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Donatella TONDI

Professore Associato
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103

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Pubblicazioni

- 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

2023 - Structure-Based Optimization of 1,2,4-Triazole-3-Thione Derivatives: Improving Inhibition of NDM-/VIM-Type Metallo-β-Lactamases and Synergistic Activity on Resistant Bacteria [Articolo su rivista]
Bersani, M.; Failla, M.; Vascon, F.; Gianquinto, E.; Bertarini, L.; Baroni, M.; Cruciani, G.; Verdirosa, F.; Sannio, F.; Docquier, J. D.; Cendron, L.; Spyrakis, F.; Lazzarato, L.; Tondi, D.
abstract

: The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-β-lactamases (MBLs) menace the efficacy of all β-lactam antibiotics, including carbapenems, a last-line treatment usually restricted to severe pneumonia and urinary tract infections. Nonetheless, no MBL inhibitor is yet available in therapy. We previously identified a series of 1,2,4-triazole-3-thione derivatives acting as micromolar inhibitors of MBLs in vitro, but devoid of synergistic activity in microbiological assays. Here, via a multidisciplinary approach, including molecular modelling, synthesis, enzymology, microbiology, and X-ray crystallography, we optimized this series of compounds and identified low micromolar inhibitors active against clinically relevant MBLs (NDM-1- and VIM-type). The best inhibitors increased, to a certain extent, the susceptibility of NDM-1- and VIM-4-producing clinical isolates to meropenem. X-ray structures of three selected inhibitors in complex with NDM-1 elucidated molecular recognition at the base of potency improvement, confirmed in silico predicted orientation, and will guide further development steps.

2023 - The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents [Articolo su rivista]
Linciano, Pasquale; Pozzi, Cecilia; Tassone, Giusy; Landi, Giacomo; Mangani, Stefano; Santucci, Matteo; Luciani, Rosaria; Ferrari, Stefania; Santarem, Nuno; Tagliazucchi, Lorenzo; Cordeiro-da-Silva, Anabela; Tonelli, Michele; Tondi, Donatella; Bertarini, Laura; Gul, Sheraz; Witt, Gesa; Moraes, Carolina B; Costantino, Luca; Costi, Maria Paola
abstract

2022 - Characterisation and Optimisation of Anti-LexA Nanobodies Targeting the SOS-response Pathway to Fight Antibiotic Resistance [Poster]
Campagnaro, Enrica; Vascon, Filippo; Maso, Lorenzo; Chinellato, Monica; Goormaghtigh, Frédéric; Bellio, Pierangelo; Van Melderen, Laurence; Ruzzene, Maria; Pardon, Els; Angelini, Alessandro; Celenza, Giuseppe; Steyaert, Jan; Tondi, Donatella; Cendron, Laura
abstract

2022 - Discovery of a Novel Allosteric Inhibitor PK-188 Targeting New Delhi Metallo-β Lactamases. [Poster]
Kavaliauskas, Povilas; Tondi, Donatella; Cendron, Laura; Grybaite, Birute; Anusevicius, Kazimieras; Mickevicius, Vytautas; Petraitiene, Ruta; Norvaisas, Povilas; Kamuntavicius, Gintautas; Bastas, Orestis; Yang, Tony; Macer, Sam; Tal, Roy; Jocys, Zygimantas; Prat, Alvaro; Craney, Arryn; Petraitis., Vidmantas
abstract

2022 - Looking for broad-spectrum inhibitors targeting carbapenemases [Relazione in Atti di Convegno]
Spyrakis, Francesca; Gianquinto, Eleonora; Bersani, M.; Failla, M.; Bertarini, Laura; Vascon, Filippo; Sannio, F.; Verdirosa, F.; Beatrice, N.; De, Luca; N., Costi; Maria, Paola.; Cruciani, G.; Docquier, J-D; Cendron, Laura.; Tondi, Donatella; Lazzarato, L.
abstract

2022 - Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway [Articolo su rivista]
Maso, Lorenzo; Vascon, Filippo; Chinellato, Monica; Goormaghtigh, Frédéric; Bellio, Pierangelo; Campagnaro, Enrica; Van Melderen, Laurence; Ruzzene, Maria; Pardon, Els; Angelini, Alessandro; Celenza, Giuseppe; Steyaert, Jan; Tondi, Donatella; Cendron, Laura
abstract

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.

2022 - Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators [Articolo su rivista]
Fagnani, Lorenza; Nazzicone, Lisaurora; Bellio, Pierangelo; Franceschini, Nicola; Tondi, Donatella; Verri, Andrea; Petricca, Sabrina; Iorio, Roberto; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe
abstract

2022 - Synthesis of novel N-substituted amino acids derivatives bearing 2-hydroxy-5-nitrophenyl moietyas promising metallo- β-lactamases inhibitors [Relazione in Atti di Convegno]
Kavaliauskas, Povilas; Grybaitė, Birutė; Grigalevičiūtė, Ramunė; Petraitis, Vidmantas; Petraitienė, Rūta; Naing, Ethan; Garcia, Andrew; Centron, Laura; Tondi, Donatella; Mickevičius, Vytautas
abstract

Growing antimicrobial resistant among Gram-negative bacterial pathogens remains one of the major healthcare associated threats worldwide [1]. The emergence of broad-spectrum metallo-β-lactamases (MBL) results in resistance to majority of β-lactam ring containing antibiotics, leading to the treatment failure and death. Therefore, it is important to search for novel inhibitors targeting MBL’s. In this study we report a characterization of synthesis and invitro inhibitory activity of novel amino acid derivatives on NDM-1 and VIM-2 metallo-β- lactamases. […]. Fig. 1. The synthesis of novel amino acid derivatives bearing 2-hydroxy-5-nitrophenyl moiety One of the most convenient methods for the preparation of N-substituted β-amino acids and 1-aryl-5-oxopyrrolidine-3-carboxylic acids is the interaction of primary amines with unsaturated acrylic and itaconic acids. For this purpose starting material was chosen 2-ydroxy-5-nitroaniline (1), which reacted with acrylic acid to form β-amino acid 2. Meanwhile the reaction of 2-hydroxy-5-nitroaniline (1) with it aconic acid gave an intermediate product 3,which is cyclized to the target compound 4 during the reaction. Obtained compounds 2,4 were used for the synthesis of methyl esters 5,6. Reactions carried out in boiling methanol in the presence of a catalytic amount of sulfuric acid. When conducting hydrazinolysis of both esters, it was observed that ester 5 not only forms the acid hydrazide, but also occurs when the nitro group is reduced with the formation of the compound 7. Table 1. The in vitro inhibitory activity of compounds 2, 4-8 on the enzymatic activity of NDM-1 and VIM-2metallo-β-lactamases. […]

2021 - – IN SILICO SCREENING OF NOVEL INHIBITORS OF BETA-LACTAMASES [Poster]
Bertarini, Laura; Verri, Andrea; Tondi, Donatella
abstract

Bacterial Resistance represents one of the greatest threats to global health leading to a worrying imbalance between bacterial infections, which are becoming more serious and severe, and available treatments gradually getting ineffective. New resistance mechanisms are emerging and spreading worldwide, making infectious diseases very difficult to treat, if not impossible: as a matter of fact, antibiotic resistance is leading us to a pre-antibiotic era. The most prevalent class of antibiotics used in therapy is represented by beta-lactams, while beta-lactamases are versatile enzymes able to hydrolyze and then to inactivate most of available beta lactams antibiotics. To face this risk and overcome resistance, there is a huge need to discover new antibiotics and effective beta-lactamases inhibitors. Our study is focused on the identification of novel inhibitors active against KPC-2 and GES-5, beta - lactamases [1-2] belonging to Class A, and NDM-1 [3-4], from Class B. We used a computational in silico approach: with the computer software Molecular Docking (GLIDE) we performed a virtual screening on a large library of commercially available compounds, previously filtered for drug likeness properties. Our studies led to novel ligands, chemically characterized by several different moiety (i.e carboxylic acids, tetrazoles and pyrrolidines etc). The performed docking studies were supported by preliminary studies on relevant waters mechanistically involved in beta-lactamase enzymes’ hydrolysis mechanism. Flexibility within the active site was also considered for key residues such as Trp105 in KPC-2 and Trp-99 in GES-5. After further docking validation, the molecules selected in silico as most promising inhibitors that have been now directed to in vitro validation.

2021 - Discovery and preliminary characterization of a novel inhibitor of the SOS response in Pseudomonas aeruginosa [Abstract in Atti di Convegno]
Vascon, Filippo; Chinellato, Monica; Genovese, Filippo; Romanyuk, Zhanna; De Felice, Sofia; Tondi, Donatella; Kavaliauskas, Povilas; Cendron, Laura
abstract

Bacterial antibiotic resistance (AR) is becoming one of the biggest threats to human health, progressively disarming the current arsenal of antimicrobial drugs. Besides efforts to develop new antimicrobial agents, strategies to avoid the onset of novel resistance mechanisms are strongly needed. The bacterial SOS response to DNA damage (a common outcome of antibiotic treatment), mainly orchestrated by LexA and RecA proteins, is one of the crucial pathways involved in AR acquisition. In previous studies, the SOS response suppression has proved to be an efficient strategy to delay the appearance of drug resistance, but currently known inhibitors of the RecA-LexA axis are limited to few compounds. From a Fluorescence Polarization (FP)-based high-throughput screening of a small molecule library, a novel hit compound (hereafter “A12”) acting as inhibitor of the Pseudomonas aeruginosa SOS system was discovered. In-vitro dose-response characterization of A12 revealed an inhibitory potency in the high micromolar range, while biophysical assays including differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) assessed RecA as the main target. We are currently producing and screening a sub-library based on the structure of compound A12 in order to select more potent derivatives to be tested on P. aeruginosa cultures and determine their effectiveness as antibiotic adjuvants to synergize with bactericidal treatment and delay the onset of resistance.

2021 - Novel targets and mechanisms in antimicrobial drug discovery [Articolo su rivista]
Tondi, D.
abstract

The spread of infections resistant to available anti-infective drugs is a serious menace to human health [...].

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 - Can We Exploit β-Lactamases Intrinsic Dynamics for Designing More Effective Inhibitors? [Articolo su rivista]
Gianquinto, E.; Tondi, D.; D'Arrigo, G.; Lazzarato, L.; Spyrakis, F.
abstract

β-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics.

2020 - Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials [Articolo su rivista]
Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Cracchiolo, Salvatore; Franceschini, Nicola; Reale, Samantha; de Angelis, Francesco; Perilli, Mariagrazia; Amicosante, Gianfranco; Spyrakis, Francesca; Tondi, Donatella; Cendron, Laura; Celenza, Giuseppe
abstract

LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage.

2020 - Targeting the Class A Carbapenemase GES-5 via Virtual Screening [Articolo su rivista]
Klein, Raphael; Cendron, Laura; Montanari, Martina; Bellio, Pierangelo; Celenza, Giuseppe; Maso, Lorenzo; Tondi, Donatella; Brenk., Ruth
abstract

The worldwide spread of β-lactamases able to hydrolyze last resort carbapenems contributes to the antibiotic resistance problem and menaces the successful antimicrobial treatment of clinically relevant pathogens. Class A carbapenemases include members of the KPC and GES families. While drugs against KPC-type carbapenemases have recently been approved, for GES-type enzymes, no inhibitors have yet been introduced in therapy. Thus, GES carbapenemases represent important drug targets. Here, we present an in silico screening against the most prevalent GES carbapenemase, GES-5, using a lead-like compound library of commercially available compounds. The most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5 leading to four derivatives active as high micromolar competitive inhibitors. For the best inhibitors, the ability to inhibit KPC-2 was also evaluated. The discovered inhibitors constitute promising starting points for hit to lead optimization.

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.

2020 - 4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-β-Lactamases [Articolo su rivista]
Linciano, Pasquale; Gianquinto, Eleonora; Montanari, Martina; Maso, Lorenzo; Bellio, Pierangelo; Cebrián-Sastre, Esmeralda; Celenza, Giuseppe; Blázquez, Jesús; Cendron, Laura; Spyrakis, Francesca; Tondi, Donatella
abstract

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available β-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of β-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine β−Lactamase (SBLs) KPC-2 and class B1 metallo β−Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

2019 - A multidisciplinary approach in the successful identification of novel carbapenemases inhibitors [Relazione in Atti di Convegno]
Tondi, D.
abstract

2019 - Acyclic boronic acids mimic β-Lactamases tetrahedral intermediates leading to broad spectrum inhibitors [Relazione in Atti di Convegno]
Tondi, D.
abstract

2019 - Development of therapeutic agents blocking SOS activation. [Poster]
Maso, L.; Bellio, P.; Linciano, P.; D' Acunto, M.; Celenza, G.; Tondi, Donatella; Cendron, L.
abstract

Multi-Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections. The situation is even more dramatic if we consider the limited number of new antimicrobial agents currently in clinical phase. SOS response, orchestrated and triggered by LexA-RecA(ssDNA-bound) interaction, has been recently validated as a key target for combating the evolution of antibiotic resistance. Blocking and/or modulation of RecA/LexA pathway have a direct effect on the expression of genes involved in SOS response and thus on the development of intrinsic resistance and acquisition of genes of resistance. The project, based on a multidisciplinary approach, focuses on the development of therapeutic agents blocking SOS activation, through the inhibition of LexA self-cleavage and/or interference with RecA/LexA interactions. Most promising molecules we are currently testing belongs to both boronic acids derivatives, identified in silico, and natural-compounds derived macrocycles. Furthermore, encouraging results have been obtained very recently in the development and screening of a nanobodies library targeting LexA-RecA complex. This option will be adopted both to rigidify the activated SOS-machinery for single particle cryo-EM studies, as well as a probe to investigate the minimal peptide scaffold able to block LexA active site. Such results will pave the way for peptide-based macrocycles building and optimization.

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 - First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase [Articolo su rivista]
Spyrakis, Francesca; Bellio, Pierangelo; Quotadamo, Antonio; Linciano, Pasquale; Benedetti, Paolo; D’Arrigo, Giulia; Baroni, Massimo; Cendron, Laura; Celenza, Giuseppe; Tondi, Donatella
abstract

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase.

2019 - Phenylboronic Acids Probing Molecular Recognition against Class A and Class C beta-Lactamases [Articolo su rivista]
Linciano, P.; Vicario, M.; Kekez, I.; Bellio, P.; Celenza, G.; Martin-Blecua, I.; Blazquez, J.; Cendron, L.; Tondi, D.
abstract

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, Β-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine Β-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect Β-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with Β-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.

2019 - Targeting Bacterial SOS-Response To Save the Antimicrobials Arsenal [Poster]
Maso, Lorenzo; Chinellato, M.; A., Mengucci; W., Xiaobo; J., Steyaert; E., Pardon; B., Shoichet; A., Angelini; Tondi, Donatella; L., Cendron
abstract

2019 - Ten years with NDM-1 metallo β-lactamase: from structural insights to inhibitor design [Articolo su rivista]
Linciano, Pasquale; Cendron, Laura; Gianquinto, Eleonora; Spyrakis, Francesca; Tondi, Donatella
abstract

The worldwide emergence of metallo β-lactamase NDM-1 as carbapenemase able to hydrolyze near all available β-lactam antibiotics has characterized the last decade, endangering efficacious antibacterial treatments. No inhibitors for NDM-1 are available in therapy, nor promising compounds are in the pipeline for future NDM-1 inhibitors. We report the studies dedicated to the design and development of effective NDM-1 inhibitors. The discussion for each agent moves from the employed design strategy to the ability of the identified inhibitor to synergize β-lactam antibiotics. A structural analysis of NDM-1 mechanism of action based on selected X-ray complexes is also reported: the intrinsic flexibility of the binding site and the comparison between penicillins/cephalosporins and carbapenems mechanisms of hydrolysis are evaluated. Despite the valuable progresses in terms of structural and mechanistic information, the design of a potent NDM-1 inhibitor to be introduced in therapy remains challenging. Certainly, only the deep knowledge of NDM-1 architecture and of the variable mechanism of action that NDM-1 employs against different classes of substrates could orient a successful drug discovery campaign.

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 - Antibiotic Drug Discovery: New Targets and Molecular Entities. [Recensione in Rivista]
Tondi, D.
abstract

Comprising a total of eight chapters and almost 300 pages, this book is the 58th volume of the appreciated Drug Discovery Series of the Royal Society of Chemistry. The book deals with one the most compelling problems medicinal chemistry is facing right now: bacterial resistance. The spread of bacteria resistant to the available antibiotic armamentarium is a serious threat to human health. The World Health Organization (WHO) predicts that by 2050, bacterial resistance will cause 50 million deaths, thus becoming the leading cause of death worldwide. The scientific community is on alert, and solutions must be found rapidly; at present, even under the resurgence of antibiotic drug discovery, the pipeline of new antibiotics under development is slim, thus not encouraging that solutions are at hand. To fight resistant bacteria, the discovery and validation of new targets is crucial, and only innovative drugs with new modes of action can be a viable solution to curb the continued emergence and spread of antibiotic‐resistant bacteria. Internationally coordinated actions as well as consistent and continuous investments to support research in antibiotic drug discovery are necessary to increase the chances of success. In this perspective, the book discusses recent results on antibacterial drug discovery with particular attention to new targets and innovative strategies implemented to combat bacterial resistance. The book will be of interest to a large audience, not only to medicinal chemists in the field, to whom it can serve as a valuable point of reference, but to anyone wishing to follow the latest approaches in antibiotic drug design. The book is also valuable as a textbook for postgraduate students in medicinal chemistry: concepts are explained clearly and concisely throughout all chapters; sections are well organized and nicely illustrated; the quality of the diagrams and schemes, especially with regard to chemical structures, is uniform throughout the book. Most chapters have a reasonable length of around 20 to 40 pages with sufficient key references and one or more color figures, comprehensive tables, and detailed lists of discussed chemical analogues. Contents are easily accessible through the table of contents, while a subject index helps the reader to navigate the volume.

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 - In silico identification and experimental validation of hits active against KPC-2 β-lactamase [Articolo su rivista]
Klein, Raphael; Linciano, Pasquale; Celenza, Giuseppe; Bellio, Pierangelo; Papaioannou, Sofia; Blazquez, Jesus; Cendron, Laura; Brenk, Ruth; Tondi, Donatella
abstract

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four fold.

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 - New weapons against antimicrobial resistance: 
Targeting SOS response to recover b
 actericidal activity of antibiotics
 [Poster]
Maso, Lorenzo; Bellio, Pierangelo; Linciano, Pasquale; D’Acunto, Maria; Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura 

abstract

Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections.The development of novel strategies fighting bacterial infections is strongly desirable. One such approach is a means of prolonging the lifespan of current antibiotics, namely, the development of antibiotic adjuvants, i.e. compounds that do not themselves kill bacteria but instead enhance the effect of an antibiotic. Adjuvants have potential application in a number of situations, with the most obvious being the case of formerly susceptible bacteria that have acquired resistance. Additionally, the relative infancy of the adjuvant approach means that there are likely a greater number of both undiscovered targets and previously unidentified chemical scaffolds.

2018 - Phenyl boronic acids development led to validated leads active in clinical strains overexpressing KPC-2: a step against bacterial resistance. [Articolo su rivista]
Celenza, Giuseppe; Vicario, Mattia; Bellio, Pierangelo; Linciano, Pasquale; Perilli, Mariagrazia; Oliver, Antonio; Blazquez, Jesús; Cendron, Laura; Tondi, Donatella
abstract

The emergence and dissemination of multi drug resistant (MDR) pathogens resistant to near all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last resort carbapenems. In this study we investigate the molecular recognition requirements in KPC-2 active site by small phenyl boronic acid derivatives. Four new phenyl boronic acid derivatives were designed and tested vs KPC-2. For the most active, despite their simple chemical structures, nanomolar affinity was achieved. New derivatives restored susceptibility to meropenem in clinical strains overexpressing KPC-2. Moreover no cytotoxicity was detected in cell viability assays, further validating the designed leads. Two crystallographic binary complexes of best inhibitors binding KPC-2 were obtained at high resolution. Kinetic descriptions of slow binding, time dependent inhibition and interactions geometries in KPC-2 were fully investigated. This study will ultimately lead toward optimization and development of more effective KPC-2 inhibitors.

2018 - Structure-based virtual screening for the discovery of novel inhibitors of New Delhi Metallo-β-lactamase-1 [Articolo su rivista]
Spyrakis, Francesca; Celenza, Giuseppe; Marcoccia, Francesca; Santucci, Matteo; Cross, Simon; Bellio, Pierangelo; Cendron, Laura; Perilli, Mariagrazia; Tondi, Donatella
abstract

Bacterial resistance has become a worldwide concern after the emergence of metallo β-lactamases MBLs. They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. In this perspective, we performed a structure-based in silico screening of a commercially available library using FLAPdock and identified several, non β-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured in vitro revealing, for some of them, low micromolar affinity towards NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem Meropenem

2017 - A multidisciplinary approach to the design of novel inhibitors for KPC-2 [Poster]
Celenza, Giuseppe; Bellio, Pierangelo; Linciano, Pasquale; Perilli, Mariagrazia; Cendron, Laura; Tondi, Donatella
abstract

Background. The emergence and dissemination of multi drug resistant (MDR) Gram-negative pathogens resistant to all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella Pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last resort carbapenems. [1-2] Materials/methods. Four boronic acid derivatives were designed and tested in vitro vs KPC-2.[3] In biological assays their ability to synergize last generation antibiotics was evaluated. X-ray crystallography was applied to confirm binding orientation and new compounds ability to reach consensus-binding sites in carbapenemases (Figure 1). Results. For the most actives active compounds nanomolar affinity was achieved. The best inhibitor has nanomolar affinity for the enzyme, a ligand efficiency of 0.78 kcal mol–1 and a molecular weight of 158 Da validating it as lead-like molecule. In biological assays against Escherichia coli overexpressing KPC-2 new derivatives restored susceptibility to cefotaxime, aztreonam and last resort carbapenems. Two crystallographic binary complexes of the best inhibitors binding KPC-2 were obtained at high resolution. Conclusion. We investigate the molecular recognition requirements in KPC-2 active site by boronic acid derivatives. Kinetic descriptions of slow binding, time dependent inhibition and interactions geometries in KPC-2 were fully investigated. This study will guide further lead optimization and development of more effective KPC-2 inhibitors. Figure 1 References [1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print). [2] Tondi, D.; Cross, S.; Venturelli, A.; Costi, MP; Cruciani, G.; Spyrakis, F. Current Drug Targets 2016 17, no. 9 (2016) [3] Tondi,D.; Venturelli,A.; Bonnet,R.; Pozzi, C.; Shoichet, BK.; Costi, M.P. JMC. 2014. 57 (12), pp 5449–5458.

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 - 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 - LexA e la risposta SOS nei batteri: design e ottimizzazione di inibitori contrastanti lo sviluppo della resistenza batterica. [Altro]
Tondi, Donatella
abstract

Progetto FAR 2017 UNIMORE PI DOnatella Tondi Abstract La diffusione di batteri resistenti agli antimicrobici rappresenta una riconosciuta emergenza mondiale che limita sempre più il trattamento delle infezioni batteriche: l’OMS considera la resistenza batterica agli antibiotici fra i principali rischi per la salute umana. Il progetto ha come oggetto di studio il repressore trascrizionale LexA coinvolto nella risposta SOS, un importante sistema di riparazione del DNA presente nei batteri. In seguito a stress esterno (es. trattamento antibiotico) nei batteri viene attivata l’auto-proteolisi di LexA e come conseguenza la risposta SOS: viene così indotta l'espressione di oltre 60 geni coinvolti, per esempio, nella riparazione e ricombinazione del DNA. E’ stato dimostrato che il blocco della risposta SOS riduce de facto l'acquisizione e l'integrazione di nuovi geni di resistenza, limitando così le capacità dei batteri di evolvere in cellule resistenti. Il progetto, avvalendosi di un approccio di Structure-Based, propone l’identificazione di inibitori di LexA e della loro successiva ottimizzazione sintetica hit-to-lead. Le molecole candidate verranno validate in vitro vs LexA e le migliori avviate a studi ex vivo vs i batteri per valutarne la capacità di bloccare la risposta SOS. Per le molecole più promettenti saranno condotti studi di cristallizzazione a raggi-x. Presso il proponente sono stati avviati studi di validazione di LexA come proteina target: è stato sviluppato un modello cinetico di analisi, la produzione della proteina transgenica è stata messa a punto ed un saggio cellulare è già disponibile. L’obiettivo finale che il progetto si prefigge è ritardare lo sviluppo di batteri resistenti intervenendo a livello del trasferimento orizzontale e verticale dei geni di resistenza. In questo contesto il blocco dell’attività di LexA e della risposta SOS consentirebbe di riabilitare in terapia numerosi antibiotici ormai abbandonati e di prolungare la vita di quelli di nuovo sviluppo.

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 - SOS response in bacteria: inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein [Articolo su rivista]
Bellio, Pierangelo; Di Pietro, Letizia; Mancini, Alisia; Piovano, Marisa; Nicoletti, Marcello; Brisdelli, Fabrizia; Tondi, Donatella; Cendron, Laura; Franceschini, Nicola; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe
abstract

Abstract Background RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although, recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Purpose Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. Methods The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100 µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition versus ATP and versus ssDNA, was evaluated. Results Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2 µM for protolichesterinic acid to 42.6 µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. Conclusion In this paper is describe the first RecA ssDNA binding site ligand. Our study set epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach natural products in general and lichen specifically, represent a successful source of active ligands and structural diversity.

2017 - Structure based drug design: the discovery of novel inhibitors active against New Delhi Metallo-β-lactamase-1 [Poster]
Cendron, Laura; Spyrakis, Francesca; Marcoccia, Francesca; Cross, Simon; Bellio, Pierangelo; Celenza, Giuseppe; Perilli, Mariagrazia; Tondi, Donatella
abstract

Background. The emergence of bacterial strains resistant to available antibiotic armamentarium is nowadays a pressing issue in clinical therapy. Among the several mechanisms of bacterial resistance, the rapid evolution and spread of carbapenemases, β-lactamases (BLs) with versatile hydrolytic capacities able to inactivate last resort carbapenems, represents a menace in treating highly resistant infections. [1-3] Carbapenem-resistant Enterobacteriaceae (CREs), reported with increased frequency, are progressively spreading throughout the World thus leaving no effective antibiotics. [1-3] In particular CREs overexpressing Metallo beta-lactamases (MBLs; i.e. NDM-1, VIM-2, IMP-1) are high-priority target pathogens for the development of novel antibacterials. Our studies focused on NDM-1: its substrate promiscuity, its resistance to available drugs, the easiness of variants appearance and transferability make NDM-1 one of the most worrisome BLs. [2] Moreover, respect to serine β-lactamases, no inhibitors for any MBLs are currently available in therapy. Materials/methods. Looking for new potential MBLs inhibitors we performed a structure-based in silico screening of commercially available library using FLAP, and identified several non β-lactam derivatives as promising candidates active against our target New Delhi metallo-beta-lactamase-1 NDM-1. Candidates were validated in vitro and investigated for their mechanism of inhibition. Results. The binding affinities of the highest scoring hits revealed, for several of them, micromolar inhibitory activity towards NDM-1. Among molecules selected for targeting NDM-1 few demonstrated an activity comparable to that provided by known inhibitors (Ki for the best-selected inhibitor equal to 0.72 μM; ). The identified inhibitors all share common non-covalent, competitive inhibition mechanism vs NDM-1. Conclusion. For the best ones, studies for improving their affinity and to investigate their potential to synergize beta-lactam antibiotics are ongoing. 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. Figure 1 References [1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print). [2] Robert A. Bonomo. “New Delhi Metallo-β-Lactamase and Multidrug Resistance: A Global SOS?”Clin Infect Dis. (2011) 52 (4): 485-487 [3] Donatella Tondi, Alberto Venturelli, Richard Bonnet, Cecilia Pozzi, Brian K Shoichet and Maria Paola Costi. “Targeting Serine Beta lactamases with a novel broad spectrum boronic acid”. Journal Medicinal Chemistry. (2014)

2017 - The SOS response in bacteria and the LexA transcriptional repressor. Boronic acids derivatives: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials [Relazione in Atti di Convegno]
Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura; Spyrakis, Francesca; Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Vicario, Mattia; Perilli, Mariagrazia; Amicosante, Gianfranco
abstract

Multi-Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections. The World Health Organization considers bacterial resistance among the three main risks for human health, and the development of new strategies for fighting bacterial infections strongly desirable [1]. In 2014 report, WHO evokes a post-antibiotic era in which "common infections and minor injuries can kill" [2]. In bacteria, the SOS response is mainly orchestrated by two proteins: RecA and LexA [3, 4]. As a consequence of damage to the DNA, RecA promotes self-cleavage of the transcriptional repressor LexA, inducing the expression of more than 40 genes involved in DNA repair and mutagenesis [5]. The inactivation of the SOS regulators, by deletion of RecA and/or by engineering a noncleavable LexA into bacteria, reduces de facto the acquisition and integration of new resistance genes and the ability of bacteria to mutate, slowing the appearance of new drug resistance phenotypes [6-8]. The development of resistant bacteria is delayed at the level of vertical and horizontal transfer of resistance genes [9]. LexA represents therefore, an attractive target: blocking its action and reducing bacterial ability to evolve antibiotic resistance, would extend the long-term viability of both known and new antibiotics. This study is focused on the development of potential inhibitors of the SOS activation, through the inhibition of LexA self-cleavage. The autoproteolysis of LexA was experimentally induced by alkaline pH, or in presence of RecA protein, ATP and ssDNA. A first structure-based in silico screening has been performed and few boronic acid derivatives (Fig 1a) have been identified as potential LexA inhibitors. In our hand, boronic acids represented a potent tool to probe, for the first time, LexA catalytic site. We have developed a quantitative UPLC-MS method to efficiently follow the kinetics of LexA self-cleavage and the inhibitory effect on autoproteolysis of boronic acids. While the first order rate constant was calculated from the time course of autohydrolysis, to quantify the “potency” of inhibition a novel kinetic model has been developed (Fig 1b). The in vivo efficacy of boronic acids was evaluated by observing the reduction of filamentation induced by levofloxacin in E. coli (Fig 1c). The results coming from this first screening were encouraging, providing critical information on the catalytic pocket and on the binding requirements necessary for the improved design of high affinity ligands. Our preliminary data suggest that boronic acids can efficaciously inhibit the autoproteolysis of LexA.

2017 - The SOS response in bacteria and the LexA transcriptional repressor: withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials [Relazione in Atti di Convegno]
Celenza, Giuseppe; Tondi, Donatella; Cendron, Laura; Spyrakis, Francesca; Bellio, Pierangelo; Mancini, Alisia; Di Pietro, Letizia; Vicario, Mattia; Perilli, Mariagrazia; Amicosante, Gianfranco
abstract

Multi-Drug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections. The World Health Organization considers bacterial resistance among the three main risks for human health, and the development of new strategies for fighting bacterial infections strongly desirable [1]. In 2014 report, WHO evokes a post-antibiotic era in which "common infections and minor injuries can kill" [2]. In bacteria, the SOS response is mainly orchestrated by two proteins: RecA and LexA [3, 4]. As a consequence of damage to the DNA, RecA promotes self-cleavage of the transcriptional repressor LexA, inducing the expression of more than 40 genes involved in DNA repair and mutagenesis [5]. The inactivation of the SOS regulators, by deletion of RecA and/or by engineering a noncleavable LexA into bacteria, reduces de facto the acquisition and integration of new resistance genes and the ability of bacteria to mutate, slowing the appearance of new drug resistance phenotypes [6-8]. The development of resistant bacteria is delayed at the level of vertical and horizontal transfer of resistance genes [9]. LexA represents therefore, an attractive target: blocking its action and reducing bacterial ability to evolve antibiotic resistance, would extend the long-term viability of both known and new antibiotics. This study is focused on the development of potential inhibitors of the SOS activation, through the inhibition of LexA self-cleavage. The autoproteolysis of LexA was experimentally induced by alkaline pH, or in presence of RecA protein, ATP and ssDNA. A first structure-based in silico screening has been performed and few boronic acid derivatives (Fig 1A) have been identified as potential LexA inhibitors. In our hand, boronic acids represented a potent tool to probe, for the first time, LexA catalytic site. We have developed a quantitative UPLC-MS method to efficiently follow the kinetics of LexA self- cleavage and the inhibitory effect on autoproteolysis of boronic acids. While the first order rate constant was calculated from the time course of autohydrolysis, to quantify the “potency” of inhibition a novel kinetic model has been developed (Fig 1B). The in vivo efficacy of boronic acids was evaluated by observing the reduction of filamentation induced by levofloxacin in E. coli (Fig 1C). The results coming from this first screening were encouraging, providing critical information on the catalytic pocket and on the binding requirements necessary for the improved design of high affinity ligands. Our preliminary data suggest that boronic acids can efficaciously inhibit the autoproteolysis of LexA.

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 - 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 - Designing novel inhibitors for Metallo Beta-lactamases [Abstract in Atti di Convegno]
Spyrakis, Francesca; Bellio, Pierangelo; Vicario, Mattia; Cendron, Laura; Celenza, Giuseppe; Tondi, Donatella
abstract

The extensive use of eta-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 Metallo BLs (MBLs)[3,4]. Their substrate promiscuity, their resistance to available drugs, the easiness of variants appearance and transferability make MBLs 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 B beta-lactamases (MBL). 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: resistance, SBDD, in silico screening, enzyme inhibition, antimicrobial activity, x-ray crystallography

2016 - Designing novel inhibitors for Metallo beta-lactamases. [Relazione in Atti di Convegno]
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 beta-lactamases (BLs), which have emerged in both Gram-positive and Gram-negative bacteria [1,2]. Medicinal chemists have introduced beta-lactam-based molecules that inhibit or are stable to their action. These molecules are themselves beta-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 beta-lactamases inhibitors in general and especially against Metallo BLs (MBLs)[3,4]. Their substrate promiscuity, their resistance to available drugs, the easiness of variants appearance and transferability make MBLs 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 B beta-lactamases (MBL). 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 beta-lactam-based inhibitors are now under evaluation. Moreover, since our inhibitors are novel, non beta-lactam based, we expect them to do not up-regulate beta-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.

2016 - SOS response in bacteria: inhibitory activity of lichen secondary metabolites against RecA protein [Abstract in Atti di Convegno]
Di Pietro, Letizia; Bellio, Pierangelo; Mancini, Alessia; Tondi, Donatella; Spyrakis, Francesca; Cendron, Laura; Nicoletti, Mario; Piovano, Mario; Perilli, Mariagrazia; Celenza, Giuseppe
abstract

implementing multiple resistant mechanisms. Among processes that bacteria implement to contribute to the evolution
of antibiotic resistance, the SOS pathway plays a central role. The SOS response is orchestrated mainly by two enzymes: the first, RecA, is the stress sensor, which promote self-cleavage of the second protein, LexA, involved in the system as transcriptional repressor and SOS regulator: LexA cleavage results in induction of the SOS effectors. The activation of the SOS response is directly related to bacteria ability to evolve into resistant bacteria by development of intrinsic resistance and/or acquisition of antimicrobial resistance genes. RecA is a highly conserved protein that plays a critical role in homologous recombination and in LexA activation. Structurally, RecA consists of three domains with a central core surrounded by smaller regulatory domains. RecA monomers can form large nucleoprotein filaments on ssDNA. RecA binds ATP, which is required for filament formation, at the monomer-monomer interface. The nucleofilament is the prerequisite for LexA activation via allosteric modulation. Thus ATP or ssDNA binding sites are potential targets for inhibition. At this purposes several secondary metabolites form lichens belonging to depside, depsidone and acyl-itaconic acid derivatives where investigated for their ability to inhibit RecA. All compounds where characterized to be pure at least at 99% as demonstrated by TLC and H1 NMR. recA gene, previously His-tagged at N-terminus, has been cloned in an overexpression vector (pET 28b) and the protein purified by Hi-Trap. ATPase activity has been monitored by following the production of free phosphate by malachite green. The percentage of inhibition was calculated at 100 µM of each compound dissolved in 0.01% of Triton-X. From 30 tested compounds only 9 showed a percentage of inhibition above 50%. The IC50 calculated for each of them was ranging from 14 µM for protolichesterinic acid to 42 µM for sphaerophorin. We further investigated protolichesterinic acid in order to assess the mechanism of inhibition and the potential site of inhibition. First of all the ATPase activity of RecA was monitored at several concentration of ATP (from 30 to 600 µM) and fix concentration of ssDNA (polydT 36 mer). The plot of IC50 as function of [ATP]/KMATP showed a non-competitive mechanism of inhibition. However, when the same experiment has been performed at different concentration of polydT (from 9.8×10-4 to 2 µM) and 600 µM of ATP, the plot of IC50 as function of [polydT]/KMpolydT showed a possible competitive mechanism of inhibition. Since RecA is able to hydrolyse ATP only when is bound to ssDNA, its reduced ATPase activity in presence of protolichesterinic acid can be ascribed to its ability to bind the ssDNA binding site. Further investigations on the mechanism of action of protolichesterinic acid and the other most active compounds are actually carried on. Keywords: SOS response; RecA protein, lichen compounds

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

2014 - CTM-M-9 in complex with the broad spectrum inhibitor 3-(2- carboxyvinyl)benzo(b)thiophene-2-boronic acid [Altro]
Tondi, Donatella
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 - 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]
Farina, Davide; Spyrakis, Francesca; Venturelli, Alberto; Cross, Simon; 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.

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 - 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.

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.

2009 - AmpC beta-lactamase in complex with a p.carboxyphenylboronic acid [Altro]
Tondi, Donatella
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.

2008 - Constrained dansyl derivatives reveal bacterial specificity of highly conserved Thymidylate Synthases A [Articolo su rivista]
Calo', S.; Tondi, Donatella; Ferrari, Stefania; Venturelli, A.; Ghelli, S.; 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 - 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 - Structural and Thermodynamic analysis of novel inhibitors of beta-lactmases. [Relazione in Atti di Convegno]
Morandi, F.; Tondi, Donatella; Venturelli, A.; Bonnet, R.; Shoichet, B. K.; Costi, Maria Paola
abstract

.

2007 - AmpC beta-lactamase in complex with 5-diformylaminomethyl-benzo[b]thiophen-2-boronic acid [Altro]
Tondi, Donatella
abstract

Benzo[b]thiophene-2-ylboronic acid, 1, is a 27 nM inhibitor of the class C beta-lactamase AmpC and potentiates the activity of beta-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 - 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 - Optimizing Cell Permeation of an Antibiotic Resistance Inhibitor for Improved Efficacy [Articolo su rivista]
Venturelli, A.; Tondi, Donatella; Cancian, Laura; Morandi, Federica; Cannazza, Giuseppe; Segatore, B.; Prati, Fabio; Amicosante, G.; Shoichet, B. K.; 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.

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

2005 - AmpC beta-lactamase in complex with 3-(3-nitro-phenylsulfamoyl)-thiophene-2-carboxylic acid [Altro]
Tondi, Donatella
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.

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 - Improving specificity vs bacterial thymidylate synthases through N-dansyl modulation of didansyltyrosine [Articolo su rivista]
Tondi, Donatella; Venturelli, A.; Ferrari, Stefania; Ghelli, S.; 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 inhibitor design vs. beta-lactamase [Abstract in Atti di Convegno]
Shoichet, B; Tondi, Donatella; Morandi, F; Babaoglu, K.
abstract

Fragment-based screens test multiple low-molecular weight molecules for binding to a target. Fragments often bind with low affinities but typically have better ligand efficiencies (DeltaG(bind)/heavy atom count) than traditional screening hits. This efficiency, combined with accompanying atomic-resolution structures, has made fragments popular starting points for drug discovery programs. Fragment-based design adopts a constructive strategy: affinity is enhanced either by cycles of functional-group addition or by joining two independent fragments together. The final inhibitor is expected to adopt the same geometry as the original fragment hit. Here we consider whether the inverse, deconstructive logic also applies--can one always parse a higher-affinity inhibitor into fragments that recapitulate the binding geometry of the larger molecule? Cocrystal structures of fragments deconstructed from a known beta-lactamase inhibitor suggest that this is not always the case.

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 - 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.

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

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

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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 β-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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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 - 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 combinatorial optimization of novel competitive inhibitors of thymidylate synthase [Abstract in Atti di Convegno]
Sholchet, Bk; Tondi, Donatella; Slomczynska, U.
abstract

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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 - Mono- and Disubstituted-3,8-diazabicyclo[3.2.1]octane Derivatives as Analgesics Structurally Related to Epibatidine: Synthesis, Activity, and Modeling. [Articolo su rivista]
Barlocco, Daniela; Cignarella, Giorgio; Tondi, Donatella; Vianello, Paola; Villa, Stefania; Bartolini, Alessandro; Ghelardini, Carla; Galeotti, Nicoletta; ANDERSON DAVID, J. KUNTZWEILER THERESA A.; Colombo, Diego; Toma, Lucio
abstract

A series of 3,8-diazabicyclo[3.2.1]octanes substituted either at the 3 position (compounds 1) or at the 8 position (compounds 2) by a chlorinated heteroaryl ring were synthesized, as potential analogues of the potent natural analgesic epibatidine. When tested in the hot plate assay, the majority of the compounds showed significant effects, the most interesting being the 3-(6-chloro-3-pyridazinyl)-3,8-diazabicyclo[3.2.1]octane (1a). At a subcutaneous dose of 1 mg/kg, 1a induced a significant increase in the pain threshold, its action lasting for about 45 min. 1a also demonstrated good protection at a dose of 5 mg/kg in the mouse abdominal constriction test, while at 20 mg/kg it completely prevented the constrictions in the animals. Administration of naloxone (1 mg/kg i.p.) did not antagonize its antinociception while mecamylamine (2 mg/kg i.p.) did, thus suggesting the involvement of the nicotinic system in its action. Binding studies confirmed high affinity for the alpha 4 beta 2 nAChR subtype (Ki = 4.1 +/- 0.21 nM). nAChR functional activity studies on three different cell lines showed that 1a was devoid of any activity at the neuromuscular junction. Finally, due to the analogy in their pharmacological profile with that of epibatidine, compounds were compared from a structural and conformational point of view through theoretical calculations and high-field 1H NMR spectroscopy. Results indicate that all of them present one conformation similar to that of epibatidine.

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 - 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 - 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.