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Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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2023 - Serum Mass Spectrometry Proteomics and Protein Set Identification in Response to FOLFOX-4 in Drug-Resistant Ovarian Carcinoma [Articolo su rivista]
D'Arca, Domenico; Severi, Leda; Ferrari, Stefania; Dozza, Luca; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Pagani, Lisa; Tagliazucchi, Lorenzo; Villani, Marco; D'Addese, Gianluca; Piga, Isabella; Conteduca, Vincenza; Rossi, Lorena; Gurioli, Giorgia; De Giorgi, Ugo; Losi, Lorena; Costi, Maria Paola

2022 - A toolbox of biophysical and analytical assays helps to confirm the activity of novel hTS dimer disrupters (Ddis) with anticancer properties [Poster]
Tagliazucchi, Lorenzo; Venturelli, Alberto; Malpezzi, Giulia; Moschella, MARIA GAETANA; Aiello, Daniele; Lopresti, Ludovica; Pozzi, Cecilia; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2022 - Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth [Articolo su rivista]
Costantino, Luca; Ferrari, Stefania; Santucci, Matteo; MH Salo-Ahen, Outi; Carosati, Emanuele; Franchini, Silvia; Lauriola, Angela; Pozzi, Cecilia; Trande, Matteo; Gozzi, Gaia; Saxena, Puneet; Cannazza, Giuseppe; Losi, Lorena; Cardinale, Daniela; Venturelli, Alberto; Quotadamo, Antonio; Linciano, Pasquale; Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; Guerrini, Remo; Pacifico, Salvatore; Luciani, Rosaria; Genovese, Filippo; Henrich, Stefan; Alboni, Silvia; Santarem, Nuno; CORDEIRO DA SILVA, Anabela; Giovannetti, Elisa; J Peters, Godefridus; Pinton, Paolo; Rimessi, Alessandro; Cruciani, Gabriele; M Stroud, Robert; C Wade, Rebecca; Mangani, Stefano; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2022 - Development of a sensitive biochemical tool to assess the expression levels of recombinant ectopic proteins in in vitro engineered cellular systems [Abstract in Atti di Convegno]
Moschella, Mg; Marverti, G; Cassanelli, Valentina; Venuti, Federica; Tagliazucchi, L; Costi, Mp; D’Arca., D

2022 - Identification of a Quinone Derivative as a YAP/TEAD Activity Modulator from a Repurposing Library [Articolo su rivista]
Lauriola, A.; Uliassi, E.; Santucci, M.; Bolognesi, M. L.; Mor, M.; Scalvini, L.; Elisi, G. M.; Gozzi, G.; Tagliazucchi, L.; Marverti, G.; Ferrari, S.; Losi, L.; D'Arca, D.; Costi, M. P.

The transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are the major downstream effectors in the Hippo pathway and are involved in cancer progression through modulation of the activity of TEAD (transcriptional enhanced associate domain) transcription factors. To exploit the advantages of drug repurposing in the search of new drugs, we developed a similar approach for the identification of new hits interfering with TEAD target gene expression. In our study, a 27member in-house library was assembled, characterized, and screened for its cancer cell growth inhibition effect. In a secondary luciferase-based assay, only seven compounds confirmed their specific involvement in TEAD activity. IA5 bearing a p-quinoid structure reduced the cytoplasmic level of phosphorylated YAP and the YAP–TEAD complex transcriptional activity and reduced cancer cell growth. IA5 is a promising hit compound for TEAD activity modulator development.

2022 - LC-MS serum proteomics reveals a panel of proteins prognostic of positive responsiveness to bevacizumab therapy in late-stages ovarian cancer patients [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; D'Addese, Gianluca; Califano, Daniela; Arenare, Laura; Mezzanzanico, Delia; Chinello, Clizia; Magni, Fulvio; Villani, Marco; Losi, Lorena; Marverti, Gaetano; D'Arca, Domenico; Chiodini, Paolo; Pignata, Sandro; Costi, Maria Paola

2022 - Lead-optimization process and characterization of the dissociative effect applied to new dimer disrupters of human Thymidylate Synthase [Abstract in Atti di Convegno]
Aiello, Daniele; Ascione, Cristian; Tagliazucchi, Lorenzo; Malpezzi, Giulia; Cassanelli, Valentina; Ponterini, Glauco; D'Arca, Domenico; Marverti, Gaetano; Moschella, MARIA GAETANA; Falchi, Federico; Venturelli, Alberto; Costi, Maria Paola

2022 - Targeting the dimer-monomer equilibrium of thymidylate synthase, to accelerate protein degradation and cancer cell growth inhibition [Abstract in Atti di Convegno]
Costi, Maria Paola; Venturelli, A; Ponterini, G; Pozzi, C; Wade, Rebecca; Giovannetti, E; Rimessi, A; Tagliazucchi, L; Moschella, M; Marverti, G; D’Arca, D

2022 - Telomere Dysfunction Is Associated with Altered {DNA} Organization in Trichoplein/Tchp/Mitostatin ({TpMs}) Depleted Cells [Articolo su rivista]
Lauriola, Angela; Davalli, Pierpaola; Marverti, Gaetano; Caporali, Andrea; Mai, Sabine; D'Arca, Domenico

Abstract: Recently, we highlighted a novel role for the protein Trichoplein/TCHP/Mitostatin (TpMs), both as mitotic checkpoint regulator and guardian of chromosomal stability. TpMs-depleted cells show numerical and structural chromosome alterations that lead to genomic instability. This condition is a major driving force in malignant transformation as it allows for the cells acquiring new functional capabilities to proliferate and disseminate. Here, the effect of TpMs depletion was investigated in different TpMs-depleted cell lines by means of 3D imaging and 3D Structured illumination Microscopy. We show that TpMs depletion causes alterations in the 3D architecture of telomeres in colon cancer HCT116 cells. These findings are consistent with chromosome alterations that lead to genomic instability. Furthermore, TpMs depletion changes the spatial arrangement of chromosomes and other nuclear components. Modified nuclear architecture and organization potentially induce variations that precede the onset of genomic instability and are considered as markers of malignant transformation. Our present observations connect the tumor suppression ability of TpMs with its novel functions in maintaining the proper chromosomal segregation as well as the proper telomere and nuclear architecture. Further investigations will investigate the connection between alterations in telomeres and nuclear architecture with the progression of human tumors with the aim of developing personalized therapeutic interventions.

2021 - Cellular uptake and metabolic degradation of a conjugate of folic acid with an anticancer peptide targeting the human Thymidylate synthase enzyme [Relazione in Atti di Convegno]
Tagliazucchi, Lorenzo; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2021 - Designing selective Cys-ligands to unpair the binding of the Human Transcription Enhancer Associated Domain 4 (hTEAD-4) with its modulators to halt cancer cell growth [Poster]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Pozzi, Cecilia; Lopresti, Ludovica; Venturelli, Alberto; D'Arca, Domenico; Marverti, Gaetano; Cecconi, Ciro; Ponterini, Glauco; Costi, Maria Paola

The Hippo Signalling cascade is an emerging target in tumour suppression regulation, neoplastic hypertrophy, and regenerative medicine. The pathway is activated by circulating anti-proliferative signals which leads to the phosphorylation of Yes Associated Protein (hYAP1) on Ser127/381, thus 14-3-3σ mediated cytosolic retention. Genetic alterations or exogenous factor may cause YAP nuclear migration and association to TEAD1-4 (Transcription Enhancer Associated Domain), triggering up-regulation of anti-apoptotic genes [1]. hTEAD is an enhancer that activates the nuclear transcription of genes as EMT’s, EGFR and cyclins, and promotes the synthesis of survivin, tyrosine kinase HER3, and mitochondrial Bcl-xL involved in cell proliferation. TEAD binds a palmitic (palm) or myristic (myr) acids, tethered at Cys367 pocket, however its biological role is still not well known. hTEAD isoform-4 is the most represented of its family in solid tumours and its overexpression or mutation leads to cancer development and metastasis. Recent studies have considered hTEAD a promising target for anticancer drugs. Its inhibition strategy includes the disruption/prevention of YAP1:TEAD4 complex formation [2]. With the aim to develop a specific cysteine-directed inhibition strategy, we studied Cys on the protein surface and investigated their reactivity. Hence, our studies focus on characterizing the recombinant hTEAD4-ybd (aa217-434) surface though the analysis of the reactivity of its four Cys thiols (Cys310, Cys335, Cys367, Cys410), all close to YAP binding area. First, myr-Cys-367 was investigated to confirm the auto-myristoilation of the E. coli recombinant hTEAD4 through RP-chromatography on UHPLC-Orbitrap Q-Ex (ThermoFisher™) by multicharged TIC deconvolution, and the total myr-TEAD was assessed around 25%. Myristate position was confirmed by FASP protein tryptic hydrolysis and tandem-MS peptide analysis. We studied hTEAD binding of a small disulphides and thiols library with different chemical properties through the exposed cysteines residues in presence of different concentration of reducing agent [3]. Top8 DDA (HCD)-MS/MS scan on the tryptic peptides suggested the ligands’ high selectivity towards Cys335. Cys367 was never found conjugated, even in the non-Myr fraction, hinting the low accessibility to the lipid pocket. The number of surface reactive Cys was confirmed by a reverse-titration of the protein against increasing amount of thiophenol; excess of unreacted thiophenol was measured by HPLC-UV-ELSD (Agilent™ 1260), suggesting a 1:1 stoichiometry. We confirmed hTEAD-ybd ligand ratio by fluoresceine labelling with absorption and fluorescence differential spectroscopy. The ongoing work engages the screening of a larger compound library to study YAP:TEAD interaction with a ligand displacement assay of labelled TEAD to a rhodamine-tagged peptidomimetic probe to achieve structural information of the heterodimer interface and to start a hit-optimization programme. REFERENCES [1] Santucci M, Vignudelli T, et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment. J Med Chem. 2015 Jun 25;58(12):4857-73. [2] Elisi G.M, Santucci M, et al. Repurposing of Drugs Targeting YAP-TEAD Functions. Cancers 2018, 10, 329. [3] Malpezzi G MSc Degree Thesis, Solvent exposure, and reactivity of the cysteines of Transcription Enhancer Associate Domain (TEAD), a potential anticancer target, 2021. University of Pavia – University of Modena and Reggio Emilia.

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

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

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

2021 - Structural insight into YAP-TEAD4 protein-protein interactions as target for cancer treatment [Abstract in Atti di Convegno]
Lopresti, Ludovica; Pozzi, Cecilia; Tagliazucchi, L; D’Arca, D; Marverti, G; Costi, Mp; Mangani, Stefano

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

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

2020 - Depletion of Trichoplein (TpMs) Causes Chromosome Mis-Segregation, DNA Damage and Chromosome Instability in Cancer Cells [Articolo su rivista]
Lauriola, Angela; Martello, Andrea; Fantini, Sebastian; Marverti, Gaetano; Zanocco-Marani, Tommaso; Davalli, Pierpaola; Guardavaccaro, Daniele; Mai, Sabine; Caporali, Andrea; D’Arca, Domenico

Mitotic perturbations frequently lead to chromosome mis-segregation that generates genome instability, thereby triggering tumor onset and/or progression. Error-free mitosis depends on fidelity-monitoring systems that ensure the temporal and spatial coordination of chromosome segregation. Recent investigations are focused on mitotic DNA damage response (DDR) and chromosome mis-segregations with the aim of developing more efficient anti-cancer therapies. We previously demonstrated that trichoplein keratin filament binding protein (TpMs) exhibits hallmarks of a tumor suppressor gene in cancer-derived cells and human tumors. Here, we show that silencing of TpMs expression results in chromosome mis-segregation, DNA damage and chromosomal instability. TpMs interacts with Mad2, and TpMs depletion results in decreased levels of Mad2 and Cyclin B1 proteins. All the genetic alterations observed are consistent with both defective activation of the spindle assembly checkpoint and mitotic progression. Thus, low levels of TpMs found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

2020 - Structural, Hirshfeld surface and in vitro cytotoxicity evaluation of five new N-aryl-N’-alkoxycarbonyl thiocarbamide derivatives. [Articolo su rivista]
Pandey, Sunil K.; Pratap, Seema; Rai, Sunil K.; Marverti, Gaetano

Five new compounds, N-(2, 4-dichlorophenyl)-N’-(methoxycarbonyl) thiocarbamide (1), N-(2, 4- dichlorophenyl)-N’-(ethoxycarbonyl) thiocarbamide (2), N-(2, 4-dichlorophenyl)-N’-(2, 2, 2-trichloroethoxycarbonyl) thiocarbamide (3), N-(2,4-dichlrophenyl)-N’-(pentoxycarbonyl) thiocarbamide (4) and N-(4-nitrophenyl)-N’-(pentoxycarbonyl) thiocarbamide (5), have been synthesized by the reaction of various alkoxy chloroformates with 2, 4-dichloroaniline/4-nitroaniline.The molecular structures of the compounds were elucidated by using spectroscopic methods (FT-IR, 1H and 13C NMR) and single-crystal X-ray structure analysis of compounds 2 and 5. Antiperiplanar orientation of C¼O and C¼S group across C–N bonds of thiocarbamide core may be due to the presence of intramolecular (N–HO–C) hydrogen bond in the crystal structure of both the compounds. The presence of intermolecular interactions (C–HS, C–HO and N–HS) in the molecular structure of the compounds has been studied in detail using Hirshfeld surfaces and their associated twodimensional fingerprint plots. In vitro cytotoxicity screening of the synthesized compounds evaluated on a panel of seven human cancer cell lines (cervical carcinoma (2008, C13), colorectal (HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and IGROV-1)) demonstrated significant inhibitory properties.

2020 - Synthesis, characterisation, Hirshfeld surface and in vitro cytotoxicity evaluation of new N-aryl-N0-Alkoxycarbonyl thiocarbamide derivatives. [Articolo su rivista]
Pandey, Sunil K.; Pratap, Seema; Rai, Sunil K.; Marverti, Gaetano; Kaur, Manpreet; P. Jasinski., Jerry

Four new compounds N-(4-nitrophenyl)-N’-(isobutoxycarbonyl) thiocarbamide (1), N-(2, 4-nitrophenyl)- N’-(isobutoxycarbonyl) thiocarbamide (2), N-(4-nitrophenyl)-N’-(ethoxycarbonyl) thiocarbamide (3) and N-(2-Chloro- 4-nitrophenyl)-N’-(ethoxycarbonyl) thiocarbamide (4) were prepared and their structures confirmed by using various spectroscopic (FT-IR, UVeVisible, 1H and 13C NMR) and single crystal X-ray studies of 1 and 3. The presence of intramolecular (NeH/O]C) hydrogen bond in the crystal structure of both the compounds causes planarity of carbonyl thiocarbamide unit and trans orientation of C]O and C]S group. The intermolecular contacts (CeH/S, CeH/O and NeH/S) present in crystal structures have been examined by Hirshfeld surface analysis and their associated 2D fingerprint plots. All the compounds were assessed for their in vitro cytotoxic properties against a panel of seven human cancer cells such as cervical carcinoma (2008, C13*), colorectal (HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and IGROV-1). Among them, compounds 2 and 4 exhibited better activity than 1 and 3 against all the cell lines tested.

2019 - Copper (I) complexes based on novel N, N'-disubstituted thiocarbamides: Synthesis, spectroscopic, in vitro cytotoxicity, DNA damage and G0/G1 cell cycle arrest studies [Articolo su rivista]
Pandey, S. K.; Pratap, S.; Pokharia, S.; Mishra, H.; Marverti, G.; Kaur, M.; Jasinski, J. P.

Four trigonal planar copper (I) complexes with novel N, N′-disubstituted isobutoxycarbonyl thiocarbamide ligands were synthesized and characterized by elemental analysis, spectroscopic (FT–IR, 1H and 13C NMR, UV–Visible), TG analysis and single crystal X-ray studies of ligands 1 and 2. The synthesized copper (I) complexes (1a–4a) bear the general formula [Cu(ROCONHCSNHR1)2Cl] where R=eCH2CH(CH3)2 and R1=2, 4- dichlorophenyl (1), 2-chloro 4-nitrophenyl (2), 2-methoxyphenyl (3), 4-chloro-2-nitrophenyl (4). All the complexes are mononuclear coordinating through thione sulfur only. Coordination through carbonyl oxygen would have not been possible owing to the presence of strong intramolecular hydrogen bonding (NeH⋯O]C) in the ligands. The proposed trigonal planar geometry of complexes has been validated by density functional theory (DFT) study of complex 1a. Computational details of theoretical calculations (DFT) of complex have been discussed. Cyclic voltammogram of complexes 1a–4a displayed quasireversible redox behaviour corresponding to Cu(I)/Cu(II) couple. In vitro cytotoxicity results of ligands and complexes against five human cancer cell lines indicated that all the complexes displayed stronger inhibitory properties than the ligands. The most effective were complexes 3a, 4a and 5a. All the complexes exhibit IC50 values even lower than cisplatin against C13* cell line (cisplatin resistant). The comet assay test of all the complexes against 2008, C13* and IGROV-1 cell lines indicated significant damage to the DNA structure. All the complexes induce apoptosis in 2008, C13* and IGROV-1 cells by blocking cell cycle progression of these cells in G0/G1 phase.

2019 - Copper (I) complexes of N-(2/4 methoxy/2-chloro-4-nitro) phenyl-N’ (methoxycarbonyl) thiocarbamides as potential anticancer agents: Synthesis, crystal structure, in vitro cytotoxicity and DNA damage studies. [Articolo su rivista]
Pandey, Sunil K.; Singh, Durga P.; Pratap, Seema; Marverti, Gaetano; J. Butcher., R.

Synthesis and structural assignment of four trigonal planar copper(I) complexes (1a–4a) having the general formula [Cu(CH3OCONHCSNHR)2Cl] where R = 2-methoxyphenyl (1), 4-methoxyphenyl (2), 2-chloro 4-nitrophenyl (3) and 2-methoxy 4-nitrophenyl (4) have been described. The characterization were done by elemental, spectroscopic (FT-IR, 1H, 13C NMR, UV–Visible), TG analysis and single crystal X-ray studies of ligands 1, 3 and complex 2a. In the complex 2a the methoxycarbonyl groups adopt cis conformation with respect to chlorine atom and are nearly coplanar with the central plane in a trigonal planar geometry. Cyclic voltammogram of complexes 1a–4a displayed quasi-irreversible redox behaviour corresponding to Cu(I)/Cu(II) couple. In vitro cytotoxicity of the ligands and their copper(I) complexes screened against five human cancer cell lines revealed that complexes were two to three times more potent than the ligands against all the cell lines. DNA damage study of complexes against 2008, C13* (cervical cancer) and IGROV-1 (ovarian cancer) cell lines indicated that cytotoxicity exerted by them is mainly through perturbation of DNA structure.

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

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

2019 - Experimental and theoretical exploration of molecular structure and anticancer properties of two N, N′–disubstituted thiocarbamide derivatives [Articolo su rivista]
Pandey, Sunil K.; Pratap, Seema; Tiwari, Manish K.; Marverti, Gaetano; Jasinski, Jerry P.

Two new compounds N-(2-chloro-4-nitrophenyl)-N’-(phenoxycarbonyl) thiocarbamide (1) and N-(2-chloro-4-nitrophenyl)-N’-(4-nitrobenzoyl) thiocarbamide (2), have been derived by the reaction of phenoxycarbonyl isothiocyanate/4-nitrobenzoyl isothiocyanate with 2-chloro-4-nitroaniline. The structures of these compounds were determined by spectroscopic (FT-IR, 1H and 13C NMR, UV–Visible) and single crystal X-ray studies. Both the crystal structures are symmetrical and planar with anti-periplanar orientation of C[dbnd]O and C[dbnd]S group. The molecular structure and vibrational properties of the compounds studied at B3LYP/6-311G ++ (d, p) level of density functional theory further concrete the experimental results. These compounds were screened for their in vitro cytotoxicity activity against seven human cancer cell lines; cervical (2008 and C13*), colorectal (HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and IGROV-1). Compound 2 exhibited significant activity against all the cell lines whereas compound 1 demonstrated appreciable activity only against ovarian carcinoma cell lines.

2019 - Exploring the biological activity of a library of 1,2,5-Oxadiazole derivatives endowed with antiproliferative activity [Articolo su rivista]
Gelain, A; Mori, M; Meneghetti, F; Porta, F; Basile, L; Marverti, G; Asai, A; Hyeraci, M; García-Argáez, An; Via, Ld; Guccione, S; Villa, S

The identification of a series of oxadiazole-based compounds, as promising antiproliferative agents, has been previously reported. The aim of this study was to explore the SAR of newly-synthesized oxadiazole derivatives and identify their molecular targets. Materials and Methods: A small library of 1,2,5-oxadiazole derivatives was synthetized and their antiproliferative activity was tested by the MTT assay. Their interaction with topoisomerase I was evaluated and a molecular docking study was performed. Results: Several candidates showed cytotoxicity towards two human tumor cell lines, HCT-116 (colorectal carcinoma) and HeLa (cervix adenocarcinoma). Some derivatives exhibited inhibitory effects on the catalytic activity of topoisomerase I and this effect was supported by docking studies. Conclusion: The enzyme inhibition results, although not directly related to cytotoxicity, suggest that a properly modified 1,2,5 oxadiazole scaffold could be considered for the development of new antitopoisomerase agents.

2019 - Synthesis, characterization, Hirshfeld surface, cytotoxicity, DNA damage and cell cycle arrest studies of N, N-diphenyl-N'-(biphenyl-4- carbonyl/4-chlorobenzoyl) thiocarbamides [Articolo su rivista]
K Pandey, Sunil; K Rai, Sunil; Marverti, Gaetano; Kaur, Manpreet; P Jasinski, Jerry; Pratap, Seema

The condensation reaction of biphenyl-4-carbonyl isothiocyanate/4-chlorobenzoyl isothiocyanate with diphenylamine yielded two new compounds; N-diphenyl-N'-(biphenyl-4-carbonyl) thiocarbamide (1) and N, N-diphenyl-N'-(4-chlorobenzoyl) thiocarbamide (2). Structure of the compounds were determined by analytical, spectroscopic (UVeVisible, FTIR, 1H, & 13C NMR), powder and single-crystal X-ray diffraction methods. Hirshfeld surface analysis and their associated two dimensional fingerprint plots of compounds were used as theoretical approach to assess driving force for crystal structure formation via the intermolecular interactions in their crystal lattices. The compounds were screened for their in vitro cytotoxicity activity against a panel of five human cancer cell lines namely; cervical (2008 and C13*) and ovarian carcinoma (A2780, A2780/CP and IGROV-1). Both the compounds exhibited promising activity against cervical and IGROV-1 cancer cells whereas for the other two cell lines appreciable activities were observed. The cell cycle arrest at G0/G1 phase is supported by the DNA damage and apoptosis studies of the compounds against 2008, C13* and IGROV-1 cell lines

2019 - Synthesis, spectroscopic, crystal structure and in vitro cytotoxicity studies of N-thiophenoyl-N'-substituted phenyl thiocarbamide derivatives [Articolo su rivista]
Pandey, Sunil K.; Pratap, Seema; Marverti, Gaetano; Kaur, Manpreet; Jasinski, Jerry P.

A series of eight biologically active N, N0-disubstituted thiocarbamide compounds (1e8) have been prepared from thiophene-2-carbonyl isothiocyanate and various substituted aromatic primary amines (2,4-dichlorophenyl aniline, 4-chloro-3-nitrophenyl aniline, 4-methoxycarbonylphenyl aniline, 3- methoxycarbonylphenyl aniline, 2-methoxycarbonylphenyl aniline, 4-methoxyphenyl aniline, 2- methoxyphenyl aniline and 2-nitrophenyl aniline). Their structures were confirmed by elemental analyses, various spectroscopic techniques ((FTeIR, 1H and 13C NMR) and single crystal X-ray analysis of compound (1). In the molecular structure of compound (1) twisted confirmation of the carbonyl and thiocarbonyl group across CeN bond of thiocarbamide moiety and an offset face-to-face pep stacking between two thiophene and two benzene ring of two molecules is observed. In vitro cytotoxicity assay of all the above compounds and five more (9e13) were carried out using seven human cancer cell lines; cervical (2008 and C13*), colorectal (HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and IGROV-1). The results revealed that compounds 1, 11, 12 and 13 displayed promising inhibitory activity against all the cell lines tested.

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

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

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

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

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

2018 - Monodentate Coordination of N, N′-Disubstituted Thiocarbamide Ligands: Syntheses, Structural Analyses, In Vitro Cytotoxicity and DNA Damage Studies of Cu(I) Complexes [Articolo su rivista]
Pandey, Sunil K.; Singh, Durga P.; Marverti, Gaetano; Butcher, R. J.; Pratap, Seema

Structural analysis of three novel substituted thiocarbamide ligands N-(naphthyl)-N′-(isobutoxycarbonyl) thiocarbamide (H2L1), N-(4-methoxyphenyl)-N′-(isobutoxycarbonyl) thiocarbamide (H2L2) & N-(2-methoxy-4-nitrophenyl)-N′-(isobutoxycarbonyl) thiocarbamide (H2L3) and their copper(I) complexes [(H2L1)2CuCl] (1), [(H2L2)2CuCl] (2) and [(H2L3)2CuCl] (3) was performed using various spectroscopic techniques (FT−IR, 1H and 13C NMR, UV-Visible),TG analysis and single crystal X-ray studies of (H2L1) and [(H2L1)2CuCl] (1). The copper(I) complexes possess trigonal planar geometry coordinating through two thione sulfur atoms from two ligand molecules and one chloride ion. Two intramolecular hydrogen bonding interactions present between (−N1H) and carbonyl oxygen (−N2H) and coordinated chlorine stabilize the trigonal planar structure of the complexes. Cyclic voltammogram of complexes 1–3 displayed quasireversible redox behaviour corresponding to CuI/CuII couple. Determination of in vitro cytotoxicity of ligands and their complexes using five human carcinoma cell lines 2008, C13* (cervical carcinoma), A2780, A2780/CP and IGROV-1 (ovarian carcinoma) revealed that copper(I) complexes were more potent inhibitors than the ligands against all the cell lines. The most effective were complexes 2 and 3. The comet assay test of complexes 2 and 3 against 2008, C13* and IGROV-1 cell lines indicated significant damage to the DNA structure.

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

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

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

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

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

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

2018 - Synthesis, molecular structure exploration and in vitro cytotoxicity screening of five novel N, N′- disubstituted thiocarbamide derivatives [Articolo su rivista]
Pandey, Sunil K.; Pratap, Seema; Gozzi, Gaia; Marverti, Gaetano; Butcher, R. J.

The synthesis of five N,N″-substituted thiocarbamides, namely N-(naphthyl)-N″-(pentoxycarbonyl) thiocarbamide (H2L1), N-(2-Chloro-4-nitrophenyl)-N″-(pentoxycarbonyl) thiocarbamide(H2L2), N-(2-methoxy-4-nitrophenyl)-N″-(pentoxycarbonyl) thiocarbamide (H2L3), N-(3-nitrophenyl)-N″-(pentoxycarbonyl) thiocarbamide (H2L4) and N-(naphthyl)-N″-(2, 2, 2-trichloroethoxycarbonyl) thiocarbamide (H2L5) was performed by the reaction of pentoxycarbonyl chloroformate with naphthyl amine, 2-chloro-4-nitroaniline, 2-methoxy-4-nitroaniline, 3-nitroaniline, respectively, for the first four and by the reaction of 2, 2, 2-trichloroethoxycarbonyl chloroformate with naphthyl amine for the last compound. These compounds were fully characterized by using various spectroscopic (FT-IR,1H and13C NMR) and single crystal X-ray studies of H2L1 and H2L5. In the crystal structure of both the compounds the (C˭S) and (C˭O) groups are trans to each other across the C−N bond. The crystal packing of H2L1 shows that the molecules form centrosymmetric dimers connected by N2−H····S hydrogen bonds. In H2L5 an offset face-to-face π–π stacking is observed between two naphthalene rings of two molecules. In vitro cytotoxicity of synthesized compounds was evaluated using five human carcinoma cell lines 2008, C13* (cervical carcinoma), A2780, A2780/CP and IGROV-1 (ovarian carcinoma). The IC50values of compounds H2L2 ─ H2L4 demonstrated them to be very promising anticancer agents.

2018 - Targeting Oxidatively Induced DNA Damage Response in Cancer: Opportunities for Novel Cancer Therapies [Articolo su rivista]
Davalli, Pierpaola; Marverti, Gaetano; Lauriola, Angela; D’Arca, Domenico

Cancer is a death cause in economically developed countries that results growing also in developing countries. Improved outcome through targeted interventions faces the scarce selectivity of the therapies and the development of resistance to them that compromise the therapeutic effects. Genomic instability is a typical cancer hallmark due to DNA damage by genetic mutations, reactive oxygen and nitrogen species, ionizing radiation, and chemotherapeutic agents. DNA lesions can induce and/or support various diseases, including cancer. The DNA damage response (DDR) is a crucial signaling-transduction network that promotes cell cycle arrest or cell death to repair DNA lesions. DDR dysregulation favors tumor growth as downregulated or defective DDR generates genomic instability, while upregulated DDR may confer treatment resistance. Redox homeostasis deeply and capillary affects DDR as ROS activate/inhibit proteins and enzymes integral to DDR both in healthy and cancer cells, although by different routes. DDR regulation through modulating ROS homeostasis is under investigation as anticancer opportunity, also in combination with other treatments since ROS affect DDR differently in the patients during cancer development and treatment. Here, we highlight ROS-sensitive proteins whose regulation in oxidatively induced DDR might allow for selective strategies against cancer that are better tailored to the patients.

2017 - Heat shock protein 90 and serine/threonine kinase B-Raf inhibitors have overlapping chemical space [Articolo su rivista]
Anighoro, A.; Pinzi, Luca; Marverti, Gaetano; Bajorath, J; Rastelli, Giulio

Heat shock protein 90 (Hsp90) and B-Raf are validated targets for anticancer drug discovery. Although there is strong evidence that concomitant inhibition of Hsp90 and B-Raf may provide significant therapeutic benefits, molecules endowed with dual activity against the two targets have not been reported. For the first time, we show that Hsp90 and B-Raf inhibitors have overlapping chemical space and we disclose the first-in-class dual inhibitors. The compounds were identified through a computational strategy especially devised for detecting ligands with dual-target activity. Although the two targets had only remote binding site similarity, we were able to identify dual inhibitors with well-balanced in vitro potencies and relatively low molecular weight. Remarkably, they also inhibited the V600E mutant form of B-Raf with similar potency. This study provides the first direct proof that designing dual ligands of Hsp90 and a kinase is possible, thus opening the way to new interesting possibilities in drug discovery.

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

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

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

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

2015 - 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

Composti destabilizzanti l’omodimero timidilato sintasi

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

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

2015 - N-(naphthyl)-N′-(methoxy carbonyl)thiocarbamide and its Cu(I) complex: synthesis, spectroscopic, X-ray, DFT andin vitrocytotoxicity study [Articolo su rivista]
Singh, Durga P.; Pratap, Seema; Pandey, Sunil K.; Butcher, Ray J.; Marverti, Gaetano

The structural characterization of two new compounds N-naphthyl-N′-methoxycarbonyl thiocarbamide (NMCT) (1) and its Cu(I) complex, bis(N-naphthyl-N′-methoxycarbonyl thiocarbamide) copper(I) chloride [(NMCT)2CuCl] (1a) have been done by spectroscopic techniques (FT-IR, 1H NMR, 13C NMR and electronic spectroscopy) and X-ray crystallography. To get a deeper insight of vibrational frequencies and electronic transitions, DFT and TD-DFT studies have also been performed. X-ray study revealed trigonal planar geometry around copper(I). The ligand coordinates through thione sulfur only. The cytotoxicity of 1 and 1a has been assayed in five human carcinoma cell lines, 2008, C13* (cervical carcinoma), A2780, A2780/CP and IGROV-1 (ovarian carcinoma). Both the compounds exhibited cytotoxicity. The inhibitory activity of copper complex was better than ligand against all the cell lines.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2013 - Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells [Articolo su rivista]
Belluti, Silvia; Basile, Valentina; Benatti, Paolo; Ferrari, Erika; Marverti, Gaetano; Imbriano, Carol

Topoisomerase-IIa (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy.

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

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

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

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

Costi, Maria Paola; Marverti, Gaetano; Pirondi, Silvia; Martello, Andrea; D'Arca, Domenico; Pela', Michela; Guerrini, R.; Marraccini, Chiara

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

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

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

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

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

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

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

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

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

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

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

2012 - Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression [Articolo su rivista]
Ligabue, Alessio; Marverti, Gaetano; Liebl, Ursula; Myllykallio, Hannu

5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*.

2011 - Characterization of the cell growth inhibitory effects of a novel DNA-intercalating bipyridyl-thiourea-Pt(II) complex in cisplatin-sensitive and-resistant human ovarian cancer cells [Articolo su rivista]
Marverti, Gaetano; Ligabue, Alessio; Montanari, Monica; Guerrieri, Davide; M., Cusumano; M. L., Di Pietro; L., Troiano; DI VONO, Elena; S., Iotti; G., Farruggia; F., Wolf; Monti, Maria Giuseppina; Frassineti, Chiara

The cellular effects of a novel DNA-intercalatingagent, the bipyridyl complex of platinum(II) with diphenylthiourea, [Pt(bipy)(Ph2-tu)2]Cl2, has been analyzed in thecisplatin (cDDP)—sensitive human ovarian carcinoma cellline, 2008, and its—resistant variant, C13* cells, in which thehighest accumulation and cytotoxicity was found among sixrelated bipyridyl thiourea complexes. We also show here thatthis complex causes reactive oxygen species to form andinhibits topoisomerase II activity to a greater extent in thesensitive than in the resistant line. The impairment of thisenzyme led to DNA damage, as shown by the comet assay.As a consequence, cell cycle distribution has also beengreatly perturbed in both lines. Morphological analysisrevealed deep cellular derangement with the presence of cellular masses, together with increased membrane permeability and depolarization of the mitochondrial membrane. Some of these effects, sometimes differentially evident between the two cell lines, might also be related to the decrease of total cell magnesium content caused by this thiourea complex both in sensitive and resistant cells, though the basal content of this ion was higher in the cDDP-resistant line. Altogether these results suggest that this compound exerts its cytotoxicity by mechanisms partly mediated by the resistance phenotype. In particular, cDDP-sensitive cells were affected mostly by impairing topoisomerase II activity and by increasing membrane permeability and the formation of reactive oxygen species; conversely, mitochondrial impairment appeared to play the most important role in the action of complex F in resistant cells.

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.

2011 - Newly synthesized curcumin derivatives: crosstalk between chemico-physical properties and biological activity. [Articolo su rivista]
Ferrari, Erika; Francesca, Pignedoli; Imbriano, Carol; Marverti, Gaetano; Valentina, Basile; Ettore, Venturi; Saladini, Monica

New curcumin analogues (ester and acid series) were synthesized with the aim to improve the chemical stability in physiological conditions and potential anticancer activity. Cytotoxicity against different tumorigenic cell lines (human ovarian carcinoma cells – 2008, A2780, C13* and A2780/CP - and human colon carcinoma cells - HCT116 and LoVo) was tested to evaluate cellular specificity and activity.Physico/chemical properties such as acidity, lipophilicity, kinetic stability and free radical scavenging activity were investigated to shed light on the structure-activity relationship and provide new attractive candidates for drug development. Most of ester derivatives show IC50 values lower than curcumin and exhibit selectivity against colon carcinoma cells. Especially they are extremely active after 24h exposure showing enhanced inhibitory effect on cell viability.The best performances of ester curcuminoids could be ascribed to their high lipophilicity that favors a greater and faster cellular uptake overcoming their apparently higher instability in physiological condition.

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

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

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

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

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

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

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

2009 - Development of new metal-chelating multi target drus derived from Curcumin [Relazione in Atti di Convegno]
Ferrari, Erika; Pignedoli, Francesca; Lazzari, Sandra; Imbriano, Carol; Marverti, Gaetano; Saladini, Monica

With the aim to develop new anti-cancer approaches, which encompass therapies based on drug combinations, we are searching for innovative anti-tumor multi-targets treatments.1 In this research Curcumin represents our referring and starting point for the design of new derivatives. Curcumin, a natural occurring molecule, was shown to inhibit growth of several types of malignant cells and its biological activity was also related to its iron chelating ability.2 Recently curcumin proceeded onto clinical trials however its use is limited by a poor bioavailability. In order to improve curcumin water solubility and drug-delivery we have synthesized new derivatives which conjugate anti-proliferative effects with metal chelating capacity. They are able to reduce free iron level and to potentially deliver a chemotherapic metal ion such as Ga(III).

2009 - Synthesis, cytotoxic and combined cDDP activity of new stable curcumin derivatives [Articolo su rivista]
Ferrari, Erika; Lazzari, Sandra; Marverti, Gaetano; Pignedoli, Francesca; Ferdinando, Spagnolo; Saladini, Monica

New curcumin derivatives are synthesized in order to improve chemical properties of curcumin. The aromatic ring glycosylation of curcumin provides more water-soluble compounds with a greater kinetic stability which is a fundamental feature for drug bioavailability. The glycosylation reaction is quite simple, low cost, with high yield and minimum waste. NMR data show that the ability of curcumin to coordinate metal ion, in particular Ga(III), is maintained in the synthesized products. Although the binding of glucose to curcumin reduces the cytotoxicity of the derivatives towards cisplatin (cDDP)-sensitive and -resistant human ovarian carcinoma cell lines, the compounds display a good selectivity since they are much less toxic against non-tumourigenic Vero cells. The combination of cDDP with the most active glycosyl-curcuminoid drug against both cDDP-sensitive and -resistant as well as against Vero cell lines is tested. The results show an improvement of cDDP efficacy with higher selectivity towards cancer cells than non-cancer cells. These studies indicate the need for developing new valid components of drug treatment protocols to cDDP-resistant cells as well.

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

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

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

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

2008 - Studies on the anti-proliferative effects of novel DNA-intercalating bipyridyl-thiourea-Pt(II) complexes against cisplatin-sensitive and -resistant human ovarian cancer cells. [Articolo su rivista]
Marverti, Gaetano; Matteo, Cusumano; Ligabue, Alessio; Maria Letizia Di, Pietro; Pasquale Antonio, Vainiglia; Ferrari, Angela; Bergomi, Margherita; Maria Stella, Moruzzi; Frassineti, Chiara

Six bipyridyl complexes of platinum(II) with thiourea, with different substituents on thiourea moiety [Pt(bipy)(R,R0NCSNR00,R000)2]Cl2(bipy = 2,20-bipyridine: R = R0 =R00 =R000 = H; R = Me, R0 =R00 =R000 =H;R=n-Bu, R0 =R00 =R000 = H; R = Et, R0 =H,R00 = Et,R000 =H; R=p-tolyl, R0 =R00 =R000 = H; R = phenyl, R0 =H, R00 = phenyl, R000 = H), rationally designed to intercalate into DNA,have been tested against a cisplatin (cDDP)-sensitive human ovarian carcinoma cell line (2008) and its -resistant variant (C13*). We showhere that the anti-proliferative efficacy of these drugs was dependent on molecular structure, since it increased with ancillary ligand bulkinessand hydrophobicity of substituents on thiourea moiety. In particular, the presence of two phenyl groups on thiourea moiety confersan outstanding cytotoxicity. The increasing cell growth inhibition along the series of complexes partially paralleled with drug accumulation,particularly in resistant cells, but not with drug intercalation into DNA since all compounds exerted comparable ethidium bromidedisplacement ability. The cDDP-resistant phenotype seems, at least in part, to be involved in the action of these compounds, since the levelof cross-resistance established for most complexes appeared to be in agreement with the observed impairment of drug accumulation in theresistant subline. These findings indicate that resistance to alkylating agents such as cDDP confers low level of cross-resistance to this classof DNA intercalators, which, however, depending on substituents on thiourea moiety may present remarkable cell growth inhibition evenof resistant cells.

2008 - Synthesis, chemical and biological studies on new Fe3þ-glycosilated b-diketo complexes for the treatment of iron deficiency [Articolo su rivista]
Beatrice, Arezzini; Marco, Ferrali; Ferrari, Erika; Frassineti, Chiara; Lazzari, Sandra; Marverti, Gaetano; Ferdinando, Spagnolo; Saladini, Monica

A simple synthetic pathway to obtain glycosilated b-diketo derivatives is proposed. These compounds show a good iron(III) affinity thereforewe may suggest the use of their Fe3þ-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH3) are characterized by means of spectroscopic (UV, 1H and 13C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t1/2 > 100 h) and show a low cytotoxicity also in high concentrations (IC50 > 400 mM). They are able to bind Fe3þ ion in acid condition (pHw2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (log P > 0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca2þ, and in particular 6-GlcOCH3 is able to inhibit lipid peroxidation.

2007 - 1H, 13C, 195Pt NMR study on platinum(II) interaction with sulphur containing Amadori compounds [Articolo su rivista]
Ferrari, Erika; Grandi, Romano; Lazzari, Sandra; Marverti, Gaetano; Rossi, Cecilia; Saladini, Monica

AbstractThe NMR study on the interaction of Pt(II) with Amadori compounds is performed. The Amadori compounds are derived from the reaction of β-d-glucose with l-cystine leading to N,N′-di-(1-deoxy-β-fructos-1-yl)-l-cystine [FruCyscys], and with l-methionine leading to N-(1-deoxy-β-fructos-1-yl)-l-methionine [FruMet].The great instability of 2-(1,2,3,4,5-pentahydroxypentyl)-1,3-thiazolidine-4-carboxylic acid [GlcCys], formed by the condensation reaction of β-d-glucose and l-cysteine, prevents the formation of its Pt(II) complexes. Differently, FruMet well reacts with K2PtCl4 in 1:1 M/L molar ratio leading to the formation of [Pt(FruMet-N,S)Cl2], in which the Amadori compound coordinates the metal ion through nitrogen and sulphur atoms. FruMet originates also a solid trans complex [Pt(FruMet-N,S)2]Cl2. Its NMR solution study allowed to identify two isomers with respect to nitrogen and sulphur atoms: R,R and S,S.In [Pt2FruCyscysCl4] species, FruCyscys molecule links two Pt(II) ions giving rise to two pentaatomic N,S-chelate rings.

2006 - Glucosyl curcuminoid derivatives: Fe(III) and Ga(III) chelating agents with anti-cancer properties [Relazione in Atti di Convegno]
Ferrari, Erika; Frassineti, Chiara; Lazzari, Sandra; Marverti, Gaetano; Grandi, Romano; Saladini, Monica


2006 - Glucosyl curcuminoid derivatives: Fe(III) and Ga(III) chelating agents with anti-cancer properties [Relazione in Atti di Convegno]
Ferrari, Erika; Frassineti, Chiara; Lazzari, Sandra; Marverti, Gaetano; Grandi, Romano; Saladini, Monica


2005 - Spermidine/spermine N1-acetyltransferase transient over-expression restores sensitivity of resistant human ovarian cancer cells to N1,N12-bis(ethyl)spermine and to cisplatin [Articolo su rivista]
Marverti, Gaetano; Monti, Maria Giuseppina; PEGG A., E; MCCLOSKEY D., E; Bettuzzi, S; Ligabue, Alessio; Caporali, A; D'Arca, Domenico; Moruzzi, Maria Stella

The limited induction of spermidine/spermine N1-acetyltransferase (SSAT) activity has been implicated as an important determinant of the reduced response, to the spermine analogue N1,N12-bis(ethyl)spermine (BESpm) by the cisplatin (cDDP)-resistant human ovarian carcinoma cell line (C13*). We checked whether under conditions of SSAT over-expression, enzyme induction and cell sensitivity to both BESpm and cDDP were restored to levels comparable with those of more responsive cDDP-sensitive 2008 cells. We transiently transfected the SSAT repressed C13* cells with two expression vectors driving human SSAT over-expression by diverse promoters, and then we analysed their responses in the absence and in the presence of BESpm. SSAT activity was promptly but briefly expressed by transfection with both pOP/SSAT and pCMV-SSAT plasmids. However, only in the presence of BESpm, did SSAT activity reach the highest levels of induction for longer times, with different time-courses for the two vectors, which paralleled the effect on cell growth. Under these conditions, growth sensitivity to BESpm of the less-responsive C13* cells was 25% reverted to cell growth inhibition displayed by 2008 cells. More interestingly, the sensitivity to cDDP cytotoxicity also increased in parallel to SSAT over-expression. BESpm induction of pCMV-SSAT-transfected cells caused a further 20-30% reduction of cell survival induced by cDDP, almost recovering the sensitivity of 2008 cells. The enhanced effectiveness of cDDP was also confirmed by the comet assay, showing an increase in the number and length of tails of damaged DNA. These findings confirm that SSAT over-expression inhibits cell growth and enhances growth sensitivity to BESpm in C13* cells, showing for the first time that restoring high inducibility of SSAT activity subverts the reduced sensitivity to cDDP of SSAT-deficient cells, making them almost indistinguishable from the responsive parental 2008 cells.

2004 - Cisplatin-resistance modulates the effect of protein synthesis inhibitors on spermidine/spermine N-1-acetyltransferase expression [Articolo su rivista]
Marverti, Gaetano; Monti, Maria Giuseppina; Bettuzzi, Saverio; A., Caporali; Astancolle, Serenella; Moruzzi, Maria Stella

Cisplatin (DDP)-resistance confers a deficient expression of spermidine/spermine N-1-acetyltransferase (SSAT) gene in response to the spermine analog N-1,N-12-bis(ethyl)spermine (BESpm) in the DDP-resistant human ovarian carcinoma cell line (C13*), compared with their parental DDP-sensitive 2008 cells. This SSAT gene deficiency is correlated with a reduced growth sensitivity to spermine analogs. This study was performed to determine whether SSAT gene expression of resistant cells was kept suppressed by labile repressor proteins developed during resistance selection. We show here that inhibitory concentrations of cycloheximide (CHX) and anisomycin (ANISO) differentially affect BESpm-induced SSAT activity in 2008 and in C13* cells in a concentration-dependent manner and allow resistant cells to reach activation levels comparable to those of the sensitive cells. Northern blot analysis revealed that both CHX and ANISO in combination with BESpm caused a synergistic BESpm-mediated accumulation of SSAT mRNA in C13* cells, with respect to each drug alone, while in 2008 cells only a slight increase was observed. The more pronounced effect of inhibitors on the SSAT activity induced by BESpm in the resistant cells was also the result of a more prolonged stabilization of SSAT mRNA and enzyme protein. By contrast, sub-inhibitory concentrations of CHX and ANISO did not significantly stimulate BESpm-induced SSAT transcription and activity. These results suggest that labile repressor proteins, related to DDP-resistance phenotype, play a regulatory role in SSAT gene expression, and further indicate that by overcoming this inhibitory control it is possible to recover BESpm response.

2004 - Polyamine depletion switches the form of 2-deoxy-D-ribose-induced cell death from apoptosis to necrosis in HL-60 cells. [Articolo su rivista]
Monti, Maria Giuseppina; Ghiaroni, Stefania; Marverti, Gaetano; Montanari, Monica; Moruzzi, Maria Stella

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy- -ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. -Rib-treated cells with normal polyamine levels, progressing through the G1 into the S and G2/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G1 phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.

2001 - Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis (ethyl)spermine involves transcriptional and post-transcriptional regulation. [Articolo su rivista]
Marverti, Gaetano; Bettuzzi, S.; Astancolle, Serenella; Pinna, C.; Monti, Maria Giuseppina; Moruzzi, Maria Stella

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analog, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study we have investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin- sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 µM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 µg/ml Act-D, suggesting that transcription plays a major role in the analog-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilized in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels.

1999 - Differential induction of spermidine/spermine N1-acetyltransferase in cisplatin-sensitive and -resistant human breast cancer cells by N1, N12-bis(ethyl)spermine. [Capitolo/Saggio]
Marverti, Gaetano; G., Monti; M. G., Piccinini; And, Moruzzi; M., S.

The cisplatin-resistant human breast cancer MCF7/CH cells have been shown to be also cross-resistant to the spermine analog N1-N12-bisethylspermine (BESPM) in comparison to their -sensitive counterparts, MCF7 cells. This cross-resistance was, at least in part, ascribable to the reduced induction of the polyamine key catabolic enzyme SSAT, which is one of the main target of this drug. As a consequence of this different induction of SSAT activity, the resistant cell line was less depleted of its polyamine content than the parental line, accounting for its better survival to the analog treatment.

1999 - 2-Deoxy-D-ribose-induced apoptosis in HL-60 cells is associated with the cell cycle progression by spermidine [Articolo su rivista]
Monti, Maria Giuseppina; S., Ghiaroni; D., Barbieri; C., Franceschi; Marverti, Gaetano; Moruzzi, Maria Stella

The presence of polyamines is required for the apoptotic program triggered by a-deoxy-D-ribose (dRib) in HL-60 cells, but their oxidative metabolites does not appear to be involved in the oxidative stress caused by the sugar. The present study points to a relationship between spermidine-induced G(1) to S phase transition and the onset of dRib-induced apoptosis. Conversely, the G(1) block induced by alpha-difluoromethylornithine (DFMO) is associated with a protective effect against dRib-induced cell suicide. Replenishment of the intracellular spermidine pool by exogenous putrescine and spermidine induces cell cycle progression and restores apoptotic levels. The present data indicate that the induction of cell cycle progression by spermidine is a condition facilitating the activation of the apoptotic process by dRib.

1998 - N-1,N-12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines [Articolo su rivista]
Marverti, Gaetano; Piccinini, Giorgio; S., Ghiaroni; D., Barbieri; Quaglino, Daniela; Moruzzi, Maria Stella

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N-1,N-12-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately IO-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC50 value of 24.6 +/- 2 mu M vs. 3.4 +/- 0.8 mu M of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of sperdimine/spermine N-1-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G(2)/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype. Int J. Cancer 78:33-40, 1998.

1998 - Polyamine depletion protects HL-60 cells from 2-deoxy-D-ribose-induced apoptosis. [Articolo su rivista]
Monti, Maria Giuseppina; Ghiaroni, S.; Pernecco, Laura Rosa; Barbieri, D.; Marverti, Gaetano; Franceschi, Claudio

We investigated the involvement of natural polyamines in HL-60 cell death triggered by exposure to 2-deoxy-D-ribose (dRib)...

1997 - Modulation of cis-diamminedichloroplatinum(II) accumulation and cytotoxicity by spermine in sensitive and resistant human ovarian carcinoma cells. [Articolo su rivista]
Marverti, Gaetano; P. A., Andrews; Piccinini, Giorgio; S., Ghiaroni; D., Barbieri; Moruzzi, Maria Stella

The effect of spermine (Sp), a natural polycationic amine, on cisplatin (CDDP) sensitivity and accumulation of a human ovarian CDDP-sensitive cell line (2008) and its resistant variant (C13*) was investigated. Survival was also studied. ...

1996 - Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression [Articolo su rivista]
Monti, M. G.; Pernecco, L.; Manfredini, R.; Frassineti, C.; Barbieri, D.; Marverti, G.; Ghiaroni, S.

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G, to S phase transition.

1996 - Stimulation of cis-diamminedichloroplatinum(II) accumulation by modulation of passive permeability with genistein: an altered responce in accumulation-defective resistant cells. [Articolo su rivista]
Marverti, Gaetano; P. A., Andrews

The effect of the tyrosine kinase inhibitor genistein onthe accumulation of cisplatin (DDP) was investigated inDDP-sensitive and -resistant human 2008 ovarian cardnomacell lines. DDP accumulation after a 1-h exposure wasmaximally increased by concurrent 40 piM genistein. Themaximal stimulation of accumulation was observed after 2 hof total genistein exposure and was 83 ± 13% (n = 5) higherthan controls. With resistant C13* cells, however, the stimulationof accumulation was delayed until 4 h and wasincreased only 46 ± 18% compared to controls. RevertantRH4 cells that retained the accumulation defect behaved likethe C13 cells. Genistein stimulated [3H]mannitol accumulation(a marker of passive permeability) by 43 ± 9% (n =3) in 2008 cells, and the effect was maximal after 2 h of totalgenistein exposure. Changes in [3Hjmannitol accumulationin 2008 parent cells were highly correlated with DDP accumulation(r = 0.9010). These experiments also revealed that[3H]mannitol accumulation after 2 h in C13* cells was reduced38% compared to 2008 cells, a decrease that reflectedthe DDP accumulation defect. Fluid-phase pinocytosis determinedwith lucifer yellow CH as a marker showed nodifference between 2008 and C13* cells and no effect ofgenistein. Genistein was demonstrated to clearly inhibit protein-tyrosine phosphorylation initiated by the epidermalgrowth factor receptor kinase. Differences were noted in thephosphotyrosine pattern between the 2008 and C13* cells.Under the conditions that had the maximal effect on DDPaccumulation in 2008 cells, genistein decreased the IC50 ofDDP 8.2-fold in 2008 cells and 4.7-fold in C13* cells. Weconclude that: (a) genistein stimulates DDP accumulation bymodulating the passive permeability of the plasma membrane;(b) C13* cells are less permeable to passively diffusingsmall molecules, which offers a mechanism for the DDPaccumulation defect without invoking carrier proteins; (C)the effect of tyrosine kinase inhibition on passive permeabilityis altered in C13 cells; and (d) pinocytosis contributes insignificantly to DDP accumulation. Genistein, a dietaryisoflavone, thus seems to be a promising clinical candidatefor combination with DDP.

1995 - The effect of spermine on calcium requirement for protein kinase C association with phospholipid vesicles. [Articolo su rivista]
Moruzzi, Maria Stella; Marverti, Gaetano; Piccinini, Giorgio; Frassineti, Chiara; Monti, Maria Giuseppina

We have previously reported that polyamines interfere with protein kinase C-membrane interactions. ...

1994 - Effect of spermine on irreversible insertion of protein kinase C into phospholipid vesicles. [Relazione in Atti di Convegno]
Moruzzi, M. S; Marverti, Gaetano; Piccinini, G; Pernecco, L; Monti, M. G.

Nelle condizioni in cui prevale la formazione del complesso caratterizzato da una molecola di SPM/molecola di PS, la poliamina sembra legarsi perpendicolarmente alla superficie delle vescicole, e mediante i gruppi amminici non coinvolti nell’interazione con i fosfolipidi legare i siti anionici dell’enzima, favorendo l’interazione della PKC con i liposomi. Questa disposizione sembra essere confermata anche da esperimenti di correlazione eteronucleare bi-dimensionale con la spettroscopia di risonanza magnetica nucleare. Poichè’ stato dimostrato che la PKC può esistere in 2 stati di associazione con la membrana, uno reversibile e Ca++-dipendente ed uno irreversibile Ca++-indipendente, avente le caratteristiche di una proteina intrinseca di membrana, abbiamo investigato l’effetto inibitorio della SPM sull’associazione reversibile o irreversibile della PKC con i liposomi in funzione della concentrazione di Ca++. I risultati ottenuti hanno dimostrato che, a concentrazioni di Ca++ inferiori a 0,1 μM, la SPM inibiva la formazione del complesso fra PKC e membrane. A concentrazioni maggiori la SPM non impediva il processo di associazione, ma faceva diminuire le molecole di enzima inserite nella membrana e insensibile ai chelanti come EGTA, senza alterare l’affinità di legame. L’aumento della concentrazione di PL aumentava i livelli di PKC inserita nei liposomi, confermando che la SPM, complessando i siti di legame sulle membrane sia in presenza che in assenza di Ca++, favorisce le condizioni di legame che ostacolano l’inserimento dell’enzima nella membrana.

1994 - Spermine protects protein kinase C from phospholipid-induced inactivation [Articolo su rivista]
Monti, Maria Giuseppina; S., Ghiaroni; Marverti, Gaetano; G., Piccinini; L., Pernecco; Moruzzi, Maria Stella

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca2+. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca2+. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca2+ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca2+ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca2+ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.

1993 - Effect of Spermine on Membrane-Associated and Membrane-Inserted Forms of Protein Kinase C [Articolo su rivista]
Moruzzi, Maria Stella; Marverti, Gaetano; G., Piccinini; Frassineti, Chiara; Monti, Maria Giuseppina

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on N(max); spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes. Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected.

1993 - Relationship Between Spermine and Calcium on the Activation Process of Protein Kinase C. [Articolo su rivista]
Moruzzi, Maria Stella; Marverti, Gaetano; Piccinini, Giorgio; Frassineti, Chiara; Monti, Maria Giuseppina

Relationship Between Spermine and Calcium on the Activation Process of Protein Kinase C.

1991 - Effetto delle poliammine sulla regolazione della proteina cinasi C. [Relazione in Atti di Convegno]
Moruzzi, M. S; Piccinini, G; Marverti, Gaetano; Monti, M. G.

Effetto delle poliamine sulla regolazione della proteina cinasi C. Nel tentativo di contribuire al chiarimento del ruolo delle poliamine sui processi di fosforilazione intracellulari, in questa fase dell’attività di ricerca abbiamo preso in esame gli effetti delle poliamine su diversi aspetti della regolazione della proteina cinasi C (PKC), serina/treonina cinasi che si presenta in diverse forme isoenzimatiche, alcune Ca++/diacilglicerolo (DAG)/fosfolipide dipendenti, ed altre con attività indipendente dal Ca++. Dal momento che l’associazione della PKC alle membrane è il prerequisito essenziale per la risposta fisiologica dell’enzima, particolare interesse è stato rivolto allo studio dell’effetto delle poliamine sul processo di associazione della PKC alle membrane. A questo scopo abbiamo utilizzato un sistema sperimentale costituito da PKC parzialmente purificata da cervello di ratto e da liposomi, costituiti da fosfolipidi acidi, soprattutto acido fosfatidico (PA) e fosfatidilserina (PS). La PKC associata all membrane è stata saggiata quantitativamente come recettore degli esteri del forbolo cioè misurando il legame dell’enzima attivato ad un estere del forbolo radioattivo (3H-PDBu) con cui l’enzima interagisce con una stechiometria di 1:1. Esperimenti eseguiti con liposomi di composizione definita hanno permesso di stabilire che nelle condizioni in cui prevale la formazione del complesso caratterizzato da una stechiometria di legame di 3/4 molecole di PL acido/molecola di SPM si ha un’inibizione nella formazione del complesso attivo. In questo caso infatti sembra che la SPM si disponga parallelamente alla superficie del liposoma impedendo l’ulteriore legame dell’enzima. Al contrario, nelle condizioni in cui prevale la formazione del complesso caratterizzato da una molecola di SPM/molecola di PS, la poliamina sembra legarsi perpendicolarmente alla superficie delle vescicole, e mediante i gruppi amminici non coinvolti nell’interazione con i fosfolipidi legare i siti anionici dell’enzima, favorendo l’interazione della PKC con i liposomi.

1990 - Effect of spermine on association of protein kinase C with phospholipid vesicles. [Articolo su rivista]
Moruzzi, Maria Stella; Monti, Maria Giuseppina; Piccinini, G; Marverti, Gaetano; Tadolini, B.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.