M.; Saxena, Puneet; Myllykallio, Hannu; Costi, Maria Paola; Ponterini, Glauco; Wade, Rebecca C.
abstract

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.


2015 - Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance [Articolo su rivista]
Taddia, Laura; D'Arca, Domenico; Ferrari, Stefania; Marraccini, Chiara; Severi, Leda; Ponterini, Glauco; Assaraf, Yahuda G.; Marverti, Gaetano; Costi, Maria Paola
abstract

Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemo-resistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug esistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.


2015 - Maxwell A: F1000Prime Recommendation of Evaluation [Neres J et al., ACS Chem Biol 2014]. In F1000Prime, 09 Jan 2015; DOI: 10.3410/f.725248139.793502885. F1000Prime.com/725248139#eval793502885 [Altro]
Maxwell, Anthony; Neres, J; Hartkoorn, Rc; Chiarelli, Lr; Gadupudi, Ramakrishna; Pasca, Mr; Mori, G; Venturelli, Alberto; Savina, S; Makarov, V; Kolly, Gs; Molteni, E; Binda, C; Dhar, N; Ferrari, Stefania; Brodin, P; Delorme, V; Landry, V; de Jesus Lopes Ribeiro, Al; Farina, Davide Salvatore Francesco; Saxena, Puneet; Pojer, F; Carta, Antonello; Luciani, Rosaria; Porta, Andrea; Zanoni, G; De Rossi, E; Costi, Maria Paola; Riccardi, G.
abstract

Tuberculosis (TB) is still a serious problem worldwide, particularly with the advent of drug-resistant strains. Using phenotypic screening, the authors have discovered quinoxaline compounds (especially Ty38c) that are active against extracellular and intracellular Mycobacterium tuberculosis, targeting DprE1, an enzyme involved in cell wall synthesis. The structural work reported in this paper will underpin further efforts to find other compounds that inhibit this target. There is no doubt that TB is a scourge of humankind, killing >1 million people each year. Current therapy is problematic owing to resistance issues and the long times over which drugs have to be taken. Current work has tended to move away from target-based screening to phenotypic screening. Using this approach, the authors have discovered the quinoxaline compound Ty38c as a TB drug candidate. Initial mutations were found in rv3405c, a transcriptional regulator that controls expression of rv3406, whose gene product inactivates Ty38c. Using an rv3406-deficient strain, the authors show that the actual target of Ty38c is DprE1, which is a known target for other TB inhibitors. Using a range of Ty38c analogues, the authors perform a series of structure-activity experiments, and using X-ray crystallography they reveal the molecular details of the Ty38c-DprE1 interactions. Taken together, the work represents a tour de force in TB drug discovery research and provides significant hope that new TB agents can be developed from such approaches.


2015 - Nanoencapsulation of an hTS inhibitor octapeptide against ovarian cancer in solid lipid matrix [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Hanuskova, Miriam; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract

New octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), cannot cross the cell membrane alone and require an appropriate delivery system. In the aim to transport hTS inhibiting LR octapeptide (LR-op) into the cells, Solid Lipid Nanoparticles (SLNs) were developed and evaluated in vitro. The optimized SLNs were formulated in the absence and presence of squalene (7S and 7Sq) both in the LR-op loaded and unloaded form. All the SLNs produced had dimensions below 150 nm, negative Zpotential and a good stability both in suspension and after freeze-drying. Only the sample obtained in the absence of squalene showed to stably incorporate the LR-op promoting its cell internalization, as demonstrated by in vitro studies on C13* ovarian carcinoma cell line.


2015 - pH sensitive PEGylated Liposomes delivering active hydrophilic peptide with anticancer activity: in vitro study on cDDP-resistant ovarian cell line [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pinetti, Diego; Genovese, Filippo; Maretti, Eleonora; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Thymidylate synthase (TS) can be considered a very interesting molecular target for the therapy of the ovarian cancer.. Recently, specific octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), have been identified. Similarly to the majority of peptides, they cannot cross the cell membrane and require a delivery system for transport into the cells and pH sensitive liposomes, destabilizing at mildly acidic pH, are considered efficient tools for delivering water-soluble drugs into the cell cytoplasm. In the present study in order to attain the peptide triggering in the cells promoting endosomal escape, stealth pH-sensitive liposomes were developed and characterized. Results suggested that pH sensitive liposomes seemed suitable carriers for the encapsulation of small hydrophilic molecules like peptides. The appreciable difference in cytotoxicity between loaded and unloaded liposomes demonstrated that the peptide, whose activity is held in the cytoplasm, was triggered in the proper biological site.


2015 - Reflection-type long period grating biosensor for detection of drug resistant bacteria: The OptoBacteria project [Relazione in Atti di Convegno]
Quero, G.; Consales, M.; Vaiano, P.; Cusano, A.; Zuppolini, S.; Diodato, L.; Borriello, A.; Giordano, M.; Venturelli, A.; Costi, M. P.
abstract

In this paper, we report the experimental results on the fabrication and validation of a multilayer coated reflection-type long period fiber grating (LPG) biosensor, useful for the detection of antibiotic resistance bacteria. A standard LPG is first transformed in a practical probe working in reflection mode, then it is coated by two layers: atactic polystyrene (aPS) and (methyl methacrylate-co-methacrylic acid) (PMMA-co-MA). This multilayer strategy allow, at same time, to increase the surrounding refractive index sensitivity of the standard LPG and create the necessary conditions for a correct bio-functionalization. Standard linkage chemistry has been applied to anchor the bioreceptors on the probe surface. The experimental results demonstrate the capability of our LPG biosensor to successfully monitor all the biological steps of the biomolecular experiments, with β-lactamase AmpC binding detection tests obtained using two different ligands characterized by two different β-lactamase affinities.


2015 - The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment [Articolo su rivista]
Santucci, Matteo; Vignudelli, Tatiana; Ferrari, Stefania; Mor, Marco; Scalvini, Laura; Bolognesi, Maria Laura; Uliassi, Elisa; Costi, Maria Paola
abstract

The Hippo pathway is an important organ size control signaling network and the major regulatory mechanism of cell-contact inhibition. Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) are its targets and terminal effectors: inhibition of the pathway promotes YAP/TAZ translocation to the nucleus, where they interact with Transcriptional Enhancer Associate Domain (TEAD) transcription factors and co-activate the expression of target genes, promoting cell proliferation. Defects in the pathway can result in overgrowth phenotypes due to deregulation of stem-cell proliferation and apoptosis; members of the pathway are directly involved in cancer development. The pharmacological regulation of the pathway might be useful in cancer prevention, treatment and regenerative medicine applications; currently, a few compounds can selectively modulate the pathway. In this review, we present an overview of the Hippo pathway, the sequence and structural analysis of YAP/TAZ, the known pharmacological modulators of the pathway and those targeting YAP/TAZ-TEAD interaction.


2015 - 2-Carboxyquinoxalines Kill Mycobacterium tuberculosis through Noncovalent Inhibition of DprE1 [Articolo su rivista]
Neres, João; Hartkoorn, Ruben C.; Chiarelli, Laurent R.; Gadupudi, Ramakrishna; Pasca, Maria Rosalia; Mori, Giorgia; Venturelli, Alberto; Savina, Svetlana; Makarov, Vadim; Kolly, Gaelle S.; Molteni, Elisabetta; Binda, Claudia; Dhar, Neeraj; Ferrari, Stefania; Brodin, Priscille; Delorme, Vincent; Landry, Valérie; de Jesus Lopes Ribeiro, Ana Luisa; Farina, Davide Salvatore Francesco; Saxena, Puneet; Pojer, Florence; Carta, Antonio; Luciani, Rosaria; Porta, Alessio; Zanoni, Giuseppe; De Rossi, Edda; Costi, Maria Paola; Riccardi, Giovanna; Cole, Stewart T.
abstract

Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.


2014 - ACS Medicinal Chemistry Letters. member of the Editorial Board [Direzione o Responsabilità Riviste]
Costi, Maria Paola
abstract

ACS Medicinal Chemistry Letters is interested in receiving manuscripts that discuss various aspects of medicinal chemistry. The journal will publish studies that pertain to a broad range of subject matter, including compound design and optimization, biological evaluation, drug delivery, imaging agents, and pharmacology of both small and large bioactive molecules. Specific areas include but are not limited to: Identification, synthesis, and optimization of lead biologically active molecules and drugs (small molecules and biologics) Biological characterization of new molecular entities in the context of drug discovery Computational, cheminformatics, and structural studies for the identification or SAR analysis of bioactive molecules, ligands and their targets, etc. Novel and improved methodologies, including radiation biochemistry, with broad application to medicinal chemistry Discovery technologies for biologically active molecules from both synthetic and natural (plant and other) sources Pharmacokinetic/pharmacodynamic studies that address mechanisms underlying drug disposition and response Pharmacogenetic and pharmacogenomic studies used to enhance drug design and the translation of medicinal chemistry into the clinic Mechanistic drug metabolism and regulation of metabolic enzyme gene expression Chemistry patents relevant to the medicinal chemistry field


2014 - Development of flavonoids derivatives as leads against Leishmaniasis and Trypanosomiasis-invited lecture [Altro]
Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Tait, Annalisa; Costantino, Luca; Pellati, Federica; Ferrari, Stefania; Costi, Maria Paola
abstract

Parasites of the family of Trypanosomatidae are the agents of serious human diseases, including African sleeping sickness, Chagas' disease and leishmaniasis. There is an urgent requirement for new effective drugs since current treatments show severe side effects, develop toxicity and drug resistance. Enzymes involved in the folate cycle, e.g. dihydrofolate reductase (DHFR)-thymidylate synthase (TS) bifunctional proteins are important drug targets for the treatment of bacterial infections, cancer and certain parasitic diseases, notably malaria. Trypanosomatids are auxotrophic for folates and pterins, and the inhibition of the enzymes involved in the salvage pathways should provide effective treatments. However, antifolates are currently not yet employed in the therapy of trypanosomatids infections also because of the pteridine reductase (PTR1) activity in the target organisms. PTR is a short-chain dehydrogenase/reductase involved in the biopterin reduction pathway, able to carry out subsequent reductions of both conjugated (folate) and unconjugated (biopterin) pterins. When classical antifolate drugs inhibit DHFR-TS, PTR1 can be over-expressed ensuring the parasite survival and causing resistance to typical antifolate. The treatment of trypanosomatidic infections through the simultaneous inhibition of DHFR and PTR1 may provide a successful treatment for leishmanisis infections and Tbrucei/cruzi. Previous work in our laboratories has contributed to demonstrate this principle. Computational studies were performed to screen through a docking approach a library of 90 natural compounds from plants of different origins and target-based screening delivered flavans as one of the interesting compound class to be explored. The flavonoid structure however has been largely explored and often pleiotropic properties can be observed leading to promiscuous inhibition. The aim of the presented work is to identifiy novel chemically tractable scaffolds that will be further developed through medicinal chemistry approaches. X-ray crystallographyc structure of some of the studied compounds have been obtained and used for further structure-based drug design. While the initial compounds showed moderate detectable biological activities even related to their low solubility, recent derivatives were more soluble and showed improved enzyme inhibitory activity. The design, synthesis and biological evaluation of the flavan derivatives will be discussed. Reference 1.Ong HB et al. J Biol Chem.2011 Mar 25;286(12):10429-38. Cavazzuti A et al. PNAS. 2008 Feb 5;105(5):1448-53. 3. Ferrari S. et al, J Med Chem. 2011 Jan 13;54(1):211-21 Acknowledgement This work was supported by “PRIN 2009-2011 to MPC and NMTrypI (New medicine for Trypanosomatidic Infections) This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no.603240.


2014 - Development of lipid nanocarriers as delivery systems for a small peptide with anti-ovarian activity [Abstract in Atti di Convegno]
Sacchetti, Francesca; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Cannazza, Giuseppe; Iannuccelli, Valentina; Maretti, Eleonora; Costi, Maria Paola; Leo, Eliana Grazia
abstract

The encapsulation of a small peptide in SLN was achieved modifying the hot high shear homogenization method. The data obtained by the comparison of SLN to standard Liposomes suggested that even if Liposomes are more efficient carrier for hydrophilic peptides, it is possible to embed this kind of molecules in a solid lipid matrix achieving carriers with higher in vitro stability and lower cytotoxicity. Moreover, the ability of the loaded carriers to reduce the cell viability more efficiently than the unloaded vectors, indicates that the peptide was released inside the cell environment being able to exert its action.


2014 - EVALUATION OF LR STABILITY BY LC CHIP Q-TOF AND QUANTITATIVE DETERMINATION OF LR PEPTIDE CELL PENETRATION BY LC-MS/MS [Articolo su rivista]
Cazzato, ADDOLORATA STEFANIA; Cannazza, Giuseppe; Ponterini, Glauco; Marraccini, Chiara; Pirondi, Silvia; Genovese, Filippo; Costi, Maria Paola
abstract

In the present work the degradation profile of an anticancer peptide in different biological matrixes like DMEM (Dulbecco’s Modified Eagle Medium) and cell lysates by LC Chip Q-TOF was shown. Subsequently, an LC-MS/MS method for the quantitative analysis of LR in cell lysates was developed and fully validated.


2014 - Internalization and stability of a thymidylate synthase peptide inhibitor in ovarian cancer cells [Articolo su rivista]
Cannazza, Giuseppe; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Pavesi, Giorgia; Pirondi, Silvia; R., Guerrini; M., Pelà; Frassineti, Chiara; Ferrari, Stefania; Marverti, Gaetano; Ponterini, Glauco; Costi, Maria Paola
abstract

Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability.


2014 - Long period fiber grating biosensor for the detection of drug resistant bacteria: The 'OPTObacteria' project [Relazione in Atti di Convegno]
Consales, M.; Quero, G.; Zuppolini, S.; Sansone, L.; Borriello, A.; Giordano, M.; Venturelli, A.; Costi, M. P.; Santucci, M.; Cusano, A.
abstract

We report on the development of a multilayer coated reflection-type long period fiber grating (LPG) biosensor, useful for the detection of antibiotic resistance bacteria. A standard LPG is first transformed in a more practical probe working in reflection mode, then it is coated by a primary layer of aPS and a secondary layer of PMMA in order to increase its surrounding refractive index sensitivity and at the same time provide the necessary conditions for a correct bio-functionalization. Standard linkage chemistry has been applied to anchor the bioreceptors on the probe surface. We show some preliminary results demonstrating the capability of our LPG biosensor to successfully monitor all the biological steps of the biomolecular experiments, including β-lactamase binding detection tests. © 2014 IEEE.


2014 - Mass Spectrometric/Bioinformatic Identification of a Protein Subset That Characterizes the Cellular Activity of Anticancer Peptides [Articolo su rivista]
Genovese, Filippo; A., Gualandi; L., Taddia; Marverti, Gaetano; S., Pirondi; C., Marraccini; P., Perco; M., Pelà; R., Guerrini; M. R., Amoroso; F., Esposito; A., Martello; Ponterini, Glauco; D'Arca, Domenico; Costi, Maria Paola
abstract

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [D-Gln4]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides’ cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.


2014 - Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth [Articolo su rivista]
M., Pelà; Saxena, Puneet; Luciani, Rosaria; Santucci, Matteo; Ferrari, Stefania; Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; Pirondi, Silvia; Genovese, Filippo; S., Salvadori; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola; R., Guerrini
abstract

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The D-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.


2014 - Peptides binding to the dimer interface of thymidylate synthase for the treatment of cancer [Brevetto]
Costi, Maria Paola; Ferrari, Stefania; Venturelli, Alberto; Ponterini, Glauco; Cardinale, Daniela; Marverti, Gaetano
abstract

The present invention relates to peptides binding to the thymidylate synthase protein, in particular to human thymidylate synthase (hTS) protein, for the treatment of cancer.The present invention relates to peptides that can bind at a binding site located at the interface of thymdylate synthase protein. These peptides range from 3 to 10, preferably 4-8 amino acids and have a sequence that binds to each subunit of the thymidylate synthase dimer at the level of dimer interface, stabilizing the dimeric inactive form of the thymdylate synthase enzyme. In addition, the present invention relates to pharmaceutical compositions comprising these compounds as active agents, and uses thereof for the treatment of cancer and to reverse or/and be active in cancer drug resistance.


2014 - Peptides binding to the dimer interface of thymidylate synthase for the treatment of cancer-US8916679 “B2 - Granted patent as second publication”. [Brevetto]
Costi, Maria Paola; Marverti, Gaetano; Cardinale, Daniela; Venturelli, Alberto; Ferrari, Stefania; Ponterini, Glauco
abstract

Peptides binding to the dimer interface of thymidylate synthase for the treatment of cancer AB (EP2507255) Provided are peptides that bind to the thymidylate synthase protein, in particular to human thymidylate synthase (hTS) protein, for the treatment of cancer. Further provided are peptides that can bind at a binding site located at the interface of thymidylate synthase protein. These peptides range from 3 to 10, preferably 4-8 amino acids and have a sequence that binds to each subunit of the thymidylate synthase dimer at the level of dimer interface, stabilizing the dimeric inactive form of the thymidylate synthase enzyme. In addition, provided are pharmaceutical compositions including these compounds as active agents, and uses thereof for the treatment of cancer and to reverse or/and be active in cancer drug resistance. (From US8916679 B2)


2014 - Representative for Italy in the COST ACTION CM1307- Management Commette [Altro]
Costi, Maria Paola
abstract

Advances in the chemotherapy against human and animal parasitic diseases remain limited largely because drug candidates have low specificity and show poor in vivo bioavailability. The Action aims at uniting scientists with different backgrounds to create synergistic interactions paving the way for antiparasitic drug discovery for diseases caused by protozoa and helminths. The scientific aim is to bundle together the identification and validation of parasite drug targets based on the established genomes, medicinal chemistry including structure-based drug design, crystallography, bioinformatics, and drug targeting using chemical and nanotechnological approaches to improve drug performance. Also, rational assessment of the potential of natural product and other compound libraries will be used to identify new leads. Crucially, the Action will create an unprecedented combined forum for human health scientists and veterinarians, because of the enormous unmet needs in treating human and animal parasitic diseases and due to methodological homogeneity of their drug design strategies. The most promising compounds and formulations will be tested in established infection models before further preclinical and clinical development with emphasis on drug safety. Expected benefits include intensified cooperations between Academia and Industry. This will be achieved through focused conferences, networking, a dedicated website and training schools for state-of-the-art technologies.


2014 - Targeting Class A and C Serine β-Lactamases with a Broad-Spectrum Boronic Acid Derivative [Articolo su rivista]
Tondi, Donatella; Venturelli, Alberto; Bonnet, Richard; Pozzi, Cecilia; Shoichet, Brian K.; Costi, Maria Paola
abstract

Production of β-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight β-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by class A and C BLs. We describe a boronic acid analogue possessing interesting and potent broad-spectrum activity vs class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-β-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of class A and C BLs. Structure-based drug design (SBDD), synthesis, enzymology data, and X-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activity vs serine-active BLs using double mutant thermodynamic cycle studies.


2014 - The Inhibition of Extended Spectrum beta-Lactamases: Hits and Leads [Articolo su rivista]
Davide, Farina; Francesca, Spyrakis; Alberto, Venturelli; Simon, Cross; TONDI, Donatella; COSTI, Maria Paola
abstract

The ongoing emergence of bacterial strains resistant to even third- and fourth-generation β-lactam antibiotics is one of the most pressing and challenging issues in clinical therapy. Furthermore, under the pressure of antibiotics used ubiquitously over the last 80 years, functional mutations and new resistances are continuously increasing. Therefore, new drugs and new approaches to the infections produced by multidrug-resistant Gram-negative bacteria are categorically necessary and expected by the scientific community. This review describes the most deleterious known extended-spectrum β-lactamases and the molecules now available for targeting bacterial infections. The active-site chemical and geometric properties that are potentially exploitable for the design of both broad-spectrum and selective compounds are described.


2014 - 2-[N-Alkyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines as anticancer agents inhibitors of folate enzymes [Articolo su rivista]
Piras, Sandra; Carta, Antonio; Briguglio, Irene; Corona, Paola; Paglietti, Giuseppe; Luciani, Rosaria; Costi, Maria Paola; Ferrari, Stefania
abstract

Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60 human tumors cell lines at National Cancer Institute of Bethesda (NCI), we have synthesized a novel series of thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1-31). The lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR enzyme, with a Ki of 0.2 μM. Docking studies were performed on compound 1 and here reported to predict its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1-31) were assayed versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds inhibited hDHFR with Ki values included between 0.2 and 11 μM, while only a few (6, 21, 24, 27, 29) showed great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50 equal to -5.49 and log LC50 equal to -4.19 and maintained significant percent of growth inhibition on seven cancer cell lines at the concentration of 1 μM. Compound 17 was the second most active and moreover showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257, Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100-0.01 μM).


2013 - A proteomic approach to investigate the mechanism of action of anticancer peptides [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Ferrari, Stefania; Marverti, Gaetano; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. The main goal of our research is to identify compounds able to inhibit hTS by interfering with its dimerization, without causing its over-expression and the onset of cellular drug resistance against the traditional hTStargeted compounds. We have recently discovered some peptides which specifically target the hTS dimer interface and inhibit the enzyme by stabilizing its di-inactive form [1]. These molecules have been recently investigated for their SAR profile. LR, our lead compound, inhibits the intracellular enzyme in both cisplatin (cDDP)-sensitive and -resistant ovarian cancer cells without causing protein overexpression, thus showing a potential for overcoming the limits of OC chemotherapy. This work aims at setting up a proteomic approach able to provide information on the changes in the protein expression profile induced in OC cells by treatment with LR with respect to a well-known folate antimetabolite, Pemetrexed (PTX) and identify key proteins that are involved in its mechanism of action.


2013 - Biochemical effects of riluzole on Leishmania parasites [Articolo su rivista]
Guerrieri, Davide; Ferrari, Stefania; Costi, Maria Paola; Michels, Paul
abstract

We have previously shown that riluzole (6-(trifluoromethoxy)benzothiazol-2-amine), an agent used to treat CNS disorders, possesses inhibitory activity against pteridine reductase (PTR1) in pathogenic protists at low micromolar concentrations. Therefore, the potential use of this drug in antiparasitic chemotherapy deserves evaluation. In this study, we report the effect of this compound on cell cultures of Leishmania mexicana and L. major. The anti-parasitic activity of riluzole was confirmed, with the largest effect observed when the drug was administered to cells during their exponential growth phase. Moreover, a remarkable decrease in PTR1 activity was observed in the lysates of cells pretreated with the compound, which is due to impairment of the enzyme’s preferential reaction with biopterin as a cofactor. In addition, the treatment increased the parasites’ susceptibility to oxidative stress, affecting the ability of Leishmania to survive under severe oxidative conditions. These results suggest that the inhibitory effect of riluzole on PTR1 is not the only mechanism through which it induces the death of Leishmania parasites.


2013 - Chairperson EUTROC- Mayo Conference-Berlin - Preliminary characterization of pharmacodynamic biomarkers for LR-peptide growth inhibition of ovarian cancer (OC) cell models. [Altro]
Costi, Maria Paola; Amoroso, MARIA ROSARIA; Marverti, Gaetano; Genovese, Filippo
abstract

Preliminary characterization of pharmacodynamic biomarkers for LR-peptide growth inhibition of ovarian cancer (OC) cell models. M.R.Amorosoa,b, G.Marvertib, F.Genovese, DS. Matassab, J.HEllemanc, E.Bernsc, F.Espositob*, MP Costib*. aDept. Biomedical Sciences and cDept. Life Science, Via Campi 287-183, University of Modena and Reggio Emilia, 41125, Modena, Italy; Dept. of Medical Oncology, Erasmus University Medical Center - Cancer Center, PO Box 2040, 3000 CA, Rotterdam, Netherlands. bDepartment of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, Naples 80131, Italy. LR-peptide is an inhibitor of human Thymidylate synthase (TS) through the stabilization of its inactive form as shown in drug-protein interaction experiments (1). Our previous findings demonstrated an inhibition of ovarian cancer cell growth at 2-4 μM in four cis-platin sensitive and resistant ovarian cancer cell-lines, i.e. A2780 and A2780/CP, 2008 and C13. The level of inhibition caused by LR-peptide is similar to that of paclitaxel, a first line drug in OC therapy. Mass spectrometry differential proteomic studies were performed on A2780 OC cell models in which untreated cells were examined in comparison with LR-peptide treated cells (2). Bioinformatic analysis of the results led to the identification of at least 10 proteins that were modulated upon LR-peptide treatment. Among them TRAP1 an antiapoptotic mitochondrial HSP75 protein was observed. This result was the starting point for a deeper understanding of the role of the combined TRAP1/DHFR/hTS modulation in ovarian cancer cells. We first studied the role of TRAP1 alone in OC cell models. We treated a panel of ovarian cancer cells (including IGROV1 and COV504) with paclitaxel, whose cytotoxic activity involves the activation of ER (endoplasmic reticulum) stress pathways. TRAP1 shows a protective role from ER stress, as reported previously (3). We observed that upon 1-hour exposure to a sub-lethal concentration of paclitaxel, cells expressing higher TRAP1 levels (IGROV1) showed a weak activation of ER stress sensors. such as phospho PERK, phospho eIf2alpha and Grp78/BiP. COV504 cell line express lower level of TRAP1, are more sensitive to paclitaxel treatment, showing an hyperactivation of ER stress markers. Higher concentrations of paclitaxel for longer times led to apoptotic processes as confirmed by stronger activation of the ER-stress induced caspase 12, in COV cells than in IGROV cell line. These data confirm the protective role of TRAP1 against paclitaxel induced ER stress, offering a possible mechanism of drug resistance in ovarian cancer. 1.Cardinale D. et al., PNAS 2011. 2. Proceedings EPS32 Athen 2013 p_466 3. Amoroso MR, et al.Cell Death Differ. 2012 Acknowledgement This work has been supported by AIRC-DROC 10474 project to MPC.


2013 - Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity [Articolo su rivista]
Natalia, Realini; Carlos, Solorzano; Chiara, Pagliuca; Daniela, Pizzirani; Andrea, Armirotti; Luciani, Rosaria; Costi, Maria Paola; Tiziano, Bandiera; Daniele, Piomelli
abstract

The expression of acid ceramidase (AC) - a cysteine amidase that hydrolyses the proapoptotic lipid ceramide - is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents.


2013 - Fragment Based Discovery of Thymidylate Synthase Dimeric Interface Inhibitors Through Mass Spectrometry. Invited lecture to the Fragment Based Lead Discovery track. [Relazione in Atti di Convegno]
Costi, Maria Paola; Ponterini, Glauco; Ferrari, Stefania; Costantino, Luca; Franchini, Silvia; Venturelli, Alberto; Genovese, Filippo
abstract

Fragment-based drug design has been applied to Thymidylate synthase.The strategy applied is Mass Spectrometry related. identification of small molecule library that can bind to the protein represents the starting point for further drug design of a novel class of TS inhibitors with high potential as anticancer agents.


2013 - Invited lecture to 18th World Congress on Advances in Oncology and 16th International Symposium on Molecular Medicine 10-12 October, 2013, Creta Maris, Hersonissos, Crete, Greece [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Marverti, Gaetano; Taddia, Laura; Glauco, Ponterini; Pirondi, Silvia; Pela', Michela; Pavesi, Giorgia; Costi, Maria Paola
abstract

Proteomic approach to the identification of early phase biomarker for anticancer peptides targeting thefolate pathway. F.Genovesea, A.Gualandia,b L.Taddiaa, G.Ponterinia, G.Marvertia, S.Pirondia, R.Guerrinic, M.Pelàa,c G.Pavesia, C.Trapellac, M.P.Costia aUniversity of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy, bCRBA, S. Orsola University Hospital, Bologna, Italy, cDepartment of Pharmaceutical Science, University of Ferrara, Italy Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. In order to investigate the effects of hTS-interface-mimicking peptides at a cellular level, we started a study in which the cellular behavior of the peptides was investigated in combination with the proteomic differential analysis of the cytoplasmatic proteins of treated vs. untreated OC cells. The same experiment was performed with pemetrexed (PTX), a well known antifolate, for control purposes. The bioinformatic analysis of the effects of our peptide drug candidate indicates that deregulations can be mainly assigned to modulation of translational initiation, termination of RNA Pol-II transcription, transport, and protein catabolic events. Although apparently folate pathway members are not directly altered at a protein level, as the selection of ions to be sequenced is stochastic and biased towards abundant peptides, the bioinformatic analysis of peptide-modulated proteins suggested cellular investigations on the proteins of the folate-associated genes showing the largest number of dependencies to the species of the core set, which is required for the phosphorylation of several deoxyribonucleosides and nucleoside analogues. Comparison with the PTX-modulated proteins shows that some proteins of the proteasome complex and ribonucleoproteins are involved in both cases. These differences suggest that the two compounds may show a different mechanism of action which is in agreement with the hypothesized pharmacological model. Detailed cellular proteins profile based on the inferred roles of the identified proteins will further clarify the biological effects. 1.A proteomic approach to investigate the mechanism of action of anticancer peptides. F. Genovese, A. Gualandi, L. Taddia, M. Caselli, G. Ponterini, S.Ferrari, G. Marverti, R. Guerrini, M. Pela, G. Pavesi, C. Trapella, M.P. Costi, Proceeding 32EPS, p.466, ISBN 978-960-466-121-3). This work is supported by AIRC-DROC IG10474. www.unimore.airc-droc.it


2013 - Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-carboxamide and sulfonamide derivatives as thymidylate synthase A inhibitors [Articolo su rivista]
Ferrari, Stefania; Ingrami, M; Soragni, Fabrizia; Wade, Rc; Costi, Maria Paola
abstract

Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A deconstruction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was identified and two compound libraries based on this scaffold were designed. The carboxamide library (Library B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)-sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant against hTS with respect to the starting lead and represent potential compounds for further lead development.


2013 - Modulation of the expression of folate cycle enzymes and polyamine metabolism by berberine in cisplatin-sensitive and -resistant human ovarian cancer cells [Articolo su rivista]
Marverti, Gaetano; Ligabue, A; Lombardi, P; Ferrari, Stefania; Monti, Maria Giuseppina; Frassineti, Chiara; Costi, Maria Paola
abstract

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.


2013 - NUOVE MOLECOLE, PER USO COME AGENTI ANTITUMORALI [Brevetto]
Costi, Maria Paola; Costantino, Luca; Sammak, Susan; Ponterini, Glauco; Ferrari, Stefania; Luciani, Rosaria; Farina, Davide Salvatore Francesco; Franchini, Silvia; Santucci, Matteo; Gabriele, Cruciani; Emanuele, Carosati
abstract

Oggetto del presente brevetto è una classe di prodotti chimici sintetizzati presso i laboratori del Dipartimento Scienze della Vita. Questi prodotti si sono dimostrati in grado di destabilizzare l’omodimero della timidilato sintasi umana, e sono i primi composti noti dotati di tale attività; quindi, come viene dettagliato nel testo del brevetto, rappresentano degli ottimi leads per lo sviluppo di farmaci antitumorali in possesso di un meccanismo di azione completamente nuovo.


2013 - Protein-protein interaction inhibitors: case studies on Small Molecules and Natural Compounds. [Capitolo/Saggio]
Costi, Maria Paola; Pellati, Federica; Ferrari, Stefania
abstract

The large size and variety of the human protein interactome and the obvious relevance of the protein–protein interactions in every physiological function, render protein–protein interactions at the same time an extremely challenging and attractive target for developing of new therapeutic substances. A further interesting aspect of targeting protein–protein interactions (PPI) for drug discovery is that, at least in some cases, molecules directed against PPI may provide a way to overcome the resistance mechanisms encountered for active site binding enzyme inhibitors. The considerations above can be extended to bacterial and viral interactome, further expanding the vastness and complexity of the subject of PPI inhibition and modulation. For these reasons, there is no surprise that targeting PPI has become a subject of intense research activity in both industry and academia over the past decade. Reviewing the literature and the available databases shows that over 150 small molecule compounds have been found to inhibit about 20 PPI targets and that some of these molecules have already reached, or are about to reach, the drug market. As usual, these figures can have a double reading: the optimistic interpretation is that these successes demonstrate the validity of the approach, while the critics might say that the huge effort dedicated to find PPI inhibitors has resulted in scarce results. The fact is that exploring PPI is a stimulating new subject of study that is relevant for the basic knowledge on the chemistry of living organisms with the important outcome to offer the possibility of opening a new era of drug discovery. This book is a collection of essays from Italian research groups from Industry and University involved in drug discovery and, although the book presents different subjects in each chapter, the unifying idea comes from our belief that only an integrated approach of the different techniques nowadays available, may overcome the challenges presented by this new frontier in drug discovery. For this reason, the book opens with reviews about the current status of the research on PPI in drug discovery and goes on by presenting the state of the art in basic techniques like computational tools, NMR, X-ray crystallography and FRET that, integrated, may give the opportunity of success in this field.


2013 - PTEROATE-PEPTIDE BIOCONJUGATE TARGETING THE FOLATE RECEPTOR IN HUMAN OVARIAN CANCER CELL LINES: TRANSPORT AND MECHANISM OF ACTION. [Abstract in Rivista]
Costi, Maria Paola; Marverti, Gaetano; Pirondi, Silvia; Martello, Andrea; D'Arca, Domenico; Pela', Michela; Guerrini, R.; Marraccini, Chiara
abstract

Objectives The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the mechanisms of resistance to cisplatin (cDDP) encountered in most of resistant human ovarian cancer cell lines, accounting for the more efficient DNA repair and synthesis. Oligopeptides were designed to inhibit TS activity by interfering with its dimerization. Among these, the LR octapeptide showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines. To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR), which enters cell by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Methods -Cell lines. The human ovarian cancer cell lines OAW28, COV504, IGROV-1, TOV112D, 2008, C13*, A2780 and A2780/CP. -Real Time PCR of FRα mRNA . -Flow cytometric analysis of FRα cell surface expression -Folic acid surface binding studies. -Uptake studies Results Real Time PCR, western blot analysis and folic acid surface binding assay indicate that IGROV-1 and OAW28 cells show high expression levels of FRα, while TOV112D, 2008 and 2008/C13* almost don't express FRα on their cell surface. The folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake. FA-LR is detected in the cell and its stability evaluated. Conclusions The chemical modification of the folate with the LR drug motif only minimally altered the intrinsic affinity the biocongiugate for FR and suggest that the pteroate-peptide conjugate exploits FR as a substrate for its internalization. Cytotoxicity of the bioconjugate will be presented.


2013 - Translational repression of thymidylate synthase by targeting its mRNA [Articolo su rivista]
D., Garg; A. V., Beribisky; Ponterini, Glauco; Ligabue, Alessio; Marverti, Gaetano; Martello, Andrea; Costi, Maria Paola; M., Sattler; R. C., Wade
abstract

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.


2013 - 2′-Deoxyuridine 5′-Monophosphate Substrate Displacement in Thymidylate Synthase through 6-Hydroxy-2H-naphtho[1,8-bc]furan-2-one Derivatives [Articolo su rivista]
Ferrari, Stefania; Calò, S; Leone, R; Luciani, Rosaria; Costantino, Luca; Sammak, Susan; Di Pisa, F; Pozzi, C; Mangani, S; Costi, Maria Paola
abstract

Thymidylate synthase (TS) is a target for antifolate-based chemotherapies of microbial and human diseases. Here, ligand-based, synthetic, and X-ray crystallography studies led to the discovery of 6-(3-cyanobenzoyloxy)-2-oxo-2H-naphto[1,8-bc]furan, a novel inhibitor with a Ki of 310 nM against Pneumocystis carinii TS. The X-ray ternary complex with Escherichia coli TS revealed, for the first time, displacement of the substrate toward the dimeric protein interface, thus providing new opportunities for further design of specific inhibitors of microbial pathogens.


2012 - Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines. [Articolo su rivista]
Giudice, Stefania; Benassi, Luisa; Bertazzoni, G; Veratti, E; Morini, D; Azzoni, Paola; Costi, Maria Paola; Venturelli, Alberto; Pirondi, Silvia; Seidenari, Stefania; Magnoni, Cristina
abstract

Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.


2012 - Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells. [Articolo su rivista]
Marverti, Gaetano; Guaitoli, Giambattista; Ligabue, Alessio; Frassineti, Chiara; Monti, Maria Giuseppina; P., Lombardi; Costi, Maria Paola
abstract

Acquired resistance to cisplatin (cDDP) is a multifactorial process that represents one of the main problems in ovarian cancer therapy. Distamycin A is a minor groove DNA binder whose toxicity has limited its use and prompted the synthesis of derivatives such as NAX001 and NAX002, which have a carbamoyl moiety and different numbers of pyrrolamidine groups. Their interaction with a B-DNA model and with an extended-TATA box model, [Polyd(AT)], was investigated using isothermal titration calorimetry (ITC) to better understand their mechanism of interaction with DNA and therefore better explain their cellular effects. Distamycin A interactions with Dickerson and Poly[d(AT)6] oligonucleotides show a different thermodynamic with respect to NAX002. The bulkier distamycin A analogue shows a non optimal binding to DNA due to its additional pyrrolamidine group. Cellular assays performed on cDDP-sensitive and -resistant cells showed that these compounds, distamycin A in particular, affect the expression of folate cycle enzymes even at cellular level. The optimal interaction of distamycin A with DNA may account for the down-regulation of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and the up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) caused by this compound. These effects seem differently modulated by the cDDP-resistance phenotype. On the contrary, NAX002 which presents a lower affinity to DNA and slightly affected these enzymes, showed a synergic inhibition profile in combination with cDDP. In addition, their combination with cDDP or polyamine analogues increased cell sensitivity to the drugs suggesting that these interactions may have potential for development in the treatment of ovarian carcinoma.


2012 - Dottorato regionale Spinner-Nuove molecole per il controllo e la differenziazione delle cellule staminali-NovaMolStam- Università di Bologna, Ferrara, Modena e Reggio Emilia, Parma-Anno 2012-2015- [Altro]
Costi, Maria Paola; Costantino, Luca; Ponterini, Glauco; Cannazza, Giuseppe
abstract

L’uso di cellule staminali rappresenta un approccio promettente nel trattamento di malattie degenerative e di altre patologie che richiedono la rigenerazione tissutale. Tuttavia, nell’applicazione di questa strategia si incontrano numerosi problemi, quali il controllo della differenziazione, le reazioni immunitarie che si scatenano nell’applicazione, la specificità della differenziazione e della produzione stessa delle cellule. Per affrontare questi problemi è necessario conoscere la biologia cellulare, e i cammini (“pathway”) metabolici più importanti che governano i processi di differenziazione. Alcuni prodotti naturali e piccole molecole di origine sintetica si sono rivelati utili nel controllo e nella differenziazione delle cellule staminali. Tra questi, ad es., l’acido retinoico, il forskolin, il desametasone, l’azacitidina, che tuttavia mancano di specificità d’azione ed in generale hanno caratteristiche non ottimali. Sulla base delle conoscenze esistenti e dei composti attualmente in uso, appare quindi necessario e possibile ricercare nuove molecole dotate di profili biologici specifici. Il presente progetto ha l’obiettivo di identificare nuovi composti dotati di specificità d’azione verso cammini metabolici importanti per il controllo e la differenziazione delle cellule staminali. Tali composti saranno molecole di origine naturale e/o sintetica e potranno essere utilizzati per ottimizzare la produzione delle staminali da usare per scopi clinici. Non sono da intendere come farmaci, ma come sostanze che supportano e promuovono il miglioramento della tecnologia di produzione delle cellule per uso clinico. Per raggiungere questo obiettivo, il progetto si articolerà in fasi successive che prevedono: a) l’identificazione dei cammini metabolici più adatti per controllare le cellule staminali e la selezione dei bersagli molecolari più adeguati all’interno dei pathway; b) la progettazione di molecole potenzialmente attive su questi bersagli mediante strategie quali il “virtual screening”, l’analogia strutturale rispetto a composti attivi esistenti e lo screening biologico diretto; c) la sintesi dei composti o il loro isolamento da fonti naturali; d) la valutazione biologica su panel di enzimi e proteine e su cellule; e) l’ottimizzazione dei prodotti migliori selezionati dalle fasi b-d mediante criteri di “drug-likeness” e successivi cicli di sintesi e valutazione biologica. Per raggiungere i suddetti obiettivi verranno applicate strategie proprie della ricerca farmaceutica. Il punto a) prevede l’utilizzo di strumenti bioinformatici per la ricerca e l’analisi di database, lo studio di proprietà strutturali e funzionali delle proteine coinvolte per la prioritizzazione dei bersagli più adeguati. Il punto b) prevede l’utilizzo di protocolli per l’identificazione di composti nuovi da banche dati di composti noti (“virtual screening”), la progettazione di composti basati sulla conoscenza delle strutture 3D dei bersagli selezionati, lo screening sperimentale di librerie molecolari disponibili nei laboratori di ricerca dei partecipanti al progetto e/o la loro valutazione diretta sulle linee cellulari staminali presso i laboratori di collaboratori interessati al progetto (Centro di Medicina Rigenerativa di Modena). Il punto c) prevede l’utilizzo di tecniche di sintesi chimica per ottenere i composti progettati, l’estrazione di prodotti naturali da piante studiate nei laboratori coinvolti nel progetto, la caratterizzazione, separazione e sintesi dei prodotti naturali identificati come interessanti. Il punto d) prevede la valutazione biologica dei nuovi composti sintetizzati verso le biomolecole bersaglio scelte. Successivamente i composti migliori saranno valutati sulle linee cellulari staminali presso i laboratori del Centro di Medicina Rigenerativa di Modena, similmente al punto b), ma con metodiche più raffinate per val


2012 - Drug approval process for ovarian cancer [Altro]
Costi, Maria Paola
abstract

Prof Maria Paola Costi talks to ecancer at the 4th EUTROC meeting in Liverpool, UK about new drug developments and the main issues surrounding the drug approval process for ovarian cancer. She highlights work being done on folate receptors and their role in ovarian cancer; in particular, a project discussed at the meeting on candidate drugs targeting this receptor and the difficulty of advancing these drugs to the clinical stages.


2012 - ESMEC invited lecture. Targeting the Biopterines Reduction Pathway for the Development of New Drugs against Leishmaniasis and Trypanosomiasis. [Altro]
Costi, Maria Paola
abstract

Targeting the Biopterines Reduction Pathway for the Development of New Drugs against Leishmaniasis and Trypanosomiasis. Premise. The discovery of new drugs in the field of neglected diseases represents a major task within the whole drug discovery area. Why is the number of drugs in the discovery pipelines so low? Why are we still using so many old drugs? Why is it more difficult to promote a drug candidate to the clinical use in the area of neglected disease? One of the answers may be the requirements of the drug profile in this area that show more constraints than in any other area. In fact the ideal drug should be very specific for the pathogen, easy to be synthetically prepared, stable at room temperature, orally available, one dose for daily use, extremely low cost and easy to combine with other existing drugs to delay resistance development. Apart from the mentioned intrinsic features, the highest barrier at the moment is due to the low level of critical mass working in the area and the low grant level. The number of groups that work on parasitic diseases is limited due to the level of financing in comparison with other therapeutic area. This has a statistical relevance on the output quality and on the pipeline filing 1. A drug discovery case in the preclinical phase within the folate pathway of the Kintetoplastidae parasites will be presented.


2012 - European COST B22 Lab for antiparasitic drug screening (http://www.icp.ucl.ac.be/cost/costB22/ screening_survey.pdf )-2008-2012. [Altro]
Costi, Maria Paola; Ferrari, Stefania
abstract

European COST B22 Lab for antiparasitic drug screening (http://www.icp.ucl.ac.be/cost/costB22/ screening_survey.pdf ). My lab is part of the new COST action on “Drug development in parasitic diseases” CM0801 (http://www.costcm0801.org/CM0801/Welcome.html ) 2008-2012. The initiative is reported in the following text: 2008 | Action Number: B22 In Vitro and In Vivo Screenings of New Antiparasitic Compounds: State-of-the-art and New Developments Pages: 92 Author(s): Reto Brun, Donatella Taramelli Publisher(s): Swiss Tropical Institute Joint meeting of COST B22 WG3 Drug Evaluation, WG4 Preclinical Research and EU-IP Antimal.


2012 - Folate Receptor Expression and Folate Receptor Targeted Chemotherapy in Cisplatin-sensitive and – resistant Human cancer cell lines Marverti G.a, Pirondi S.a, MarracciniC.a, Frassineti C.a, Helleman J.b, Berns E.M.J.J. b, and Costi, M.P. cThe 4th International Symposium on Folate Receptors and Transporters Cozumel, Mexico October 7 - 11, 2012 [Poster]
Marverti, Gaetano; Pirondi, Silvia; Marraccini, Chiara; Frassineti, Chiara; Costi, Maria Paola
abstract

Folate Receptor Expression and Folate Receptor Targeted Chemotherapy in Cisplatin-sensitive and – resistant Human cancer cell lines Marverti G.a, Pirondi S.a, MarracciniC.a, Frassineti C.a, Helleman J.b, Berns E.M.J.J. b, and Costi, M.P. c aDept. Biomedical Sciences and cDept. Pharmaceutical Sciences, Via Campi 287-183, University of Modena and Reggio Emilia, 41125, Modena, Italy; Dept. of Medical Oncology, bErasmus University Medical Center - Daniel den Hoed Cancer Center, PO Box 2040, 3000 CA, Rotterdam,The Netherlands. Targeted drug delivery systems promise to expand the therapeutic windows of drugs by increasing delivery to the target tissue as well as the target–non-target tissue ratio. Interest in exploiting the folate receptor (FR) for drug targeting applications has rapidly increased because it is a tumor-associated antigen that is overex- pressed in greater than 90% of human ovarian carcinomas and associated with increased biological aggressive- ness of this tumor [1]. The most significant advantage to emerge from studies of FR-mediated delivery has been the surprisingly low level of toxicity to normal tissues which constitute one of the most significant merits of the FR-targeting strategy. Therefore, FR presents an attractive target for tumor-selective drug delivery. The final goal of this research is to analyze the effects of oligopeptides, designed to inhibit thymidylate syn- thase (TS) activity by interfering with its dimerization, without causing TS over-expression and the develop- ment of cellular drug resistance against the traditional TS-targeted compounds. The over-expression of TS and the others folate cycle enzymes, is one of the major mechanisms of resistance to cisplatin (cDDP), encountered in most of resistant human ovarian cancer cell lines [2], accounting for the more efficient DNA repair and syn- thesis. To this aim, we have chosen two cisplatin-sensitive human ovarian cancer cell lines, 2008 and A2780, and their –resistant counterparts, C13* and A2780/CP cell lines, respectively, in order to display and study possi- ble different responses modulated by cDDP-resistance. At first, the cell lines have been tested for their total levels of FR and for functionally active receptors (FR or also other receptors?). The quantification of functional FR by the microfiltration method [3] and by an alterna- tive method, which deduces FR amount from the folic acid (FA) binding to FR [4] indicate that 2008 and C13* cells present a 3-4 fold higher expression of FR than A2780 and A2780/CP cells. However, time-dependent and concentration-dependent studies revealed that despite their higher expression of FR and the higher [3H]folic acid binding capacity, 2008 and C13* cell lines appeared to saturate earlier than A2780 and A2780/CP cell lines since they accumulated less [3H]folic acid at concentrations higher than 150 nM. Substrate specificity studies of the saturable uptake process, revealed that the 30 nM [3H]Folic acid uptake in A2780 cells was more affected than in the resistant line A2780/CP to the presence of 20μM of unlabeled folic acid (FA) and methotrexate (MTX), respectively (I do not understand this sentence, A2780 is more sensitive to unlabeled folic acid and mtx after labeled FA treatment? How do you measure this sensitivity? Do you mean affected/decreased uptake?). On the contrary, 30 nM [3H]Folic acid uptake was unaffected by unlabeled FA and MTX in both 2008 and C13* cell lines.  To evaluate the presence of ATP-dependent process affecting FA uptake, experiments were also performed at 4°C and compared with those at 37°C. The accumulation of [3H]folic acid was greatly reduced at 4°C in comparison to 37°C both in 2008 and C13* cells, whereas only about 6- and 4-fold reductions were detected in A2780 and A2780/CP cells, respectively . These results are, at least partly, in accordance with the competitive uptake studies, since they suggest that in t


2012 - Inhibitor of Ovarian Cancer Cells growth by Virtual Screening: A New Thiazole Derivative Targeting Human Thymidylate Synthase [Articolo su rivista]
Emanuele, Carosati; Anna, Tochowicz; Marverti, Gaetano; Giambattista, Guaitoli; Paolo, Benedetti; Ferrari, Stefania; Robert M., Stroud; Janet Sue Finer, Moore; Luciani, Rosaria; Davide, Farina; Gabriele, Cruciani; Costi, Maria Paola
abstract

Human Thymidylate Synthase (hTS) was targeted through a virtual screening approach. The most optimal inhibitor identified, 2-{4-hydroxy-2-[(2-hydroxy-benzylidene)-hydrazono]-2,5-dihydro-thiazol-5-yl}-N-(3-trifluoromethyl-phenyl)-acetamide (5), showed a mixed-type inhibition pattern. The inhibitor exhibited a Ki of 1.3 µM and was active against four ovarian cancer cell lines with the same potency as cisplatin. The co-crystal structure with hTS revealed that the inhibitor binds the inactive enzyme conformation. This study is the first example of a non-peptidic inhibitor that binds the inactive hTS and exhibits anticancer activity against ovarian cancer cells.


2012 - Laboratory of reference for trypanosomatidic infections -In-Vitro-and-In-Vivo-Screenings-of-New-Antiparasitic-Compounds-State-of-the-art-and-New-Developments-COST B22 [Altro]
Costi, Maria Paola; Ferrari, Stefania
abstract

2008 | Action Number: B22 In Vitro and In Vivo Screenings of New Antiparasitic Compounds: State-of-the-art and New Developments Pages: 92 Author(s): Reto Brun, Donatella Taramelli Publisher(s): Swiss Tropical Institute Joint meeting of COST B22 WG3 Drug Evaluation, WG4 Preclinical Research and EU-IP Antimal.


2012 - MITO-group Multicenter italian trials in ovarian cancer and gynecological malignancies Borad of directors-translational research group [Altro]
Costi, Maria Paola
abstract

group MITO "Multicenter Italian Trials in Ovarian cancer and gynecologic malignancies" The Association is a non-profit organization, and aims to bring together lovers of Gynecologic Oncology in order to promote progress in the clinical, experimental, and social welfare, to foster relations between the gynecology oncology specialists, general practitioners and specialists in other disciplines, to establish scientific relations with similar associations and Italian and foreign institutional organizations to participate in national, regional and local. The Association also aims to promote experimental and clinical research, the quality of cancer care through the preparation of Guidelines for the surgical and medical therapy cancer, and interdisciplinarity. Translational research has been recently included in the main disciplines and research approaches.


2012 - Proteomic Approach to the Detection of the Mechanism of Action of Anticancer Peptides [Abstract in Rivista]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Marverti, Gaetano; Pirondi, Silvia; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

A label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. Structure-activity relationships (SAR) have been performed to improve the lead peptide pharmacodynamics. All the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of OC cell growth.


2012 - Structure-Based Selectivity Optimization of Piperidine–Pteridine Derivatives as Potent Leishmania Pteridine Reductase Inhibitors [Articolo su rivista]
Paola, Corona; Gibellini, Federica; Saxena, Puneet; Luciani, Rosaria; Guerrieri, Davide; Nerini, Erika; Ferrari, Stefania; Costi, Maria Paola
abstract

The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasite’s folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.


2012 - Targeting the trypanosomatidic enzymes pteridine reductase and dihydrofolate reductase. [Capitolo/Saggio]
Ferrari, Stefania; Valeria, Losasso; Saxena, Puneet; Costi, Maria Paola
abstract

Drugs currently in use against Leishmania and Trypanosoma infections have limitations in terms of efficacy, safety, duration of the treatment, toxicity and resistance. It therefore is mandatory to identify molecular targets to be specifically inhibited. Folate is an essential cofactor in the biosynthesis of DNA and amino acids. The inhibition of its metabolism leads to the alterations of cell replication and function. Only a few trypanosomatid enzymes of the folate pathway are presently discussed as potential targets, among them the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase (PTR). The identification of a specific enzyme such as PTR1, able to reduce folates other than biopterins, allowed the understanding of the resistance of trypanosomatids against known antifolate drugs. In most cases only the inhibition of both enzymes, DHFR-TS and PTR would fully arrest the pathway’s metabolic function. The proposed combination therapy opens up a novel approach: re-positioning of the well established antifolate strategy for the treatment of trypanosomatid diseases by the discovery of novel antifolates that complement the efficacy profile of known drugs. The present article compiles the existing medicinal chemistry approaches specifically targeting the folate pathway in trypanosomatids, in particular PTR and the DHFR activity of DHFR-TS. It covers the structural biology of the targets, related computational studies, core structures synthesis and biological inhibitor characterization.


2012 - The structure of Enterococcus faecalis thymidylate synthase provides clues about folate bacterial metabolism [Articolo su rivista]
C., Pozzi; Ferrari, Stefania; D., Cortesi; Luciani, Rosaria; R. M., Stroud; A., Catalano; Costi, Maria Paola; S., Mangani
abstract

Drug resistance to therapeutic antibiotics poses a challenge to the identification of novel targets and drugs for the treatment of infectious diseases. Infections caused by Enterococcus faecalis are a major health problem. Thymidylate synthase (TS) from E. faecalis is a potential target for antibacterial therapy. We have determined the X-ray crystallographic structure of the E. faecalis thymidylate synthase (EfTS), obtained as a native binary complex composed of EfTS and 5-formyltetrahydrofolate (5-FTHF). The structure provide evidence that EfTS is a half-the-sites reactive enzyme as 5-FTHF is bound to two of the four independent subunits present in the crystal asymmetric unit. 5-FTHF is a metabolite of the one-carbon transfer reaction catalysed by 5-formyltetrahydrofolate cycloligase. Kinetic studies show that 5-FTHF is a weak inhibitor of EfTS, suggesting that the EfTS-5-FTHF complex may function as a source of folates and/or may regulate one-carbon metabolism. The structure represents the first example of endogenous 5-FTHF bound to a protein involved in folate metabolism.


2012 - Unusual Targeting of the human Thymidylate synthase interface: a tethering approach with mass-spectrometric detection. [Relazione in Atti di Convegno]
Costi, Maria Paola; Franchini, Silvia; Ferrari, Stefania; R., Wade; S., Henrich; O., Salo; Genovese, Filippo; S., Mangani; Pozzi, Cristina; M., Santucci; Costantino, Luca; Sammak, Susan; G., Cruciani; E., Carosati; Ponterini, Glauco
abstract

Ovarian cancer is the fifth most common cause of death from cancer in women. The standard first-line treatment is a combination of paclitaxel and carboplatin (DDP) or carboplatin alone. In the case of progressive disease or drug resistance treatment with platinum, either alone or in combination, investigational compounds should be used (1). In the human ovarian carcinoma cell DDP-resistance was associated with cross-resistance to the thymidylate synthase (TS) inhibitor 5-fluorouracil (prodrug of 5FdUMP, fluorodeoxyuridine monophosphate) and methotrexate (2). We aim at restoring the sensibility to Platinum-based drugs through direct inhibition of Thymidylate synthase (TS) changing the mechanism of action from active site binders (classical TS inhibitors) to inhibitors of the regulatory function of monomeric TS through small molecule cellular perturbation. To this aim we applied a multidisciplinary approach to identify new molecules that could bind to specific pocket at the protein interface. We applied the tethering approach (3) that leads to the selection of disulfide compounds to anchor at the cystein on the monomeric interface (gray coloured balls on the left in the picture). Mass Spectrometry (MS) identification of the covalent TS-thiol complexes, and medicinal chemistry development of the identified hits (coloured structure on the right in the picture). We included cystein mutants design, site directed mutagenesis, disulfide library selection, tethering of thiol ligands at the protein interface through Mass spectrometry, X-ray crystallography, structure-based drug design and synthetic chemistry. The validation of the TS-interface inhibitor binders was accomplished trough FRET (Fluorescence resonance energy transfer) and enzyme kinetic assay. The strategies adopted and the midpoint/final results of the discovery processes will be presented. 1. Ozols RF et al. Lancet 2002;. 2. D.Cardinale et al, CMC, 2010, D.Cardinale et al, PNAS, 2011 ; 3. Stroud R et al PNAS 2000 and www.light-eu.org. The project is supported by FP6 european grant (LIGHTS, www.light-eu.org), LSH 038752 and AIRC-DROC-2012(http://www.droc-airc.unimore.it/site/home.html).


2012 - Zing meeting on Medicinal Chemistry 2012-Lanzarote-invitated lecture [Relazione in Atti di Convegno]
Costi, Maria Paola; Ferrari, Stefania; Ponterini, Glauco; Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella
abstract

Allosteric modulators of Thymidylate synthase, an anticancer drug target Costi MP, Wade Rc, Ferrari Sa, Ponterini Gd, Cardinale Da, Guaitoli Ga, Tondi Da, Marverti Ge, Myllikallio H.f Mangani S.g Thymdylate synthase (TS) plays a key role in the biosynthetic supply of thymidylate, an essential precursor for DNA replication and repair. Indeed, in human cells, downregulation of this pathway halts cellular replication and leads to apoptosis of cancer cells so, hTS active-site inhibitors are largely used in cancer chemotherapy. On the other hand, resistance frequently sets in during treatment with these drugs. While the molecular bases of this phenomenon are complex, there are evidences that it is correlated with protein overexpression presumably connected with the loss of RNA-binding capacity when the protein is bound to its inhibitors. It is therefore desirable to identify inhibitors that act through new mechanisms, such that RNA regulation is not altered1. The design of peptides that mimic portions of the monomer-monomer interface of multimeric proteins has been shown to be a useful approach for the discovery of inhibitors that bind at such interfaces2. In this contribution, we wish to show how these peptides interact with hTS in vitro: the main thermodynamic and structural aspects of this interaction, as well as its consequences on the enzyme catalytic efficiency, have been determined through a multidisciplinary approach of computational design, synthesis, X-ray crystallography and other biophysical techniques. These findings suggest a new mechanism of action and provide useful information for testing against ovarian cancer cells. We have tested the most potent peptides against ovarian cancer cells sensitive and resistant to cis-platin. The peptides were able to inhibit cancer cell growth and, differently from the classical inhibitors, do not show protein over expression. References. 1. E. Chu, et al. Identification of an RNA binding site for human thymidylate synthase. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 517-521. 2. D. Cardinale, and M.P.Costi et al. Homodimeric enzymes as drug targets. Curr Med Chem. 2010,17, 826-46. The project is supported by FP6 european grant (LIGHTS, Small ligands to interfere with Thymidylate synthase dimer formation as new tools for development of anticancer agents against ovarian carcinoma. www.light-eu.org), LSH 038752. 


2011 - Crystal Structure of histidine-tagged human thymidylate synthase [Altro]
Pozzi, Cristina; Cardinale, Daniela; Guaitoli, G.; Tondi, Donatella; Luciani, Rosaria; Myllykallio, H.; Ferrari, Stefania; Costi, Maria Paola; Mangani, S.
abstract

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.


2011 - Crystal Structure of human thymidylate synthase bound to a peptide inhibitor [Altro]
Pozzi, Cristina; Cardinale, Daniela; Guaitoli, G.; Tondi, Donatella; Luciani, Rosaria; Myllykallio, H.; Ferrari, Stefania; Costi, Maria Paola; Mangani, S.
abstract

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.


2011 - Erratum: Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase (Proceedings of the National Academy of Sciences of the United States of America (2011) 108, 34 (E542-E549) DOI: 10.1073/pnas.1104829108) [Articolo su rivista]
Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, O. M. H.; Ferrari, S.; Marverti, G.; Guerrieri, D.; Ligabue, A.; Frassineti, C.; Pozzi, C.; Mangani, S.; Fessas, D.; Guerrini, R.; Ponterini, G.; Wade, R. C.; Costi, M. P.
abstract


2011 - Identification of the binding modes of N-phenylphthalimides inhibiting bacterial thymidylate synthase through X-ray crystallography screening. [Articolo su rivista]
Mangani, S; Cancian, Laura; Leone, R; Pozzi, C; Lazzari, Sandra; Luciani, Rosaria; Ferrari, Stefania; Costi, Maria Paola
abstract

To identify specific bacterial thymidylate synthase (TS) inhibitors, we exploited phenolphthalein (PTH), which inhibits both bacterial and human enzymes. The X-ray crystal structure of Lactobacillus casei TS (LcTS) that binds PTH showed multiple binding modes of the inhibitor, which prevented a classical structure-based drug design approach. To overcome this issue, we synthesized two phthalimidic libraries that were tested against TS enzymes and then we performed X-ray crystallographic screening of the active compounds. Compounds 6A, 8A, and 12A showed 40-fold higher affinity for bacterial TS than human TS. The X-ray crystallographic screening characterized the binding mode of six inhibitors in complexes with LcTS. Of these, 20A, 23A, and 24A showed a common unique binding mode, whereas 8A showed a different, unique binding mode. A comparative analysis of the LcTS X-ray complexes that were obtained with the pathogenic TS enabled the selection of compounds 8A and 23A as specific compounds and starting points to be exploited for the specific inhibition of pathogen enzymes.


2011 - Permeation through the cell membrane of a boron-based β-lactamase inhibitor. [Articolo su rivista]
Minozzi, M.; Lattanzi, G.; Benz, R.; Costi, M. P.; Venturelli, A.; Carloni, P.
abstract

Bacteria express beta-lactamases to counteract the beneficial action of antibiotics. Benzo[b]-thiophene-2-boronic acid (BZB)derivatives are b-lactamase inhibitors and, as such, promising compounds to be associated with b-lactam antibacterialtherapies. The uncharged form of BZB, in particular, is suggested to diffuse through the outer membrane of Gram negativebacteria. In this study, through the combination of electrophysiological experiments across reconstituted PC/n-decanebilayers and metadynamics-based free energy calculations, we investigate the permeation mechanism of boroniccompounds. Our experimental data establish that BZB passes through the membrane, while computer simulations providehints for the existence of an aqueous, water-filled monomolecular channel. These findings provide new perspectives for thedesign of boronic acid derivatives with high membrane permeability.


2011 - Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase [Articolo su rivista]
Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; S., Henrich; O. M. H., Salo Ahen; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; C., Pozzi; S., Mangani; D., Fessas; R., Guerrini; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola
abstract

Human thymidylate synthase is a homodimeric enzyme that playsa key role in DNA synthesis and is a target for several clinicallyimportant anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The “LR” peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.


2011 - Pteridinereductase (PTR1);dihydrofolatereductase (DHFR) [Articolo su rivista]
Costi, Maria Paola; Wade, R.
abstract

In vitro studies identified antifolate inhibitors ofLeishmania major Ptr1 that could be used withDhfr inhibitors to help treat parasitic infection. Incultured L. major, five Ptr1-targeting compoundsshowed efficacy against the parasite in combinationwith the Dhfr inhibitor pyrimethamine. One of thePtr1-targeting compounds also showed efficacy asmonotherapy. Ongoing studies include developingcompounds to use in tandem with the inhibitors.


2011 - Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase [Articolo su rivista]
Ferrari, Stefania; Morandi, Federica; Motiejunas, D.; Nerini, Erika; Henrich, S.; Luciani, Rosaria; Venturelli, Alberto; Lazzari, Sandra; Calo', Samuele; Gupta, S.; Hannaert, V.; Michels, P. A. M.; Wade, R. C.; Costi, Maria Paola
abstract

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential anti-parasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-Chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies was active in combination and is suitable for early pre-clinical evaluation of its potential for label extension as a PTR1 inhibitor and anti-parasitic drug candidate.


2010 - Allosteric Inhibition of human Thymidylate Synthase. [Abstract in Atti di Convegno]
S., Henrich; O., Salo Ahen; D., Garg; Cardinale, Daniela; Ferrari, Stefania; Franchini, Silvia; Genovese, Filippo; Guaitoli, Giambattista; Lazzari, Sandra; Venturelli, Alberto; S., Mangani; Costi, Maria Paola; R. C., Wade
abstract

Thymidylate synthase (TS) takes part in the folate pathway and is a dimeric enzyme that catalyzes the conversion from dUMP to dTMP. This reaction step is essential for all cells, but especially for fast growing cancer cells, making TS an important target for cancer treatment. Most of the clinical drugs for inhibiting TS are substrate or cofactor analogues that give rise to resistance after a certain time. The aim of the LIGHTS EU project is to overcome such resistance to inhibitors by interfering with TS dimerizationusing low molecular weight compounds and peptides binding to allosteric sites.


2010 - Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding. [Articolo su rivista]
Cardinale, Daniela; O. M. H., Salo Ahen; Guaitoli, Giambattista; Ferrari, Stefania; Venturelli, Alberto; Franchini, Silvia; Battini, Renata; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola
abstract

Due to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, and we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.


2010 - Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescenceresonance energy transfer. [Articolo su rivista]
Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco
abstract

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43' with an excitation energy donor/acceptor pair. The dimer-monomer equilibrium of the enzyme is then characterized through steady-state fluorescence determination of the inter-subunit resonance energy transfer efficiency.


2010 - Drug resistance in ovarian cancer: biomarkers andtreatments Highlights from the DROC meeting held in Modena (Italy) on the 19th and 20th of February 2009. Scientific topics discussed at the meeting are reported in the present issue. [Articolo su rivista]
Costi, Maria Paola; R., Zeillinger
abstract

Ovarian cancer is the fifth most common cause of death from cancer and the most common cause of death from gynecologic cancer in women of all ages in the Western world. This primarily reflects the fact that there are no early warning signs, and most patients with epithelial ovarian cancer present at an advanced stage of the pathology at the time of diagnosis. Efforts to improve survival continue to focus on the development of more effective systemic therapies, by the identification of biomarkers for early diagnosis, improving drug efficacy and contrasting drug resistance. Early diagnosis is crucial, marking the difference between a 95 percent survival rate for cancers found at the earliest stage and 20 percent survival among patients diagnosed with advanced disease. New and higher quality therapies are at the frontiers. The area is fragmented and needs to be more coordinated with the clinical aspects. Drug discovery, drug safety, drug delivery, proteomics, system biology, pharmacogenetics, diagnostics for patient selection and therapy monitoring in clinical trials are essential and strictly interconnected.In this event we would like to point out these aspects and the need to technically and scientifically link the different disciplines under the same aim of identifying a compact strategy to combat ovarian cancer. Biomakers and treatments are the topics of the meeting and experts from different area will present their work and will create a platform to avenue translational research in this area.


2010 - Enzyme inhibition in drug discovery and development. The good and the bad. [Recensione in Rivista]
Costi, Maria Paola
abstract


2010 - Homodimeric Enzymes as Drug Targets [Articolo su rivista]
Cardinale, Daniela; O. M. H., Salo Ahen; Ferrari, Stefania; Ponterini, Glauco; G., Cruciani; E., Carosati; A. M., Tochowicz; S., Mangani; R. C., Wade; Costi, Maria Paola
abstract

Many enzymes and proteins are regulated by their quaternary structure and/or by their association in homoand/or hetero-oligomer complexes. Thus, these protein-protein interactions can be good targets for blocking or modulatingprotein function therapeutically. The large number of oligomeric structures in the Protein Data Bank (http://www.rcsb.org/) reflects growing interest in proteins that function as multimeric complexes. In this review, we consider the particularcase of homodimeric enzymes as drug targets. There is intense interest in drugs that inhibit dimerization of a functionallyobligate homodimeric enzyme. Because amino acid conservation within enzyme interfaces is often low compared to conservationin active sites, it may be easier to achieve drugs that target protein interfaces selectively and specifically. Twomain types of dimerization inhibitors have been developed: peptides or peptidomimetics based on sequences involved inprotein-protein interactions, and small molecules that act at hot spots in protein-protein interfaces. Examples include inhibitorsof HIV protease and HIV integrase. Studying the mechanisms of action and locating the binding sites of such inhibitorsrequires different techniques for different proteins. For some enzymes, ligand binding is only detectable in vivo orafter unfolding of the complexes. Here, we review the structural features of dimeric enzymes and give examples of inhibitionthrough interference in dimer stability. Several techniques for studying these complex phenomena will be presented.


2010 - Ligand-Based Virtual Screening and ADME-Tox Guided Approach to Identify Triazolo-quinoxalines as Folate Cycle Inhibitors. [Articolo su rivista]
E., Carosati; G., Sforna; M., Pippi; Marverti, Gaetano; Ligabue, Alessio; Guerrieri, Davide; S., Piras; Guaitoli, Giambattista; Luciani, Rosaria; Costi, Maria Paola; G., Cruciani
abstract

In the process of drug discovery the lead-identification phase may be critical due to the likely poor safety profile of the candidates, causing the delay or even the abandonment of a certain project. Nowadays, combining molecular modeling and in vivo cellular evaluation can help to identify compounds with an enhanced safety profile. Previously, two quinoxalines have been identified as inhibitors of the folate-dependent proteins belonging to the thymidylate synthase cycle. Unfortunately, cytotoxic activity against a panel of cisplatin(cDDP)-sensitive ovarian carcinoma cell lines and their resistant counterparts was coupled with toxicity to non-tumorigenic Vero cells. Here we describe the application of a ligand-based virtual screening, and several [1,2,4]triazolo[4,3-a]quinoxalines were optimized to improve their ADME-tox profile. The resulting 4-(trifluoromethyl)-1-p-tolyl-[1,2,4]triazolo[4,3-a]quinoxaline (24), which interferes intracellularly with DHFR and TS reducing the protein levels like 5-FU, but without inducing TS ternary complex formation, was 2-times less toxic in vitro than cisplatin and 5-FU.


2010 - Novel Approaches for Targeting Thymidylate Synthase to Overcome the Resistance and Toxicity of Anticancer Drugs [Articolo su rivista]
Garg, Divita; Henrich, Stefan; Salo Ahen, Outi; Myllykallio, Hannu; Costi, Maria Paola; Wade, Rebecca
abstract

The antifolate drug, methotrexate, was introduced to the clinic as an anticancer agent in the early 1950s.1 Subsequently, its mechanism of action was elucidated and it was found to bind in mono- and poly-glutamated forms to dihydrofolate reductase (DHFR),2,3 thymidylate synthase (TS)4 and amino-imidazolecarboxamide-ribonucleotide transformylase (AICARTF).5 A fluoropyrimidine, 5-fluorouracil (5FU), was conceived in 19576 following the observation that uracil was utilised preferentially in malignant over non-malignant cells,7 and has since been a first line drug in cancer chemotherapy. Subsequently, 5-fluoro-2'-deoxyuridine-5’-monophosphate (5FdUMP), an active metabolite of 5FU, was found to inhibit TS by forming a covalent ternary complex with the enzyme and 5,10-methylenetetrahydrofolate (mTHF).8 These discoveries marked the dawn of exploiting TS as an anticancer target.TS (EC 2.1.1.45), which is encoded by the TYMS gene in humans, catalyses the conversion of 2’-deoxyuridine-5’-monophosphate (dUMP) to 2’-deoxythymidine-5’-monophosphate (dTMP) by using mTHF as a cosubstrate. dTMP is then phosphorylated by thymidylate kinase to 2’-deoxythymidine diphosphate (dTDP) and then to 2’-deoxythymidine triphosphate (dTTP) by nucleoside-diphosphate kinase for use in the synthesis of new DNA. Thus, in human cells, TS plays a key role in the biosynthetic pathway that provides the sole de novo source of thymidylate, an essential precursor required for DNA replication and repair.9 In addition to its catalytic function, TS acts as a regulator of translation for some mRNAs. One of these is its own mRNA,10 and others include p53,11 which is a tumour suppressor, and c-myc,12 which is oncogenic. Binding of the TS protein to its own mRNA leads to the formation of an autoregulatory feedback loop for repression of the translation of TS mRNA (Fig 1). A 36 nucleotide sequence (75-110, Site I),13 encompassing the start codon, and a 70 nucleotide sequence (480-550, Site II)14 within the coding region have been identified as the most essential regions in the TS mRNA for binding to the TS protein. On the p53 mRNA, the nucleotide sequence from 531-1020 in the protein coding region,15 and for the c-myc mRNA, the C terminal coding region covering nucleotide positions 1625-1790,12 have been identified to be important for binding to the TS protein. Based on the observation that overexpression of TS sets the cells into a neoplastic phenotype, oncogenic behaviour is a novel role that has been attributed to TS recently.16


2010 - Ovarian Cancer Drug Resistance [Voce in Dizionario o Enciclopedia]
Costi, Maria Paola; Ferrari, Stefania; Green, J.
abstract


2010 - Spermidine/spermine N1-acetyltranferasemodulation by novel folate cycle inhibitors in cisplatin-sensitive and -resistanthuman ovarian cancer cell lines [Articolo su rivista]
Marverti, Gaetano; Ligabue, Alessio; Guerrieri, Davide; Paglietti, G; Piras, S; Costi, Maria Paola; Farina, Davide Salvatore Francesco; Frassineti, Chiara; Monti, Maria Giuseppina; Moruzzi, Maria Stella
abstract

Polyamines have been shown to play a role in the growth and survival of several solid tumors, including ovarian cancer. Intracellular polyamine depletion by the inhibition of biosynthesis enzymes or by the induction of the catabolic pathway leads to antiproliferative effects in many different tumor cell lines. Recent studies showed that the thymidylate synthase inhibitor 5-fluorouracil (5-FU) affects polyamine metabolism in colon carcinoma cells through the induction of the key catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). METHODS: We therefore examined whether combinations of novel folate cycle inhibitors with quinoxaline structure and drugs that specifically target polyamine metabolism, such as diethylderivatives of norspermine (DENSPM) or spermine (BESpm), have synergistic effect in killing cisplatin-sensitive and drug-resistant daughter human ovarian cell lines. RESULTS: Our results showed that simultaneous drug combination or quinoxaline pre-treatment synergistically increased SSAT expression, depleted polyamines, increased reactive oxygen species production, and produced synergistic tumor cell killing in both cell lines. Of note, this combined therapy increased the chemosensitivity of cisplatin-resistant cells and cross-resistant to the polyamine analogues. On the contrary, some pre-treatment regimens of Spm analogues were antagonistic. CONCLUSIONS: These results show that SSAT plays an important role in novel folate cycle inhibitors effects and suggest that their combination with analogues has potential for development as therapy for ovarian carcinoma based on SSAT modulation. Copyright © 2009 Elsevier Inc. All rights reserved.


2010 - Structural study of phenyl boronic acid derivatives as AmpC beta-lactamase inhibitors [Articolo su rivista]
TONDI, Donatella; Calò, Samuele; Shoichet, BK; COSTI, Maria Paola
abstract

A small set of boronic acids acting as low nanomolar inhibitors of AmpC beta-lactamase were designed and synthesized in the effort to improve affinity, pharmacokinetic properties, and to provide a valid lead compound. X-ray crystallography revealed the binary complex of the best inhibitor bound to the enzyme, highlighting possibilities for its further rational derivatization and chemical optimization.


2010 - Tethering low affinity ligands to the dimeric interface of human thymidylate synthase. [Relazione in Atti di Convegno]
Costi, Maria Paola; Genovese, Filippo; Franchini, Silvia; Venturelli, Alberto; Lazzari, Sandra; Farina, Davide Salvatore Francesco; Pirondi, Silvia; R. C., Wade; S., Mangani; C., Pozzi; S., Henrich; Ferrari, Stefania; Ponterini, Glauco; Guaitoli, Giambattista; G., Cruciani
abstract

...


2009 - Biochemistry: Anchors away [Articolo su rivista]
Costi, Maria Paola; Ferrari, Stefania
abstract

The mechanism of action of the enzyme Flavin dependent Thymidylate synthase is presented and discussed.


2009 - Collateral sensitivity to novel thymidylate synthase inhibitors correlates with folate cycle enzymes impairment in cisplatin-resistant human ovarian cancer cells [Articolo su rivista]
Marverti, Gaetano; Ligabue, Alessio; G., Paglietti; P., Corona; S., Piras; G., Vitale; Guerrieri, Davide; Luciani, Rosaria; Costi, Maria Paola; Frassineti, Chiara; Moruzzi, Maria Stella
abstract

The cytotoxicity of two novel folate cycle inhibitors with quinoxalinic structure, 3-methyl-7-trifluoromethyl-2(R)-[3,4,5-trimethoxyanilino]-quinoxaline (453R) and 3-piperazinilmethyl-2[4(oxymethyl)-phenoxy]quinoxaline (311S), was tested against a panel of both cisplatin(cDDP)-sensitive and -resistant carcinoma cell lines. Interestingly, the cisplatin-resistant human ovarian line, C13 cells, exhibited collateral sensitivity towards the two compounds when compared to its sensitive parental 2008 cells. In this resistant line, which showed elevated expression of the folate cycle enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), due to cisplatin-resistance phenotype, collateral sensitivity correlated with the greater reduction of enzyme expression. In addition, TS and DHFR expression of the other resistant lines, the human ovarian carcinoma A2780/CP cells and the human breast cancer MDA/CH cells, were decreased in accordance with the similar sensitivity or the low level of cross-resistance to these compounds in comparison to their respective parental lines. Noteworthy, unlike 5-fluorouracil, both drugs reduced the level of TS without inducing ternary complex formation with the co-substrate and the nucleotide analogue. Median effect analysis of the interactive effects of cisplatin with the two quinoxalines mainly showed additive or synergistic cell killing, depending on schedules of drug combinations. In particular, synergistic effects were more often obtained, even on the resistant cells, when cisplatin was added at the beginning of the treatment. These results indicate that, despite the possibility of other mechanisms being involved, inhibition of TS cycle enzymes plays an important role in the pharmacology of these compounds, which might also represent a useful component in drug treatment protocols against cDDP-resistant cells.


2009 - International Congress.Drug resistance in ovarian cancer:bomarkers andtreatments. [Esposizione]
Costi, Maria Paola; Ferrari, Stefania
abstract

Ovarian cancer is the fifth most common cause of death from cancer and the most common cause of death from gynecologic cancer in women of all ages in the Western world. This primarily reflects the fact that there are no early warning signs, and most patients with epithelial ovarian cancer present an advanced stage of the pathology at the time of diagnosis. Efforts to improve survival continue to focus on the development of more effective systemic therapies and on the identification of biomarkers for early diagnosis and drug efficacy. Early diagnosis is crucial, marking the difference between a 95 percent survival rate for cancers found at the earliest stage and 20 percent survival among patients diagnosed with advanced disease. New and higher quality therapies are at the frontiers The area is fragmented and need to be more coordinated with the clinical aspects. Disciplines such as drug discovery, drug safety, technological aspects, proteomics, system biology, pharmacogenetics, patient selection, clinical validations and applications are essential and strictly connected.In this event we would like to point out these aspects and the need to technically and scientifically link the different disciplines under the same aim of identifying a compact strategy to combat ovarian cancer. Biomakers and treatments are the guidelines of the meeting and expert from different area will present their work and will create a platform to avenue translational research in this area. LIGHTS and OVCAD EU projects of the 6th framework program will guide the events and will take the opportunity to present their results in a special session.Target audience is every scientist working in the area in the surrounding of chemistry, biology and clinic focused on OC.


2009 - Italian representative substitute of the COMMETTE MANAGEMENT for the COST CM1307 ANNI 2009-2013 [Altro]
Costi, Maria Paola
abstract

New drugs for neglected diseases Descriptions are provided by the Actions directly via e-COST. The Action will pave the way for the development of novel drugs to treat neglected diseases such as African sleeping sickness, Chagas disease and Leishmaniasis. Related approaches of molecular genetics, biochemistry, medicinal chemistry, crystallography and bioinformatics will be coordinated and complemented with industrial experience. Established genomes are used to identify drug targets essential to the parasites but absent or different in the host, since inhibitors thereof hold promise as safe and efficacious therapeutics. Validated drug targets will serve as tools in drug discovery processes using complementary strategies: i) high-throughput screening of natural product and other compound libraries and ii) in silico screening of virtual libraries to identify novel leads; iii) chemical synthesis and optimization of identified leads; and iv) structure-based inhibitor design based on established structures or molecular models. The potential therapeutic profile of novel compounds active in vitro will be worked out by techniques of high prognostic value in respect to drug safety. The most promising compounds will be tested in established infection models for all the diseases to choose the most attractive candidates for preclinical and clinical development. The Action Actions are networks centred around nationally funded research projects in fields that are of i... members will cover all expertises required for the multidisciplinary early drug discovery phase. Keywords: Drug discovery, medicinal chemistry, African sleeping sickness, Chagas disease, Leishmaniasis Chemistry and Molecular Sciences and Technologies COST Action CM0801 Description Parties Management Committee CMST Icon General Information* Chair of the Action: Prof. Leopold FLOHE (DE) Vice Chair of the Action: Dr Theodora CALOGEROPOULOU (EL) Science officer of the Action: Dr Lucia FORZI Administrative officer of the Action: Ms Svetlana VOINOVA Downloads* Action Fact Sheet Download AFS as .RTF Memorandum of Understanding Download MoU as PDF Progress Report Download Progress Report as PDF Poster Download Poster as PDF Websites* Action website: http://www.costcm0801.org/CM0801/Welcome.html Domain website: http://www.cost.eu/cmst * content provided by e-COST. Data is synchronised once per night. Management Commette substitute for Italy- COST Action CM0801 List of all management Commette substitues-Country MC Substitute Greece Dr Anastasia DETSI Israel Prof. Joseph SHLOMAI Israel Prof. Hanoch SENDEROWITZ Italy Prof. Sergio ROMEO Italy Prof Maria Paola COSTI Portugal Dr Helena CASTRO Spain Dr Francisco GAMARRO Sweden Prof. Salam AL-KARADAGHI Switzerland Prof. Andreas MAYER United Kingdom Prof. Simon CROFT


2009 - MALDI-TOF MS as a tool for rapid Identification of Low Protein Affinity Drug Fragments. [Abstract in Atti di Convegno]
Genovese, Filippo; Lazzari, Sandra; G., Cruciani; Franchini, Silvia; S., Henrich; R. C., Wade; Venturelli, Alberto; Costi, Maria Paola
abstract

...


2008 - Anti-Parasitic drugsDiscovery of potent pteridine reductase inhibitors to guide antiparasite drug development. [Altro]
Costi, Maria Paola; Ferrari, Stefania; Cavazzuti, Antonio
abstract

Inhibition of pteridine reductase (PTR1) — an enzyme essential for parasitic trypanosomatid survival — might represent a therapeutic approach for African sleeping sickness. Cavazzuti and colleagues combined a rapid-screening strategy using a folate-based library with structure-based design to identify PTR1 inhibitors with biological activity. An additive profile was observed when PTR1 inhibitors were used in combination with known dihydrofolate reductase inhibitors, indicating the potential of targeting two enzymes for the development of previously undescribed antiparasitic drugs.


2008 - Collateral sensitivity to novel folate cycle inhibitors enhances cisplatin effectiveness against human ovarian cancer cells. [Abstract in Atti di Convegno]
Marverti, Gaetano; Ligabue, Alessio; Guerrieri, Davide; Costi, Maria Paola; G., Paglietti; Frassineti, Chiara; Moruzzi, Maria Stella
abstract

Presentazione della prima evidenza del binding dei peptidi inibitori della Timidilato sintasi all'interfaccia del dimmelo della Proteina.


2008 - Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against beta-Lactamase. [Articolo su rivista]
KERIM, BABAOGLU; JOHN J., IRWIN; ANTON, SIMEONOV; MICHAEL, NELSON; CRAIG, THOMAS; BRIAN, FENG; CANCIAN, Laura; COSTI, Maria Paola; DAVID A., MALTBY; JAMES, INGLESE; CHRISTOPHER P., AUSTIN; AND BRIAN K., SHOICHET
abstract

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate “hit lists”; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against β-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 µM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.


2008 - Constrained dansyl derivatives reveal bacterial specificity of highly conserved Thymidylate Synthases A [Articolo su rivista]
S., Calo'; Tondi, Donatella; Ferrari, Stefania; A., Venturelli; S., Ghelli; Costi, Maria Paola
abstract

The elucidation of the structural/functional specificities of highly conserved enzymes remains a challenging area of investigation, and enzymes involved in cellular replication are important targets for functional studies and drug discovery. Thymidylate synthase (TS, ThyA) governs the synthesis of thymidylate for use in DNA synthesis. The present study focused on Lactobacillus casei TS (LcTS) and Escherichia coli TS (EcTS), which exhibit 50 % sequence identity and strong folding similarity. We have successfully designed and validated a chemical model in which linear, but not constrained, dansyl derivatives specifically complement the LcTS active site. Conversely, chemically constrained dansyl derivatives showed up to 1000-fold improved affinity for EcTS relative to the inhibitory activity of linear derivatives. This study demonstrates that the accurate design of small ligands can uncover functional features of highly conserved enzymes.


2008 - Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development [Articolo su rivista]
Cavazzuti, Antonio; Paglietti, G.; Hunter, W. N.; Gamarro, F.; Piras, S.; Loriga, M.; Alleca, S.; Corona, P.; Mcluskey, K.; Tulloch, L.; Gibellini, Federica; Ferrari, Stefania; Costi, Maria Paola
abstract

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.


2008 - FRET-based assay for sensing a cancer key protein functional state and inhibition by oligopeptides: Sitespecific Protein Chemical Modifications [Abstract in Rivista]
Genovese, Filippo; M., Vargiu; M. A., Ariza Mateos; Guaitoli, Giambattista; Ferrari, Stefania; Ponterini, Glauco; Costi, Maria Paola
abstract

FRET-based assay for sensing a cancer key protein functional state and inhibition by oligopeptides: Sitespecific Protein Chemical Modifications


2008 - Merging disulphide bonds for drug design Interfering with a cancer key protein [Abstract in Atti di Convegno]
Lazzari, Sandra; Venturelli, Alberto; G., Cruciani; Franchini, Silvia; S., Henrich; Genovese, Filippo; R. C., Wade; Costi, Maria Paola
abstract

...


2008 - Metodo per la funzionalizzazione sito specifica di molecole proteiche [Brevetto]
Genovese, Filippo; Ferrari, Stefania; Costi, Maria Paola; Ponterini, Glauco
abstract

Viene qui fornito un metodo in soluzione per le modifiche chimiche sito-specifiche di proteine, sfruttando l’affinità di cavità strutturate di proteine (come le cavità catalitiche degli enzimi) verso miscele di inibitori (ir)reversibili/(co)substrati o ligandi per mascherare i residui coinvolti nella formazione del complesso, permettendo la funzionalizzazione chemoselettiva di residui opportunamente selezionati al di fuori della tasca. Pertanto, grazie al mascheramento di residui di cavità strutturate, è possibile studiare ed esplorare siti allosterici a bassa affinità sia chimicamente, come porzioni funzionalizzabili, che funzionalmente. Alcune delle applicazioni in campo diagnostico, analitico e terapeutico dell’enzima ingegnerizzato risultante vengono qui discusse. Questa piattaforma di coniugazione potrebbe portare alla progettazione di un kit di coniugazione per eseguire modifiche chimiche sito-specifiche di proteine per applicazioni di ricerca, diagnostiche e terapeutiche.


2008 - Most Downloaded x-ray structure (1xgj) from Protein Data Bank between 07/2007-08/2008 [Altro]
Tondi, Donatella; Morandi, F.; Bonnet, R.; Costi, Maria Paola; Shoichet, Bk
abstract

Bacterial expression of beta-lactamases is the most widespread resistance mechanism to beta-lactam antibiotics, such as penicillins and cephalosporins. There is a pressing need for novel, non-beta-lactam inhibitors of these enzymes. One previously discovered novel inhibitor of the beta-lactamase AmpC, compound 1, has several favorable properties: it is chemically dissimilar to beta-lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 microM its activity is modest. Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. The most active of these, compound 10, had a K(i) of 1 microM, 26-fold better than the lead. To understand the origins of this improved activity, the structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 A, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to beta-lactam-based inhibitors clavulanate and cefoxitin, compound 10 did not up-regulate beta-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to beta-lactam antibiotics.


2008 - Naphthofuranone derivatives as specific inhibitors of thymidylate synthases [Brevetto]
VENTURELLI, Alberto; COSTI, Maria Paola; PECORARI, Piergiorgio; ROSSI, Tiziana; CASOLARI, CHIARA; TONDI, Donatella; BARLOCCO, DANIELA
abstract

Synthetic compounds of 1, 2 -naphthalein molecules (I) having specific inhibitory properties of the enzymatic activity of thymidylate synthases of bacterial species, their preparation, their pharmaceutical composition and use in the treatment and prophylaxis of infectious pathologies are disclosed.


2008 - Opening Opportunities for New Drugs Against Neglected Diseases [Articolo su rivista]
Costi, Maria Paola; Taramelli, D; GONZÁLEZ PACANOWSKA, D.
abstract

---no abstract available


2008 - Sequence-based Identification of Specific Target Domains in the Thymidylate Synthase Enzyme Family. [Articolo su rivista]
Ferrari, Stefania; Losasso, Valeria; Costi, Maria Paola
abstract

Thymidylate synthases from pathogenic organisms: Sequence analysis and knowledge-based grouping have provided a step forward in the identification of potential drug target regions in thymidylate synthases. This provides a unique approach toward blocking the growth of pathogenic organisms.


2008 - SSAT modulation by putative regulators of folate cycle enzymes expression in cisplatin-sensitive and –resistant human ovarian cancer cell lines. [Abstract in Atti di Convegno]
Marverti, G.; Ligabue, A.; Costi, M. P.; Lombardi, P.; Guerrieri, D.; Frassineti, C.; Monti, M. G.; Moruzzi, M. S.
abstract

SSAT modulation by putative regulators of folate cycle enzymes expression in cisplatin-sensitive and –resistant human ovarian cancer cell lines.Cisplatin (DDP)-resistance confers a deficient expression of spermidine/ spermine N(1)-acetyltransferase (SSAT) gene in response to the spermine analog N(1),N(12)-bis(ethyl)spermine (BESpm) in the DDP-resistant human ovarian carcinoma cell line (C13*), compared with their parental DDP-sensitive 2008 cells.


2008 - Structural and Thermodynamic analysis of novel inhibitors of beta-lactmases. [Relazione in Atti di Convegno]
F., Morandi; Tondi, Donatella; A., Venturelli; R., Bonnet; B. K., Shoichet; Costi, Maria Paola
abstract

.


2008 - Synthesis of N-(5,7-diamino-3-phenyl-quinoxalin-2-yl)-3,4,5-substituted anilines and N-[4[(5,7-diamino-3-phenylquinoxalin-2-yl)amino]benzoyl]-l-glutamic acid diethyl ester: evaluation of in vitro anti-cancer and anti-folate activities [Articolo su rivista]
P., Corona; M., Loriga; Costi, Maria Paola; Ferrari, Stefania; G., Paglietti
abstract

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI50 < 0.01 μM) and the ovarian cancer cell line OVCAR-4 (GI50 = 0.03 μM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.


2007 - Congress on - Medicinal Chemistry in parasitology: new avenues in drug discovery-organised in Modena in 2007. [Altro]
Costi, Maria Paola; Ferrari, Stefania; Taramelli, D.; D., Gonzales Pacnowska
abstract

WG3 Meeting on Medicinal Chemistry in Modena, Italy 19-20 February, 2007The environment and tools for R&D for neglected infectious diseases are changing. There is political attention now given not only to HIV, tuberculosis and malaria, but also to other, less known diseases with a lower overall burden; here is more money and new funding agencies; and new mechanisms, including a bewildering array of public-private partnerships (PPPs). However, the R&D pipeline varies considerably for the various diseases, and remains hugely inadequate for the most neglected diseases like the different forms of leishmaniasis and trypanosomiasis, and helminthic diseases. Here, discovering lead compounds with the potential to become usable drugs is a critical step to ensuring a sustainable pipeline for innovative products. We will discuss different approaches to drug discovery and emphasize an integrated strategy that involves networks and partnerships between academia, industry in the developed and developing countries. Specific examples will be drawn from the work of TDR covering multiple diseases and those of other organizations.


2007 - Interfering with Human Thymidylate Synthase Dimerization against ovarian human carcinoma [Abstract in Atti di Convegno]
Costi, Maria Paola; Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Ferrari, Stefania; Salo Ahen, O; Wade, R.
abstract

.


2007 - Interfering with Human Thymidylate Synthase Dimerization anti peptide design in the search of anticancer agents against ovarian human carcinoma [Abstract in Atti di Convegno]
Cardinale, Daniela; Salo Ahen, O; Guaitoli, Giambattista; Tondi, Donatella; Ferrari, Stefania; Wade, R; Costi, Maria Paola
abstract

.N/A


2007 - New thymidylate synthase inhibitors induce apoptosis in melanoma cell lines [Articolo su rivista]
Giudice, Stefania; Benassi, Luisa; Bertazzoni, Giorgia; Costi, Maria Paola; A., Gelain; Venturelli, Alberto; Bernardi, Chiara; Gualdi, Giulio; A., Coppi; Rossi, Tiziana; Giannetti, Alberto; Magnoni, Cristina
abstract

Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z-VAD-FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling. (c) 2006 Elsevier Ltd. All rights reserved.


2007 - Optimizing Cell Permeation of an Antibiotic Resistance Inhibitor for Improved Efficacy [Articolo su rivista]
A., Venturelli; Tondi, Donatella; Cancian, Laura; Morandi, Federica; Cannazza, Giuseppe; B., Segatore; Prati, Fabio; G., Amicosante; B. K., Shoichet; Costi, Maria Paola
abstract

Abstract:Benzo[b]thiophene-2-ylboronic acid, 1, is a 27 nM inhibitor of the class C -lactamase AmpC and potentiates the activity of -lactam antibiotics in bacteria that express this and related enzymes. As is often true, the potency of compound 1 against the enzymes is much attenuated in cell culture against Gram negative bacteria, where the minimum inhibitor concentration of compound 1 is in the mid-micromolar range. Here, we modulated the properties of this lead to enhance its ability to cross the membrane, using a combination of X-ray crystallography, structure-based design, and application of physical models of outer membrane crossing. This strategy led us to derivatives with substantially improved permeability. Also, the greater solubility of these compounds allowed us to measure their efficacy at higher concentrations than with the lead 1, leading to higher maximum potentiation of the antibiotic effect of ceftazidime on resistant bacteria.


2007 - Pirrole-amidine oligopeptide–DNA interaction studies through Isothermal Titration Calorimetry [Relazione in Atti di Convegno]
Venturelli, Alberto; Guaitoli, Giambattista; Cancian, Laura; P., Lombardi; Costi, Maria Paola
abstract

Pirolidine derivatives such as Distamicine and derivatives can interact with TATA box that are present in DNA of plasmodium phalciparum, therefore these compounds may be studied as potential antimalarial compounds.


2006 - Alternative strategies in medicinal chemistry to face drug resistance in anticancer therapy. [Abstract in Atti di Convegno]
Cardinale, Daniela; Ferrari, Stefania; Franchini, Silvia; Corsini, Enrico; Battini, Renata; Costi, Maria Paola
abstract

...


2006 - Antibacterial agent discovery using thymidylate synthase biolibrary screening [Articolo su rivista]
Costi, Maria Paola; A., Gelain; D., Barlocco; S., Ghelli; Soragni, Fabrizia; F., Reniero; Rossi, Tiziana; Ruberto, Ippazio Antonio; C., Guillou; A., Cavazzuti; Casolari, Chiara; Ferrari, Stefania
abstract

Thymidylate synthase (TS, ThyA) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate to 2'-deoxythymidine 5'-monophosphate, an essential precursor for DNA synthesis. A specific inhibition of this enzyme induces bacterial cell death. As a second round lead optimization design, new 1,2-naphthalein derivatives have been synthesized and tested against a TS-based biolibrary, including human thymidylate synthase (hTS). Docking studies have been performed to rationalize the experimentally observed affinity profiles of 1,2-naphthalein compounds toward Lactobacillus casei TS and hTS. The best TS inhibitors have been tested against a number of clinical isolates of Gram-positive-resistant bacterial strains. Compound 3,3-bis(3,5-dibromo-4-hydroxyphenyl)-1H, 3H-naphtho[1,2-c]furan-1-one(5) showed significant antibacterial activity, no in vitro toxicity, and dose-response effects against Staphylococcus epidermidis (MIC = 0.5-2.5 mu g/mL) clinical isolate strains, which are resistant to at least 17 of the best known antibacterial agents, including vancomycin. So far this compound can be regarded as a leading antibacterial agent.


2006 - Combination of suboptimal doses of inhibitors targeting different domains of LtrMDR1 efficiently overcomes resistance of Leishmania spp. to miltefosine by inhibiting drug efflux [Articolo su rivista]
JM Perez, Victoria; F., Cortes Selva; A., Parodi Talice; Bi, Bavchvarov; FJ Perez, Victoria; F., Munoz Martinez; M., Maitrejean; Costi, Maria Paola; D., Barron; A., Di Pietro; S., Castanys; F., Gamarro
abstract

Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-beta-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.


2006 - NAPHTHOFURANONE DERIVATIVES AS SPECIFIC INHIBITORS OF THYMIDYLATE SYNTHASES [Brevetto]
Costi, Maria Paola; Rossi, Tiziana; Casolari, Chiara; Tondi, Donatella; Barlocco, Daniela; Pecorari, Piergiorgio; Venturelli, Alberto
abstract

Synthetic compounds of 1, 2 -naphthalein molecules (I) having specific inhibitory properties of the enzymatic activity of thymidylate synthases of bacterial species, their preparation, their pharmaceutical composition and use in the treatment and prophylaxis of infectious pathologies are disclosed


2006 - New Thymidylate synthase inhibitor induces apoptosis in melanoma cell lines. [Abstract in Atti di Convegno]
Giudice, Stefania; Benassi, Luisa; Magnoni, Cristina; Bertazzoni, Giorgia; Costi, Maria Paola; Rossi, Tiziana; Bernardi, Chiara; Gualdi, Giulio; Giannetti, Alberto
abstract

Malignant melanoma is an aggressive disease and its rapidly increasing world-wide. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the last years has been developed a variety of a new chemotherapeutic agents. We investigated the action in vitro of four experimental antifolate substances (MR7, MR2, MR36 and NMR707) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. These compounds behave as antifolate but show a new structure with respect to all know antifolato agents. We studied how these substances may influence apoptosis in melanoma cell lines SK-MEL-2 (derived from malignant melanoma metastasis) and SK-MEL-28 (derived from primary malignant melanoma).The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by TUNEL technique and Comet Assay. Western-blot analysis showed down regulation of Bcl-2 protein level, up-regulation of p21 and Bax protein level and PARP cleavage especially for MR36, MR7 and MR21. Moreover, these antifolate-induced apoptosis was accompanied by caspase-9 and -8 activation. In addition, a different sensibility for apoptosis was observed for the two cell lines. In fact, in primary melanoma cells, apoptosis occurred also at low concentrations especially for MR7 and MR36. Our results show that TS inhibitors are able to induce apoptosis and for this reason may play a potential role as new therapeutic agents for melanoma, but further studies are necessary to understand their molecular mechanism.


2006 - Novel 3-benzoyl-2-piperazinylquinoxaline derivatives as potential antitumor agents [Articolo su rivista]
S., Piras; M., Loriga; A., Carta; G., Paglietti; Costi, Maria Paola; Ferrari, Stefania
abstract

[ A series of new benzoylquinoxaline derivatives (7-26) was synthesized and evaluated for antitumor activity against a panel of 60 human cell lines at the NO of Bethesda. Among the compounds which have passed the preliminary screening, compound 23 exhibited the best profile and growth inhibition activity at 100-10 mu M. The compounds were then tested towards a folate-dependent enzymes bio-library including Thymidylate synthases enzymes and human Dihydrofolate reductase at 10 mu M. The most of compounds exhibited a moderate inhibitory activity towards all or some of the enzymes tested with detectable inhibition constants (K-i) values in the range of 0.6-70 mu M. Compounds 21, 23, 24 showed K-i in the range of 10-38 mu M against both hDHFR and hTS.


2006 - Pharmacological and toxicological evaluation of a new series of thymidylate synthase inhibitors as anticancer agents [Articolo su rivista]
Benassi, Luisa; Magnoni, Cristina; Giudice, Stefania; Bertazzoni, Giorgia; Costi, Maria Paola; Rinaldi, Marcella; Venturelli, Alberto; A., Coppi; Rossi, Tiziana
abstract

Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-]H,3H-naphtho[1,8cdjpyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)IH,3H-naphtho[1,8-cd]pyran-l-one (MR35) and 6-bromo-3,3bis(3-chloro-4-hydro.xyphenyl)-1H,3H-naphtho[1,8-cd]pyran-l- one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [H-3]thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [H-3]-thymidine test was the best in predicting toxicity against melanoma cells.


2006 - Special Issue Plenary and Invited Lectures of joint Italian-Swiss Medicinal Chemistry Meeting 2005 [Curatela]
Costi, Maria Paola
abstract

Arkat USA Inc was established in 2000 through a personal donation from Alan and Linde Katritzky. It is duly registered as a charity. ARKAT USA is a not-for-profit entity designed to enhance chemical research worldwide and hence to benefit researchers, students, commercial and noncommercial enterprises and humanity in general by increasing opportunity and efficiency and reducing waste of materials, time and effort.


2006 - TYDOCK PHARMA s.r.l. Spin Off dell'Università di Modena e Reggio Emilia che ha come fine l'attività di ricerca e sviluppo nel settore delle biotech farmaceutiche. [Spin Off]
Costi, Maria Paola; Venturelli, Alberto; P., Pecorari
abstract


2005 - Crystallographic studies of novel inhibitors of [beta]-lactamases [Abstract in Rivista]
Tondi, Donatella; Venturelli, Alberto; Morandi, Federica; Bonnet, Richard; Shoichet, Brian K.; Costi, Maria Paola
abstract

Bacterial expression of -lactamases is the most widespread resistance mechanism to -lactam antibiotics. There is a pressing need for novel, non- -lactam inhibitors of these enzymes [1]. Our efforts to overcome bacterial resistance mechanisms have been directed towards novel, non -lactam inhibitors of AmpC -lactamase, a class C enzyme responsible of resistance to antibiotics treatment in gram- negative bacteria. Through a structure-based approach, we discover novel inhibitors for this enzyme, with covalent mechanism of action such as boronic acid derivatives and with no-covalent, competitive mechanism of action, such as thiophene-2-carboxylic acid derivative [2]. In one case we were able to extend the inhibitory activity towards class A -lactamases, obtaining a broad spectrum, highly potent inhibitor. Some inhibitors were active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC and did not up-regulate -lactamase expression in cell culture. The structure-based design, synthesis, biological evaluation and the crystallographic studies of such novel inhibitors will be described. [1] Cosgrove S., Carmeli Y., Clin. Infect. Dis., 2003, 36, 1433-1437. [2] Tondi D., Morandi F., Bonnet R., Costi M. P., Shoichet B. K., J. Am. Chem. Soc., 2005, 127(13), 4632-4639. Keywords: enzyme inhibition, drug resistance, X-ray complexes


2005 - Drugs Against Protozoan Parasites: Target Selection, Structural Biology and Medicinal Chemistry, Copper Mountain, CO, USA, 9-13 April 2005 [Curatela]
Costi, Maria Paola
abstract

The International meeting on Drugs against protozoan parasites is organized regularly every three years. This time the Congress, organized by Margaret A.Phillips, Pradipsinh K.Rathod and William N.Hunter, has been held in Copper Mountain Resort and is part of the Keystone symposia series. It was appropriately structured as a multidisciplinary approach to the problem of Drug discovery and development against neglected diseases. Top-ranking scientist from the different areas of functional genomics, structural biology, medicinal chemistry and clinical development had the opportunity to meet and actively discuss of the most important issues related to the field of Malaria, African trypanosomiasis and Leishmaniasis such as new drug targets identification, lacking of “shuttle funding” from lead discovery identification to clinical phases evaluation, funding opportunities from the pharmaceutical companies and govern involvement. The crucial issue of intellectual property and effective cost of the final treatment was discussed. A discrete portfolio of candidate drug, with respect to the past, is at the moment entering the clinical phase III, but most of the new chemical entities are still at the lead level or phase I and II. Among the recent compounds on the market Cancidas, Chlorproguanildapsone, (Lapdap™), Artesunate(CDA) Pediatri, in Phase I, II and III Artekin ™ (dihydroartemisinin-piperaquine), Coartem™ (MMV). The sessions dedicated to the clinical trials and details on the phase I-III were missed, and many problems to the practical approach to the clinical phase evaluation were raised.


2005 - Improving specificity vs bacterial thymidylate synthases through N-dansyl modulation of didansyltyrosine [Articolo su rivista]
Tondi, Donatella; A., Venturelli; Ferrari, Stefania; S., Ghelli; Costi, Maria Paola
abstract

N,O-Didansyl-L-tyrosine (DDT) represented the starting lead for further development of novel non-substrate-like inhibitors of bacterial thymidylate synthase. The N-dansyl structure modulation led to a submicromolar inhibitor of Lactobacillus casei TS (LcTS), which is highly specific with respect to human TS (hTS). Using molecular dynamics simulation, a binding mode for DDT vs LcTS was predicted, explaining activity and species-specificity along the series.


2005 - Second Joint Italian-Swiss Meeting on Medicinal Chemistry , Italy, Modena 19-20 February 2005. [Relazione in Atti di Convegno]
Brasili, Livio; Benvenuti, Stefania; Costantino, Luca; Costi, Maria Paola; Philipp, Floersheim; Franchini, Silvia; Gamberini, Gianfranco; Parenti, Carlo; Rastelli, Giulio; Rustichelli, Cecilia; Tait, Annalisa; Tondi, Donatella
abstract

The Second Joint Italian-Swiss Meeting on Medicinal Chemistry (ITCHMC2005) was held in Modena, (Italy) from September 12 to 16, 2005, under the auspices of the European Federation of Medicinal Chemistry (EFMC).This year, the annual meeting of the Division of Medicinal Chemistry of the Italian Chemical Society was co-organized with the Division for Medicinal Chemistry of the Swiss Chemical Society and it followed the first, successful one held in Torino in September 1997. This important event in the field of medicinal chemistry brought together scientists from both academia and industry to discuss different aspects of modern medicinal chemistry. Top-ranking scientists from medicinal chemistry and clinical development had the opportunity to meet and discuss the following topics: Carbohydrate Chemistry in Drug Design, Nuclear Receptors, Progress in Design and Development of Protease Inhibitors, Progress in Oncology Research and finally, Pain and Neurodegenerative Diseases.IntroductionThe conference was attended by 300 scientists from 13 countries, with most of the participants from Italy and Switzerland. The meeting allowed an extensive exchange of information and widespread networking. Of the 134 posters on display, 19 were selected for short oral presentations and two were selected for the Farminidustria awards. The meeeting was organized in six sessions with 6 Plenary (PL), 16 Main Lectures (ML) and 19 short communications (SC).The scientific sessions were held at the Forum Guido Monzani, a modern complex with multifunctional facilities; the opening ceremony took place at Accadenia Militare di Modena, housed in the seventeenth century Palazzo Ducale.Modena, city of art, culture and prosperous economy, offered an exciting background for stimulating scientific interactions. Participants were mainly from academia and other research centers together with 46 pharmaceutical companies; among them, six presented their work, namely Novartis Basel , Roche Basel, Novartis East Hannover USA, IRBM Pomezia Italy, Santhera Pharmaceutical AG, Heidelberg, GlaxoSmithKline (GSK) Verona, S-IN Soluzioni Informatiche, Vicenza. The areas covered by the meeting were advanced medicinal chemistry including computational chemistry, established drug targets, libraries and screens, inhibitor design and clinical advances. There were two poster sessions, with presentations given mainly by young scientists.


2005 - Structure-based optimization of a non-beta-lactam lead results in inhibitors that do not up-regulate beta-lactamase expression in cell culture [Articolo su rivista]
Tondi, Donatella; Morandi, Federica; R., Bonnet; Costi, Maria Paola; Bk, Shoichet
abstract

Bacterial expression of beta-lactamases is the most widespread resistance mechanism to beta-lactam antibiotics, such as penicillins and cephalosporins. There is a pressing need for novel, non-beta-lactam inhibitors of these enzymes. One previously discovered novel inhibitor of the beta-lactamase AmpC, compound 1, has several favorable properties: it is chemically dissimilar to beta-lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 mu M its activity is modest. Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. The most active of these, compound 10, had a K-I of 1 mu M, 26-fold better than the lead. To understand the origins of this improved activity, the structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 angstrom, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to beta-lactam-based inhibitors clavulanate and cefoxitin, compound 10 did not up-regulate beta-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to beta-lactam antibiotics.


2005 - Structure-Based Optimization of a Non-β-lactam Lead Results in Inhibitors That Do Not Up-Regulate β-Lactamase Expression in Cell Culture [Poster]
Tondi, Donatella; Federica, Morandi; Richard, Bonnet; Costi, Maria Paola; Brian K., Shoichet
abstract

Bacterial expression of β-lactamases is the most widespread resistance mechanism to β -lactam antibiotics. There is a pressing need for novel, non-β-lactam inhibitors of these enzymes. Our lead, compound 1, is chemically dissimilar to β -lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 μM its activity is modest (Figure 1). Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. Among these, compound 10, had a Ki of 1 μM, 26-fold better than the lead. The structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 Å, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to β-lactam-based inhibitors compound 10 did not up-regulate β-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to β -lactam antibiotics.


2005 - The structure of Cryptococcus neoformans thymidylate synthase suggests strategies for using target dynamics for species-specific inhibition [Articolo su rivista]
JS Finer, Moore; Ac, Anderson; Rh, O'Neil; Costi, Maria Paola; Ferrari, Stefania; J., Krucinski; Rm, Stroud
abstract

The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1 angstrom structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS-dUMP-inhibitor complexes with a greater range of antifolates than human TS. 3',3 ''-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (K-i=4 mu M) over human TS (K-i>> 245 mu M). The E. coli TS-dUMP-GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered [Phan et al. (2001), J. Biol. Chem. 276, 14170-14177] may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery.


2005 - Thymidylate synthase structure, function and implication in drug discovery [Articolo su rivista]
Costi, Maria Paola; Ferrari, Stefania; Venturelli, A; Calo, S; Tondi, Donatella; Barlocco, D.
abstract

Recent methodologies applied to the drug discovery process, such as genomics and proteomics, have greatly implemented our basic understanding of drug action and are giving more input to medicinal chemists, in finding genuinely new targets and opportunities for the development of drugs with original mechanisms of action. In this paper, an example of the successful application of some new techniques to the target enzymes with the Thymidylate Synthase (TS) function is given. The improved knowledge of the complex mechanism of the biological pathways in which thymidylate synthase is involved represents a unique chance to find new mechanism-based inhibitors, aimed to treat not only cancerous diseases, but also infectious pathologies. Thymidylate synthase (TS or ThyA) has long been considered as one of the best-known drug targets in the anti-cancer area, after which old and new drugs, such as 5-fluoro uracil and the anti-folate ZD1694, have been introduced into chemotherapy to treat solid tumours. Only a few attempts have been made to find non-classical anti-folate inhibitors that are dissimilar to the folate co-factor, with the aim of finding unshared protein target domains on the enzyme structure, in order to specifically inhibit TS enzymes from pathogens. Only recently from omic studies, a new Thymidylate Synthase Complementing Protein (TSCP or ThyX) has been identified in a number of pathogens, showing a different structure with respect to human TS, thus opening new avenues to specific inhibitions. A depiction of the most recent progress in the study of Thymidylate Synthase enzymes is presented in the following sections.


2005 - TSI INDUCES APOPTOSIS WITH A P53 INDIPENDENT PATHWAY IN MELANOMA CELL LINES AND IN NORMAL HUMAN MELANOCITES [Abstract in Rivista]
Benassi, Luisa; Magnoni, Cristina; Bertazzoni, Giorgia; Giudice, Stefania; Costi, Maria Paola; Rossi, Tiziana; Bernardi, Chiara; Gualdi, Giulio; Giannetti, Alberto
abstract

Melanoma patients have a very poor prognosis and this type of tumor is particular resistant toconventional chemotherapy and radiotherapy. For this reason in the last years has beendeveloped a variety of new therapeutic agents. We investigated four experimental antifolatesubstances (MR7, MR36, MR21 and NMR707) with a critical target for thymidylate synthase(TS), an essential enzyme for DNA synthesis and repair. All the compounds were tested fortheir inhibitory properties against TS enzymes from different species: Lactobacillus casei,Pneumocystis carinii, Cryptococcus neoformans and human. These compounds behave asantifolates but show new structure with respect to all known antifolate agents. We studiedhow these substances may influence apoptosis in melanoma cell lines SKmel 2 (derived frommalignant melanoma metastasis) and SKmel 28 (derived from primary malignant melanoma)and normal human melanocytes. The antifolate agents induced apoptosis in Skmel 2 andSkmel 28 cells. These results were confirmed by Comet Assay. Western blot analysis showeddown regulation of the level of Bcl2 and PARP clivage especially for MR36, MR7 and MR21.Moreover a different apoptotic behaviour for the two cell lines was observed, in fact inprimary melanoma cells apoptosis occurred also at low concentrations especially for MR7.After chemotherapeutic treatment with all the tested compounds p53 was not modulatedindicating a p53 independent pathway for apoptosis. Caspases 8 and 9 clearly mediateantifolate agents apoptosis independent of the status of p53. Our results show that TSinhibitors inducing apoptosis may play a future potential role as new therapeutic agents formelanoma, but further studies are necessary to understand their molecular mechanism.


2004 - “A step further in the discovery of phthalein derivatives as Thymidylate Synthase inhibitors” [Articolo su rivista]
Calo', Samuele; Tondi, Donatella; A., Venturelli; Ferrari, Stefania; Pecorari, Piergiorgio; Rinaldi, Marcella; S., Ghelli; Costi, Maria Paola
abstract

Phenolphthalein (Pth) was discovered as a low micromolar inhibitor of the enzyme ThymidylateSynthase (TS), an important target for anticancer chemotherapy. In the present work, a newseries of Pth derivatives have been designed and synthesized. All the compounds have beencharacterized through NMR techniques. A set of twelve Pth derivatives has been tested againstthree TS enzymes and their bio-profiles obtained. The bio-profiling studies suggest that theinhibitory potency of the compounds has been improved of about fifty times againstLactobacillus casei TS (LcTS) and five times against humant TS (hTS) with respect to the lead.The most active compound shows an inhibition constant (Ki) of 70 nM against Escherichia coliTS (EcTS).


2004 - Aza-boronic acids as non-beta-lactam inhibitors of AmpC-beta-lactamase [Articolo su rivista]
V., Buzzoni; J., Blazquez; Ferrari, Stefania; S., Calo; A., Venturelli; Costi, Maria Paola
abstract

With the aim of improving the ability of non-p-lactam inhibitors to inhibit AmpC-beta-tactamase, a series of 3-aza-phenyl-boronic acid derivatives was obtained using in parallel synthesis. The molecules were tested against Escherichia coli AmpC-beta-lactamase. The best inhibitors, 3-(2-hydroxy-naphthalen-1-ylazo)-phenyl-boronic acid (12) and 3-(2,4-dihydroxy-naphthalen-1-ylazo)-phenyl-boronic acid (14), showed apparent inhibition constant values (K-i) of 0.3 and 0.45 muM and increased the potency of the semi-synthetic cephalosporin antibiotic, ceftazidime, lowering its minimum inhibitory concentration (MIC) value of 50%, against Gram-negative bacteria strains, producing high levels of AmpC-p-lactamase.


2004 - Medicinal chemistry in parasitology [Esposizione]
Costi, Maria Paola; Ferrari, Stefania; Rastelli, Giulio
abstract

"Medicinal Chemistry in Parasitology", January 23, 2004. Modena(Italy).The congress has been organized to give an overview of the medicinal chemistry in the area of parasitic/tropical diseases. The aim of the workshop is to stimulate coordination, research initiatives and collaborations among different groups working in the area.


2004 - Rapid-Throughput Technologies Applied to the Discovery of New Hits against Thymidylate synthase Enzymes [Abstract in Atti di Convegno]
Ferrari, Stefania; Malaguti, E; Calò, S; Tondi, Donatella; Venturelli, Alberto; Costi, Maria Paola
abstract

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2003 - Developing New beta-Lactamase Inhibitors Through Structure-Based Design and Pharmacokinetic Improvement [Abstract in Atti di Convegno]
Venturelli, Alberto; B., Shoichet; Prati, Fabio; Cannazza, Giuseppe; Costi, Maria Paola
abstract

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2003 - From ligand-based design to Hit validation of new thymidylate synthase inhibitors [Abstract in Atti di Convegno]
Costi, Maria Paola; Ferrari, Stefania; Rinaldi, Marcella; Tondi, Donatella; Wade, R. C.
abstract

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2003 - Inhibitor Specificity via Protein Dynamics. Insights from the Design of Antibacterial Agents Targeted Against Thymidylate Synthase [Articolo su rivista]
Ferrari, Stefania; Costi, Maria Paola; Wade, R. C.
abstract

Structure-based drug design of species-specific inhibitors generally exploits structural differences in proteins from different organisms. Here, we demonstrate how achieving specificity can be aided by targeting differences in the dynamics of proteins. Thymidylate synthase (TS) is a good target for anticancer agents and a potential target for antibacterial agents. Most inhibitors are folate-analogs that bind at the folate binding site and are not species specific. In contrast, alpha156 is not a folate-analog and is specific for bacterial TS; it has been shown crystallographically to bind in a nonconserved binding site. Docking calculations and crystal structure-based estimation of the essential dynamics of TSs from five different species show that differences in the dynamics of TSs make the active site more accessible to alpha156 in the prokaryotic than in the eukaryotic TSs and thereby enhance the specificity of alpha156.


2003 - Ortho-halogen naphthaleins as specific inhibitors of Lactobacillus casei thymidylate synthase. Conformational properties and biological activity [Articolo su rivista]
S., Ghelli; M., Rinaldi; D., Barlocco; A., Gelain; P., Pecorari; Tondi, Donatella; Rastelli, Giulio; Costi, Maria Paola
abstract

Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the development of anticancer and antinfective agents. Recently, we described a series of phthalein and naphthalein derivatives as TS inhibitors. These compounds have structures unrelated to the folate (Non-Analogue Antifolate Inhibitors, NAAIs) and were selective for the bacterial versus the human TS (hTS). In particular, halogen-substituted molecules were the most interesting. In the present paper the halogen derivatives of variously substituted 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[2,3-c]furan-1-one (1-5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-c,d]pyran-1-one (6-14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational properties of the naphthalein series were explored in order to highlight possible differences between molecules that show species-specific biological profile with respect to non species-specific ones. With this aim, the conformational properties of the synthesized compounds were investigated by NMR, in various solvents and at different temperatures, and by computational analysis. The apparent inhibition constants (K-i) for Lactobacillus casei TS (LcTS) were found to range from 0.7 to 7.0 muM, with the exception of the weakly active iodo-derivatives (4, 10, 13); all] the compounds were poorly active against hTS. The di-halogenated compounds 7, 8, 14 showed the highest specificity towards LcTS, their specificity index (SI) ranging between 40 and > 558. The di-halogenated 1,8-naphthalein derivatives (7-10) exhibited different conformational properties with respect to the tetra-haloderivatives. Though a clear explanation for the observed specificity by means of conformational analysis is difficult to find, some interesting conformational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme.


2003 - Quinoxaline chemistry. Part 15. 4-[2-Quinoxalylmethylenimino]-benzoylglutamates and -benzoates, 4-[2-quinoxalylmethyl-N-methylamino]-benzoylglutamates as analogues of classical antifolate agents. Synthesis, elucidation of structures and in vitro evaluati [Articolo su rivista]
Loriga, M.; Piras, S.; Paglietti, G.; Costi, Maria Paola; Venturelli, Alberto
abstract

We report on an extension of our previous discovery of in vitro anticancer activity of trifluoromethylquinoxalines as analogues of classical and non-classical antifolic methotrexate and trimetrexate. In this case a small number of Schiff bases were obtained from the reaction of 2-bromethyl-3-R-6(7)trifluoromethylquinoxaline and ethyl p-aminobenzoylglutamate, ethyl p-aminobenzoate, p-toluidine instead of the expected 4-[2-quinoxalyl]methyl-N-methylanilino derivatives, which in turn formed with N-methylanilino derivatives. The reaction mechanism has been put forward. Structure elucidation of both Schiff bases and N-methylanilino analogues was achieved by a combination of 1H and 13C NMR spectra and hetcor experiments. Compounds 3a, 3b, 3c, 8, 11, 12, 13, Ie were tested in antifolic enzyme assay [Lactobacillus casei (LcTS), Leishmania major (LmTs), human Thymidylate synthase (hTs), human TS, human dihydrofolate reductase (hDHFR)] while compounds 3a, 3b, 3c were tested for anticancer activity. These results seem to indicate that the Schiff bases are somewhat active either as anticancer or as folate inhibitors, while compound Ie was selectively active against hDHFR with an inhibition constant (Ki) of 200 nM with a specificity of about 1000-folds with respect to hTS.


2003 - Saturation site-directed mutagenesis of Lactobacillus casei Thymidylate synthase. Mutagenesis strategy and replacement sets of Pro-196 and Pro-197 [Articolo su rivista]
Paconowska, D. G; Carreras, M. A.; Costi, Maria Paola; Santi, D. V.
abstract

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated


2002 - Dal Ligand based design di inibitori della Timidilato Sintasi all'Analisi Comparativa dei Complessi Enzima-Inibitore [Abstract in Atti di Convegno]
Costi, Maria Paola; Ferrari, Stefania; Pecorari, Piergiorgio; Rinaldi, Marcella; Tondi, Donatella; Venturelli, Alberto; Wade, R. C.
abstract

.n/a


2002 - Drug resistance - Developing targets and strategies in drug resistance - Preface [Articolo su rivista]
Costi, Maria Paola
abstract

Introduction to the issue focused on drug resistance.


2002 - Structure-based design and in parallel synthesis of boronic acid inhibitors of AmpC beta-lactamase [Relazione in Atti di Convegno]
Tondi, Donatella; Costi, Maria Paola; B. K., Shoichet
abstract

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2002 - Structure-based studies on species-specific inhibition of thymidylate synthase [Articolo su rivista]
Costi, Maria Paola; Tondi, Donatella; Rinaldi, Marcella; Barlocco, Daniela; Pecorari, Piergiorgio; Soragni, Fabrizia; Venturelli, Alberto; Stroud, Rm
abstract

Thymidylate synthase (TS) is a well-recognized target for anticancer chemotherapy. Due to its key role in the sole de novo pathway for thymidylate synthesis and, hence, DNA synthesis, it is an essential enzyme in all life forms. As such, it has been recently recognized as a valuable new target against infectious diseases. There is also a pressing need for new antimicrobial agents that are able to target strains that are drug resistant toward currently used drugs, In this context, species specificity is of crucial importance to distinguish between the invading microorganism and the human host, yet thymidylate synthase is among the most highly conserved enzymes. We combine structure-based drug design with rapid synthetic techniques and mutagenesis, in an iterative fashion, to develop novel antifolates that are not derived from the substrate and cofactor, and to understand the molecular basis for the observed species specificity. The role of structural and computational studies in the discovery of nomanalog antifolate inhibitors of bacterial TS, naphthalein and dansyl derivatives, and in the understanding of their biological activity profile, are discussed.


2001 - Enhancement of the drug discovery process by integration of Structure-based drug design and combinatorial chemistry [Capitolo/Saggio]
Tondi, Donatella; Costi, Maria Paola
abstract

This text traces developments in rational drug discovery and combinatorial library design with contributions from 50 leading scientists in academia and industry who offer coverage of basic principles, design strategies, methodologies, software tools and algorithms, and applications. It outlines the fundamentals of pharmacophore modelling and 3D Quantitative Structure-Activity Relationships (QSAR), classical QSAR, and target protein structure-based design methods.


2001 - Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase [Articolo su rivista]
Ta, Fritz; Tondi, Donatella; J., FINER MOORE; Costi, Maria Paola; Rm, Stroud
abstract

Background: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species.Results: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2-deoxyuridine-5-monophosphate (dUMP). The 2.0 Å structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzymes active site with movement of C carbons up to 6 Å relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDTs specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations.Conclusions: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.


2001 - Probing Molecular Recognition of Dansyl Derivatives vs Bacterial Thymidylate Synthase (TS) [Abstract in Atti di Convegno]
Tondi, Donatella; Calò, S.; Fritz, T.; Stroud, R.; Costi, Maria Paola
abstract

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2001 - Structural and thermodynamic combined studies for inhibitors binding to Thymidylate Synthase [Abstract in Atti di Convegno]
Venturelli, Alberto; Tondi, Donatella; Skinner, M. A.; Brown, K. A.; Ponterini, Glauco; Costi, Maria Paola
abstract

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2001 - Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase [Articolo su rivista]
Tondi, Donatella; Ra, Powers; Caselli, Emilia; Mc, Negri; J., Blazquez; Costi, Maria Paola; Bk, Shoichet
abstract

Background: Group I p-lactamases are a major cause of antibiotic resistance to beta -lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta -lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta -lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta -lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. Results: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K-i 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with iii values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 Angstrom resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Conclusions: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta -lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K-i 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta -lactamase.


2001 - Structure-based design and solid phase synthesis of novel AmpC b-lactamase inhibitors [Abstract in Atti di Convegno]
Tondi, Donatella; Powers, R.; Caselli, E.; Costi, M. P.; Shoichet, B. K.
abstract

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2001 - Update on antifolate drugs targets [Articolo su rivista]
Costi, Maria Paola; Ferrari, Stefania
abstract

Antifolate drugs are molecules directed to interfere with the folate metabolic pathway at some level. They can be recognized among the first rationally designed compounds applying the principle of structural analogy with the substrate developing the antimetabolite strategy. This strategy has taken advantage of the basic different features of the microbial and human folate metabolism and therefore allows targeting the pathway at different steps generating a specificity tools for Medicinal Chemists. Two main problems are giving renewed importance to such targets and therefore improving the efforts to discover new targets in the folate metabolism area. The first one is the increasing resistance to the present drugs due to different mechanisms such as the enzyme modification and the increased production of enzymes with not well recognized importance. The second one is the development of techniques directed to highlight the interference at genetic level of molecular probes as antifolate drug to develop new enzymes previously unknown. This approach is defined as genetic approach to drug discovery, from gene to drugs. The present article describes the importance in drug design and discovery of some antifolate targets among the best known at the present status of research such as thymidylate synthase (TS), dhydrofolate reductases, (DHFR) serine hydroxymethyltransferase (SHMT), folyilpolyglutamyl synthetase (FPGS), gamma-glutamyl hydrolase (gamma-GH), glycinamide-ribonucleotide transformylase (GARTfase), amino-imidazole-carboxamide-ribonucleotide transformylase (AICARTfase) and Folate transporters. Discovery, known functions, structure/function studies and inhibition will be described


2000 - Phenylboronic acid derivative inhibitors of ?-lactamases, their preparation, pharmaceutical compositions, and therapeutic use. [Monografia/Trattato scientifico]
Shoichet, B. K.; Costi, Maria Paola; Tondi, Donatella
abstract

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2000 - Structure-Based Design and In Parallel Synthesis of Boronic Acid Inhibitors of AmpC 61538;-Lactamase [Abstract in Atti di Convegno]
Tondi, Donatella; Powers, R.; Caselli, Emilia; Costi, Maria Paola; Shoichet, B. K.
abstract

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2000 - Structures and reactivities of 1-oxo-cycloalkan-2-ylidenacetic acids. A 1H-NMR, modeling and photochemical study [Articolo su rivista]
S., Ghelli; L., Toma; D., Barlocco; Costi, Maria Paola; Ponterini, Glauco
abstract

Four 1-oxo-cycloalkan-2-ylideneacetic acids, differing in the size of the aliphatic ring and in the nature of the condensedunsaturated cycle, exhibit different reactivities towards hydrazine, leading in only one case to the desired tryciclic pyridazinone. The observed differences in behaviour cannot be ascribed to different configurations at the exocyclic double bond of the four acids: 1H NMR experiments, combined with UV photolysis, have shown that all compounds have been prepared in the E form and are stable in the absence of light and catalysts. The photochemically obtained Z isomers show an increasing tendency to form tricyclic lactones with increasing size ofthe aliphatic ring. A theoretical structural analysis of the E and Z isomers, the tricyclic lactones and some of the hypothetical intermediates of the reaction with hydrazine suggests the size of the aliphatic ring and the associated flexibility to be crucial in modulating the ability of these compounds to form tricyclic products.


2000 - Studi termodinamici e strutturali dell’interazione tra derivati densilici e Timidilato Sintasi Batteriche [Abstract in Atti di Convegno]
Tondi, Donatella; Venturelli, Alberto; Skinner, M. A.; Brown, K.; Costi, Maria Paola
abstract

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2000 - 3-Aminophenylboronic acid derivative inhibitors of β-lactamases, their preparation, and their therapeutic use [Brevetto]
Shoichet, B. K.; Costi, Maria Paola; Tondi, Donatella
abstract

Non-β-lactam inhibitors of β-lactamases are provided.  In particular, the invention provides such inhibitors which are aminophenylboronic acid derivs. I [R1 = (un)substituted lower alkyl, (un)substituted (hetero)cyclic alkene; Z = bond, O, S, lower (hetero)alkyl], or a pharmaceutically acceptable salt thereof.  These compds. may be used with β-lactam antibiotics to treat β-lactam-antibiotic-resistant bacterial infections.  Finally, the invention provides a pharmaceutical compn. comprising these compds.


1999 - Amide Boronic Acid Inhibitors of AmpC b-lactamase to Reverse Bacterial Resistance to b-lactam Antibiotics. [Brevetto]
Shoichet, B. K.; Tondi, Donatella; Costi, Maria Paola; M., P.
abstract

The invention provides novel non-$g(b)-lactam inhibitors of $g(b)-lactamases. In particular, the invention provides such inhibitors which are boronic acids of formula (1) which is set forth in the specification. These compounds may be used with $g(b)-lactam antibiotics to treat $g(b)-lactam-antibiotic-resistant bacterial infections. Finally, the invention provides a pharmaceutical composition comprising these compounds.


1999 - Phenylboronic acid derivative inhibitors of β-lactamases, their preparation, pharmaceutical compositions, and therapeutic use [Brevetto]
Shoichet, B K; Costi, Maria Paola; Tondi, Donatella
abstract

Non-β-lactam inhibitors of β-lactamases are provided.  In particular, the invention provides such inhibitors which are phenylboronic acids I [R1 = N-lower alkyl, (un)substituted (hetero)cyclic alkene] or a pharmaceutically acceptable salt thereof.  These compds. may be used with β-lactam antibiotics to treat β-lactam-antibiotic-resistant bacterial infections.  Finally, the invention provides a pharmaceutical compn. comprising these compds.


1999 - Phthalein derivatives as a new tool for selectivity in thymidylate synthase inhibition [Articolo su rivista]
Costi, Maria Paola; Rinaldi, Marcella; Tondi, Donatella; Pecorari, Piergiorgio; Barlocco, Daniela; Ghelli, Stefano; Rm, Stroud; Dv, Santi; Tj, Stout; C., Musiu; Em, Marangiu; A., Pani; D., Congiu; Ga, Loi; P., La Colla
abstract

A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were designed to specifically complement structural features of a bacterial form of thymidylate synthase (Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds were screened for their activity and their specificity against TS enzymes from different species, namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and human thymidylate synthase (hTS). Apparent inhibition constants (Ki) for all the compounds against LcTS were determined, and inhibition factors (IF,ratio between the initial rates of the enzymatic reaction in the presence and absence of each inhibitor) against each of the four TS species were measured. A strong correlation was found between the two activity parameters, IF and Ki, and therefore the simpler IF was used as a screening factor in order to accelerate biological evaluation. Compounds 5b, 5c, 5ba, and 6bc showed substantial inhibition of LcTS while remaining largely inactive against hTS, illustrating for the first time remarkable species specificity among TSs. Due to sequence homology between the enzymes, several compounds also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H,3H-naphtho[1,8-c,d]pyran-1-one, (6bc) showed an IF < 0.04 for CnTS (K-i = 0.45 mu M) while remaining inactive in the hTS assay at the maximum solubility concentration of the compound (200 mu M). In cell culture assays most Of the compounds were found to be noncytotoxic to human cell lines but were cytotoxic against several species of Grampositive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several compounds also had selective activity against Cr. neoformans in cell culture assay. In general, the most active and selective compounds against the Gram-positive bacteria were those designed and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound was least selective against most of the cell lines tested. To our knowledge these compounds are the first TS inhibitors selective for bacterial TS with respect to hTS.


1999 - Separation, structural determination and biological evaluation of the thymidylate synthase inhibitor 3,3-di(4'-hydroxyphenyl)-6(7)-chloro-1-oxo-1H,3H-naphtho[1,8-cd]pyran [Articolo su rivista]
Costi, Maria Paola; Tondi, Donatella; Pecorari, Piergiorgio; Rinaldi, M; Celentano, G; Ghelli, Stefano; Antolini, L; Barlocco, Daniela
abstract

The chloro substituted 3,3-di-(4'-hydroxyphenyl)-1-oxo-1H,3H-naphtho[1,8-cd]pyran was synthesized in a 40/60 mixture of C-6 or C-7 substituted isomers, respectively. The two isomers were separated by hplc. The X-ray crystal structure of the mixture was obtained. Both the mixture and the single isomers were tested against Lactobacillus Casei thymidylate synthase. The X-ray analysis clearly revealed co-crystallization of the two isomeric species. The apparent Ki of the mixture was 0.8 muM, while those of the C-6 and C-7 substituted isomers were 0.42 and 0.52 muM, respectively, thus showing that the position of the chlorine in the naphthalene ring was not critical for enzymatic activity.


1999 - Structure based design and combinatorial optimization of novel Thymidylate synthase inhibitors. Meeting Abstract : 217th ACS National Meeting, Chicago March 1999 [Abstract in Atti di Convegno]
Tondi, Donatella; Slomcynska, U.; Ghelli, Stefano; Watterson, D. M.; Costi, Maria Paola; Shoichet, B. K.
abstract

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1999 - Structure-based design of inhibitors specific for bacterial thymidylate synthase [Articolo su rivista]
Tj, Stout; Tondi, Donatella; Rinaldi, Marcella; Barlocco, Daniela; Pecorari, Piergiorgio; Dv, Santi; I., Kuntz; Rm, Stroud; Costi, Maria Paola; Bk, Shoichet
abstract

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.


1999 - Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase [Articolo su rivista]
Tondi, Donatella; U., Slomczynska; Costi, Maria Paola; Dm, Watterson; Ghelli, Stefano; Bk, Shoichet
abstract

Background: The substrate sites of enzymes are attractive targets for structure-based inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging, Results: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied, The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (K-i 65 mu M). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a K-i of 1.3 mu M. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes, Conclusions: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged.


1999 - Sulfonamide Boronic Acid Inhibitors of AmpC b-lactamase to Reverse Bacterial Resistance to b-lactam Antibiotics. [Brevetto]
Shoichet, B. K.; Tondi, Donatella; Costi, Maria Paola; M., P.
abstract

The invention provides novel non-$g(b)-lactam inhibitors of $g(b)-lactamases. In particular, the invention provides such inhibitors which are boronic acids of formula (1) which is set forth in the specification. These compounds may be used with $g(b)-lactam antibiotics to treat $g(b)-lactam-antibiotic-resistant bacterial infections. Finally, the invention provides a pharmaceutical composition comprising these compounds


1999 - Thymidylate Synthase as a potential new target in drug resistance antimicrobial therapy. [Abstract in Atti di Convegno]
Costi, Maria Paola; A., Gelain; I., Cardilicchio; Soragni, Fabrizia; Rinaldi, Marcella; Pecorari, Piergiorgio; Tondi, Donatella; D. V., Santi
abstract

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1998 - Erratum: Thymidylate synthase inhibition: A structure-based rationale for drug design (Medicinal Research Reviews 18 (21-42)) [Articolo su rivista]
Costi, M. P.
abstract


1998 - Quinoxaline chemistry. Part 11. 3-phenyl-2[phenoxy- and phenoxymethyl]-6(7) or 6,8-substituted quinoxalines and N-[4-(6(7)-substituted or 6,8-disubstituted-3-phenylquinoxalin-2-yl)hydroxy or hydroxymethyl]benzoylglutamates. Synthesis and evaluation of in [Articolo su rivista]
Corona, P; Vitale, G; Loriga, M; Paglietti, G; Costi, Maria Paola
abstract

Twenty-four out of twenty-nine quinoxalines were selected at the National Cancer Institute, Bethesda, Md, USA, for in vitro anticancer screening. Among these, 10 derivatives exhibited high values of percent tumor growth inhibition at a concentration of 10(-4) M in all cancer cell lines. Four of these compounds maintained these values at 10(-5) M, whereas a certain number exhibited significant values of percent inhibition at the most diluted concentrations (10(-8)-10(-6) M). Inhibitory activity against dihydrofolate reductase (DHFR) (bovine and rat liver) was determined for the most active compounds. This test showed that this type of quinoxaline exhibited an appreciable activity in comparison with the previously described aza analogues. In the other test (Lactobacillus casei, thymidylate synthase (TS), human HTS) no or poor activity was detected in both series of compounds. (C) 1998 Elsevier Science S.A. All rights reserved.


1998 - Thymidylate synthase inhibition: A structure-based rationale for drug design [Articolo su rivista]
Costi, Maria Paola
abstract

Thymidylate synthase (TS) is a very interesting target in antiproliferative diseases. Its inhibition causes thimineless death of the cells and compounds inhibiting TS are widely used in anticancer therapy. The classical antifolate TS inhibitors are structural analogs of the folate co-factor; they often share the same metabolic pathways and this causes the development of resistance inside the cells, A detailed analysis of the available x-ray crystal structures of the complexes of the enzyme with different substrates and inhibitors support the finding of a structural basis of their biological activity. TS inhibitors nonstructural analog of folate, non-analog antifolate inhibitors (NAAI), are welcome as a new interesting research topic. Among the most recent and interesting ones, compounds from Agouron related to the indole structure, are independent on the folate metabolism, highly active and specific for human TS. Other compounds, phthalein derivatives, can inhibit TS enzymes from various sources and show an interesting biological activity profile: they inhibit better bacterial and fungal TS than human TS. The x-ray crystal structures of some of these inhibitors with TS show that they bind in a different binding site from that of the classical folate TS inhibitors. This indicates a potential allosteric binding site useful for future drug discovery studies.


1998 - 3-Aminophenylboronic acid derivative inhibitors of beta-lactamases, their preparation, and their therapeutic use. [Brevetto]
Shoichet Brian, K.; Costi, Maria Paola; Tondi, Donatella
abstract

The invention provides novel non-/9-lactam inhibitors of ^-lactamases. In particular, the invention provides such inhibitors which are boronic acids of formula (I) which is set forth in the specification. These compounds may be used with /Mactam antibiotics to treat /J-lactam-antibiotic-resistant bacterial infections. Finally, the invention provides a pharmaceutical composition comprising these compounds.


1997 - "Structure-Based Discovery & In parallel Optimization of Novel Inhibitors of Thymidylate Synthase" [Relazione in Atti di Convegno]
Tondi, D.; Slomczynska, U.; Ghelli, S.; Wattweson, D. M.; Costi, M. P.; Shoichet, B. K.
abstract


1997 - 1H-NMR conformational studies of some phtalein derivatives acting as thymidylate synthase inhibitors [Articolo su rivista]
S., Ghelli; Costi, Maria Paola; D., Barlocco; M., Rinaldi; Tondi, Donatella; P., Pecorari; Rastelli, Giulio
abstract


1996 - Asparagine 229 mutants of thymidylate synthase catalyze the methylation of 3-methyl-2'-deoxyuridine 5'-monophosphate [Articolo su rivista]
Costi, Maria Paola; Liu, L; Finermoore, Js; Stroud, Rm; Santi, Dv
abstract

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, L., gr Santi, D, V, (1993) Proc. Natl. Acad. Sci, U.S.A, 90, 8604-8608] but is a major determinant in substrate specificity [Liu, L., & Santi, D. V. (1993) Biochemistry 32, 9263-9267], 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have k(cat) values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of JUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be mon active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis, In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar K-m values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C-3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP ill the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric Interference of the 3-methyl group with the side chain of Asn 229.


1996 - Conformational analysis of phthalein derivatives acting as thymidylate synthase inhibitors by means of 1H NMR and quantum chemical calculations [Articolo su rivista]
S., Ghelli; Rastelli, Giulio; D., Barlocco; M., Rinaldi; Tondi, Donatella; P., Pecorari; Costi, Maria Paola
abstract

The conformations of a set of phthalein derivatives with bacterial thymidylate synthase (TS) inibitory activity were investigated by H-1 NMR spectra, performed at both room and low temperature, and by quantum chemical calculations. Since the crystal structure of the binary complex of phenolphthalein with the enzyme is known, we set out to study the conformation of various of its analogues in solution in order to observe the effects of the substituents on the phenolic rings, of the alpha-naphthol derivative and of the rigid analogue, fluorescein, and compare the results with the X-ray crystal structure studies. A relationship between the chemical shift of the proton on C4 (H4) of the phthalidic ring and the averaged angle formed by the phthalidic and the aromatic ring planes was found in which the most perpendicular conformations have the lowest H4 chemical shift values. At room temperature, the rotational freedom of all the studied compounds was similar, while at lower temperature the naphthol derivative assumed a partially blocked conformation. Finally, a qualitative relationship between the inhibitory properties of the compounds and their conformations is discussed. Copyright


1996 - Model chemical studies of thymidylate synthase. Nucleophilic addition of hydroxylamine to uracil and 5-fluorouracil [Articolo su rivista]
Costi, Maria Paola; Rastelli, Giulio; M., Rinaldi; M., Cevolani; P., Pecorari
abstract

To explain the reaction of 5-fluoro-2'-deoxyuridine-5'-monophosphate towards Thymidylate Synthase (TS), a chemical model formed by hydroxylamine and 5-fluorouracil (FU) or uracil (U) was suggested. The reaction mechanism would involve a nucleophilic attack of the nitrogen of the free base to C-6 of the uracil ring; the reaction is catalyzed by the cationic form of the base. The kinetic constants, the activation energies, the entropies of activation for the forward and the reverse reactions and the equilibrium constants for the reactions of FU and U with hydroxylamine were measured. The value of the activation energy for the forward reaction of FU with hydroxylamine is 1.95 Kcal/mol lower than with U. The value of the equilibrium constant for the reaction of FU with hydroxylamine is 23 times higher than that of U at 25 degrees C. Therefore the product of the first reaction is the most stable one. The reactivity of FU towards hydroxylamine is, in its complex, higher with respect to the reactivity of U in the same conditions, because the reaction proceeds largely towards higher Keg values; the activation energy of the forward reaction is lower for FU and the entropy of activation too. Thus indicating that the activated complex is more ordered than that of U. The results indicate that the increased reactivity and stability of the complex with FU in the model reaction may account for the competition observed in the biological reaction.


1996 - Naphthalimido Derivatives as Antifolate Thymidylate Synthase Inhibitors [Articolo su rivista]
Costi, Mp; Tondi, Donatella; Rinaldi, M; Barlocco, D; Cignarella, G; Santi, Dv; Musiu, C; Pudu, I; Vacca, G; Lacolla, P.
abstract

A new series of N-(substituted)benzyl-1,8-naphthalimides 4, structurally related to the previously reported thymidylate synthase (TS) inhibitor naphthaleins 3, were synthesized and compounds tested for their inhibition of several species of TS. Moreover, their in vitro cytotoxicity together with antimycotic and antibacterial properties were assayed. While no activity was detected in the antibacterial tests, the m-nitro (4ae) and the p-nitro (4af) derivatives were found able to partially inhibit TS at low micromolar concentrations. Introduction of nitro or (substituted)-amino groups in position 4 of the naphthalic ring always led to less active compounds.


1995 - Theoretical analysis of the addition of hydroxylamine to uracil and 5-fluorouracil as a model for the Thymidylate synthase reaction [Articolo su rivista]
Rastelli, Giulio; Costi, Maria Paola
abstract

In the present paper we report a quantum chemical (PM3) investigation of reagents, transition structures, intermediates and final products of the nucleophilic addition of hydroxylamine to uracil (U) and 5-fluorouracil (FU). This reaction serves as a model for the more complex enzymatic methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) by thymidylate synthase. From the analysis of the frontier orbitals of the isolated and complexed species, as well as from the calculation of activation barriers, we propose that nucleophilic attack usually proceeds after formation of an initial complex between U (or FU) and one neutral and one protonated molecule of hydroxylamine. Our results give some insight into the mechanism of these reactions and account for the higher rate of addition of hydroxylamine to FU, compared to U. The main connection between the chemical simulation and the biological scheme is that in both reactions hydrogen bonding residues are found to be necessary to assist catalysis.


1994 - A condensed thiadiazolo-pyrimidine as a new efficient fluorophore. Theoretical and experimental investigation of the electronic spectra and photophysics [Articolo su rivista]
Baraldi, Ivan; Carnevali, A.; Caselli, Monica; Costi, Maria Paola; Pecorari, P.; Ponterini, Glauco; Rinaldi, M.
abstract

A recently synthesized condensed thiadiazolo-pyrimidine (MTP) has been found to exhibit a very intense fluorescence emission in aprotic solvents. The origins of such an unusual property for a compound containing many different heteroatoms (four nitrogens, two sulphurs and one oxygen) have been investigated by a combined theoretical and experimental approach. The nature and peculiarity of the MTP chromophore have been analysed using both computed electron densities and crystallographic data from the literature. The singlet and triplet spectra have been measured and calculated, and a good agreement has been found between the two sets of results. On this basis, we have attempted to interpret the rather peculiar photophysical behaviour of MTP.


1994 - Heterodimeric thymidylate synthases with C-terminal deletion on one subunit. [Articolo su rivista]
Carreras, Cw; Costi, Maria Paola; Santi, Dv
abstract

We have combined site directed mutagenesis with the technique of reversible unfolding and subunit dissociation to construct heterodimeric thymidylate syntheses that lack the C-terminal valine from only one subunit of the dimer. Removal of this residue either from both subunits of the dimer by mutagenesis (V316Am mutation) or from only one subunit by treatment with carboxypeptidase has been reported to result in an inactive enzyme (Carreras, C. W, Climie, S. C., and Santi, D. V. (1992) Biochemistry 31, 6038-6044; Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). Arg-178 is an essential active site residue of thymidylate synthese that is donated from the opposing subunit of the dimer. The R178F-V316Am heterodimer was formed by the unfolding and refolding of a mixture of inactive R178F and V316Am mutants. This enzyme has one intact active site and was found to have half of the activity and the same K-m values as wild-type thymidylate synthase that was unfolded and refolded as a control. We have also formed the V316Am-WT heterodimer and report that this heterodimeric enzyme is also active, has a k(cat) value that is approximately half of that of the wild-type thymidylate synthase dimer, and binds substrate and cofactor with K-m values similar to those of the wild-type enzyme.


1993 - Crystal and molecular structures of 5-Fluoro-3-methyl, 5-chloro-3-methyl and 5-1,3-dimethyl -uracil hydrogen bond patterns through X-ray and molecular orbital analysis [Articolo su rivista]
G., Vampa; L., Antolini; Rastelli, Giulio; Costi, Maria Paola; P., Pecorari; M., Rinaldi
abstract

CRYSTAL AND MOLECULAR-STRUCTURES OF 5-FLUORO-3-METHYL-URACIL, 5-CHLORO-3-METHYL-URACIL AND 5-CHLORO-1,3-DIMETHYL-URACIL - HYDROGEN-BOND PATTERNS THROUGH X-RAY AND MOLECULAR-ORBITAL ANALYSIS


1992 - NEW HETEROCYCLIC STRUCTURES - [1,2,5]THIADIAZOLO[3',4'-4,5]PYRIMIDO[2,1-B][1,3]THIAZINE AND THIAZOLO[3,2-A][1,2,5]THIADIAZOLO[3,4-D]PYRIMIDINE [Articolo su rivista]
Pecorari, P; Rinaldi, M; Costi, Maria Paola; Antolini, L.
abstract

Derivatives of two new molecular structures, namely, 7,8-dihydro-6H,10H-[1,2,5]thiadiazolo[3',4':4,5]pyrim-ido[2,1-b][1,3]thiazin-10-one and 6,7-dihydro-9H-thiazolo[3,2-a][1,2,5]thiadiazolo[3,4-d]pyrimidin-9-one, and derivatives of N-substituted sulfamic acid, namely, (8-amino-3,4-dihydro-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-on-7-yl)sulfamic acid and (7-amino-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidin-5-on-6-yl)sulfamic acid, were separated out as by-products in the reduction reaction of 8-amino-3,4-dihydro-7-nitroso-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-one and 7-amino-2,3-dihydro-6-nitroso-5H-thiazolo[3,2-a]pyrimidin-5-one derivatives, respectively, with sodium hydrosulfite. A mechanism of reaction, which hypothesizes the action of sodium hydrosulfite in an asymmetic form, is proposed. The results of single-crystal X-ray investigation on 7,8-dihydro-6H,10H-[1,2,5]thiadiazolo[3',4':4,5]pyrim-ido[2,1-b][1,3]thiazin-10-one (R = 0.032 for 863 reflections) and (8-amino-3,4-dihydro-2H,6H-pyrimido[2,1-b]-[1,3]thiazin-6-on-7-yl)sulfamic acid, sodium salt (R = 0.028 for 3507 reflections) are reported.


1991 - CYCLIZATION REACTIONS OF 1,3-DIBROMOPROPAN-2-OL, 2,3-DIBROMOPROPAN-1-OL AND 1-BROMOMETHYLOXIRANE WITH 6-AMINO-2,3-DIHYDRO-2-THIOXO-4(1H)-PYRIMIDINONE [Articolo su rivista]
Pecorari, Piergiorgio; Rinaldi, Marcella; Costi, Maria Paola; Antolini, Luciano
abstract

The cyclization reactions, carried out in strongly- or weakly-basic media, are described. Sometimes, 7-amino-2,3-dihydro-3-hydroxymethyl-5H-thiazolo[3,2-a]pyrimidin-5-one is separated out, together with 8-amino-3,4-dihydro-3-hydroxy-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-one, as the principal product. A mechanism of reaction, during which the cyclizating agents are changed into oxirane derivatives, is proposed. The results of single-crystal X-ray investigations on 8-amino-3,4-dihydro-3-hydroxy-7-nitroso-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-one (R = 0.035 for 1013 reflections), and on 7-hydroxymethyl-6,7-dihydrothiazolo[3,2-a][1,2,3]triazolo[4,5-d]pyrimidin-9(1H)-one (R = 0.027 for 1607 reflections) are reported.


1990 - Theoretical study of electronic spectra and photophysics of uracil derivatives. [Articolo su rivista]
Baraldi, Ivan; Bruni, Mc; Costi, Maria Paola; Pecorari, Piergiorgio
abstract

The changes that the UV absorption spectrum and the photophysics of uracil undergo under hydrogen substitution or deprotonation, were studied theoretically within the CS-INDO/CI scheme. First of all this method was tested on uracil. It was then used for the calculation of the electronic structure of excited states (Sn, Tn) of a large number of uracil derivatives (1-, 3- and 5-methyluracil; 1,3-, 1,5- and 3,5-dimethyluracil; 5-fluoro- and 5-chlorouracil), including some anions (1- and 3-methyluracil anion). The excited states were obtained in the singly-excited configuration interaction approximation (S-CI) and the correlation effects on (pi pi*) states were studied by including the most important doubly- and triply-excited configurations in the CI. The S-CI wavefunctions were used for the calculation of the most important electronic matrix elements for spin-orbit coupling. The photophysics of these compounds is discussed using Jablonski diagrams.


1989 - NEW HETEROCYCLIC STRUCTURES - [1,3]THIAZINO[3,2-A]PURINE AND [1,2,3]TRIAZOLO[4,5-D][1,3]THIAZINO[3,2-A]PYRIMIDINE [Articolo su rivista]
Pecorari, P; Rinaldi, M; Costi, Maria Paola
abstract

The syntehtic pathway of the [1,3]THIAZINO[3,2-A]PURINE AND [1,2,3]TRIAZOLO[4,5-D][1,3]THIAZINO[3,2-A]PYRIMIDINE has been described and the antiviral activity evaluated.


1989 - Preparation and physicochemical properties of uracil derivatives with potential biological activity [Articolo su rivista]
Pecorari, Piergiorgio; Vampa, Gabriella; Albasini, Albano; Rinaldi, Marcella; Melegari, Michele; Costi, Maria Paola
abstract


1988 - Antimycotic and spectral properties of some thiouracilderivatives [Articolo su rivista]
Pecorari, P.; Melegari, M.; Rinaldi, M.; Costi, M. P.; Provvisionato, A.
abstract


1987 - Antimycotic action of alkyl derivatives of 5-(benzenesulfonamide)-1,2,3,4-tetrahydro-2-thioxo-4-pyrimidinone [Articolo su rivista]
Pecorari, P.; Melegari, M.; Albasini, A.; Rinaldi, M.; Costi, M. P.; Provvisionato, A.
abstract