LIGHTS EU project (FP6) and the PPI inhibitors (2006-2010) The project delivered three patents (1 granted) and a PNAS publication (D. Cardinale et al, PNAS 2011). We discovered the first ligand (peptide) that could bind to the interface of TS, at the di-inactive form of the protein. The new peptides (PPI inhibitor) showed a mechanism of action different from that of the existing drugs targeting TS. It works as a dimeric protein stabilizer, by increasing the intracellular concentration of the inactive form of the TS dimeric protein. The EU project paved the way to a new concept in the area of anticancer drug design, lead to the organization of the Congress “Biomarkers in ovarian cancer” (11/2009) and to funding EUTROC, the translational research network on ovarian cancer (https://goo.gl/Wdr3Kg, https://goo.gl/eFXdxY) and participation to the Translational study Committee of the MITO group.
DROC-AIRC project and the proteomic perspective (2010-2013). After LIGHTS, the AIRC foundation supported the next step in the development of the peptide compounds (PPI peptide): the study of the mechanism of action and the identification of a proteomic signature that characterizes the peptide activity at the cellular level. By combining the early drug design work with the clinical research to closely monitor the biological activity of the new candidates, we proposed to screen a protein panel that could be specific for the peptide and drug effect at the cellular level thus working as an early phase pharmacodynamic biomarker. (Genovese F., JPR, 2014). In this context some collaborations and inclusion in clinical trials for the translational proteomic study were established: 1. Dr. U. de Giorgi at the IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.) Meldola (Conteduca V. et al, Oxaliplatin plus leucovorin and 5-fluorouracil (FOLFOX-4) n heavily- pretreated platinum-resistant ovarian cancer, BMC cancer, submitted)2. Dr. J. Sheouli of the Mayo Clinic of Berlin (L.Severi et al. 2018, Frontiers in Pharm.). 3. MITO group Phase 3 CT. (Bevacizumab in OC). Two papers have been cited by AIRC. Inthesame field contribution to Encyclopedia of Cancer, http://www.springerreference .comon the Chapter “Drug resistance to platinum drugs in Ovarian Cancer”DOI: https://doi.org/10.1007/978-3-642 -27841-9_6990-5.
Abstract.
Targeting ovarian cancer drug resistance
Ovarian cancer (OC) is considered a rare diseases and represents the fifth most common cause of death from cancer in women. The standard first-line treatment is a combination of paclitaxel and carboplatin (DDP) or carboplatin alone. In the case of progressive disease or drug resistance treatment with platinum drug, either alone or in combination, investigational compounds should be used. The mechanisms behind acquired resistance to DDP and its derivatives are not clear yet, although it is evident that the process is multifactorial including, enhanced DNA repair. Some peptides designed from the interface subunit of the human Thymidylate synthase (TS) have been identified recently by the Principal Investigator, as effective anticancer agents against sensitive and resistant OC cells. One of them was also able to recover the cellular sensitivity towards cis-platinum in cis-platinum resistant OC cells in the μM range. X-ray crystallographic studies showed that the peptide binds at the interface of the two subunits thus destabilizing the TS dimer and potentially affecting the dimer-monomerequilibrium at the intracellular level. This compound(s) show new anticancer mechanism of action against OC cancer cells, with respect to classical TS inhibitors such as 5-FU, Raltitrexed and Pemetrexed. In fact it inhibits TS in cell extracts, decreases/do not alter protein levels in sensitive/ cDDP resistant OC cells while inhibiting both types of cancer cells growth. The innovative mechanism of action and potency of the mentioned compounds suggested to advance in the discovery process, and the expectation of promising results. To forward the most active compounds to the pre-clinical evaluation, two main strategies should be adopted: ì) peptide optimization and development of peptidomimetic from the active peptides, ìì) conjugation of the peptide/peptidomimetic with folic acidcarrier to deliver specifically (targeting) the candidate to the Folate Receptors (FR) membrane protein overexpressed in the OC membranes. A multidisciplinary project has been proposed in which different expertise are combined ranging form the computational chemistry for peptide/peptidomimetic design, peptide chemistry, delivery studies, molecular mechanisms study in living cells and molecular biology, biophysical and structural approaches. In some fortunate cases of high affinity complexes of the compounds with the TS-protein X-ray crystal structures could also be considered as available from external collaborations .
Specific efforts will be devoted to achieve the following objectives:
1.Understanding the effect on Thymidylate protein forms balance of the peptides at the cellular level.
2.Translate the peptides in peptidomimetics thus overcoming the intrinsic problem related to stability and delivery of peptide drugs.
3.Conjugate the peptide/peptidomimetic with folic acid (vitamin) or folate acid analogue to facilitate the specific targeting of the compound into the ovarian cancer cells that naturally overexpress FRα.
4.Profiling the effect of the peptide/peptidomimetic conjugation system on folate related pathway, including FRα through Mass Spectrometry.
5. Evaluate the effect of the combination of cDDP with the peptide/petidomimetic engineered conjugated system on OC sensitive and resistant cells and toxicity on human cells models and human Ovarian cells .
6.Provide a profile of the biological effect of the designed conjugated system on sensitive, resistant OC cells and normal ovarian cells.
At the end of the three years effective targeting complexes will be selected and provided for preclinical studies with the aim to overcome platinum drug resistance in ovarian cancer. The PI has demonstrated capacity to carry out collaborative project and provide the necessary motivation and scientific improvements to reach the expected objectives.
Publications related to the topic.
Costi, MP, S. Ferrari, J. Green, Ovarian Cancer Drug Resistance. Encyclopaedia. Schwab, M. (Ed.), Springer. 2018.
Video on the web-htttp//ecancer.org/tv/pubdate1730. MP Costi.Reference Live: Cancer.
Cardinale D, Salo-Ahen OM, Guaitoli G, Ferrari S, Venturelli A, Franchini S, Battini R, Ponterini G, Wade RC, Costi MP. Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding. Protein Eng Des Sel. 2010 Feb;23(2):81-9. doi: 10.1093/protein/gzp075. Epub 2009 Dec 2. PubMed PMID: 19955218.
Marverti G, Ligabue A, Guerrieri D, Paglietti G, Piras S, Costi MP, Farina D, Frassineti C, Monti MG, Moruzzi MS. Spermidine/spermine N1-acetyltranferase modulation by novel folate cycle inhibitors in cisplatin-sensitive and -resistant human ovarian cancer cell lines. Gynecol Oncol. 2010 May;117(2):202-10. doi:10.1016/j.ygyno.2009.11.030. Epub 2010 Jan 19. PubMed PMID: 20031193.
Cardinale D, Salo-Ahen OM, Ferrari S, Ponterini G, Cruciani G, Carosati E, Tochowicz AM, Mangani S, Wade RC, Costi MP. Homodimeric enzymes as drug targets. Curr Med Chem. 2010;17(9):826-46. Review. PubMed PMID: 20156173.
Genovese F, Ferrari S, Guaitoli G, Caselli M, Costi MP, Ponterini G. Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer. Protein Sci. 2010 May;19(5):1023-30. doi: 10.1002/pro.379. PubMed PMID: 20306493; PubMed Central PMCID: PMC2868244.
Costi MP, Zeillinger R. Drug resistance in ovarian cancer: biomarkers and treatments Highlights from the DROC meeting held in Modena (Italy) on the 19th and 20th of February 2009. Scientific topics discussed at the meeting are reported in the present issue. Gynecol Oncol. 2010 May;117(2):149-51. doi: 10.1016/j.ygyno.2010.03.006. PubMed PMID: 20363442.
Garg D, Henrich S, Salo-Ahen OM, Myllykallio H, Costi MP, Wade RC. Novel approaches for targeting thymidylate synthase to overcome the resistance and toxicity of anticancer drugs. J Med Chem. 2010 Sep 23;53(18):6539-49. doi:10.1021/jm901869w. Review. PubMed PMID: 20527892.
Carosati E, Sforna G, Pippi M, Marverti G, Ligabue A, Guerrieri D, Piras S, Guaitoli G, Luciani R, Costi MP, Cruciani G. Ligand-based virtual screening andADME-tox guided approach to identify triazolo-quinoxalines as folate cycle inhibitors. Bioorg Med Chem. 2010 Nov 15;18(22):7773-85. doi: 10.1016/j.bmc.2010.09.065. Epub 2010 Oct 1. PubMed PMID: 20951595.
Cardinale D, Guaitoli G, Tondi D, Luciani R, Henrich S, Salo-Ahen OM, Ferrari S, Marverti G, Guerrieri D, Ligabue A, Frassineti C, Pozzi C, Mangani S, FessasD, Guerrini R, Ponterini G, Wade RC, Costi MP. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):E542-9. doi: 10.1073/pnas.1104829108. Epub 2011 Jul 27. Erratum in: Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):16133.PubMed PMID: 21795601; PubMed Central PMCID: PMC3161595.
Marverti G, Guaitoli G, Ligabue A, Frassineti C, Monti MG, Lombardi P, Costi MP. Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and-resistant ovarian cancer cells. Amino Acids. 2012 Feb;42(2-3):641-53. doi: 10.1007/s00726-011-1039-3. Epub 2011 Aug 4. PubMed PMID: 21814787.
Giudice S, Benassi L, Bertazzoni G, Veratti E, Morini D, Azzoni P, Costi MP, Venturelli A, Pirondi S, Seidenari S, Magnoni C. Biological evaluation of MR36, anovel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines. Invest New Drugs. 2012 Aug;30(4):1484-92.doi: 10.1007/s10637-011-9733-2. Epub 2011 Sep 1. PubMed PMID: 21881917.
Carosati E, Tochowicz A, Marverti G, Guaitoli G, Benedetti P, Ferrari S,Stroud RM, Finer-Moore J, Luciani R, Farina D, Cruciani G, Costi MP. Inhibitor ofovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase. J Med Chem. 2012 Nov 26;55(22):10272-6. doi: 10.1021/jm300850v. Epub 2012 Nov 5. PubMed PMID: 23075414.
Ferrari S, Ingrami M, Soragni F, Wade RC, Costi MP. Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-carboxamide and sulfonamidederivatives as thymidylate synthase A inhibitors. Bioorg Med Chem Lett. 2013 Feb 1;23(3):663-8. doi: 10.1016/j.bmcl.2012.11.117. Epub 2012 Dec 7. PubMed PMID: 23273520.
Realini N, Solorzano C, Pagliuca C, Pizzirani D, Armirotti A, Luciani R, Costi MP, Bandiera T, Piomelli D. Discovery of highly potent acid ceramidaseinhibitors with in vitro tumor chemosensitizing activity. Sci Rep. 2013;3:1035. doi: 10.1038/srep01035. Epub 2013 Jan 8. PubMed PMID: 23301156; PubMed CentralPMCID: PMC3539145.
Garg D, Beribisky AV, Ponterini G, Ligabue A, Marverti G, Martello A, Costi MP, Sattler M, Wade RC. Translational repression of thymidylate synthase bytargeting its mRNA. Nucleic Acids Res. 2013 Apr;41(7):4159-70. doi: 10.1093/nar/gkt098. Epub 2013 Feb 18. PubMed PMID: 23423353; PubMed Central PMCID: PMC3627590.
Marverti G, Ligabue A, Lombardi P, Ferrari S, Monti MG, Frassineti C, Costi MP. Modulation of the expression of folate cycle enzymes and polyamine metabolismby berberine in cisplatin-sensitive and -resistant human ovarian cancer cells. Int J Oncol. 2013 Oct;43(4):1269-80. doi: 10.3892/ijo.2013.2045. Epub 2013 Jul31. PubMed PMID: 23903781.
Ferrari S, Calò S, Leone R, Luciani R, Costantino L, Sammak S, Di Pisa F, Pozzi C, Mangani S, Costi MP. 2'-Deoxyuridine 5'-monophosphate substratedisplacement in thymidylate synthase through 6-hydroxy-2H-naphtho[1,8-bc]furan-2-one derivatives. J Med Chem. 2013 Nov 27;56(22):9356-60. doi: 10.1021/jm4014086. Epub 2013 Nov 13. PubMed PMID:24147825.
Pelà M, Saxena P, Luciani R, Santucci M, Ferrari S, Marverti G, Marraccini C, Martello A, Pirondi S, Genovese F, Salvadori S, D'Arca D, Ponterini G, Costi MP, Guerrini R. Optimization of peptides that target human thymidylate synthase to inhibit ovarian cancer cell growth. J Med Chem. 2014 Feb 27;57(4):1355-67. doi:10.1021/jm401574p. Epub 2014 Feb 12. PubMed PMID: 24450514.
Piras S, Carta A, Briguglio I, Corona P, Paglietti G, Luciani R, Costi MP, Ferrari S. 2-[N-Alkyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines as anticancer agents inhibitors of folate enzymes. Eur J Med Chem. 2014 Mar21;75:169-83. doi: 10.1016/j.ejmech.2014.01.048. Epub 2014 Jan 30. PubMed PMID:24531230.
Genovese F, Gualandi A, Taddia L, Marverti G, Pirondi S, Marraccini C, Perco P, Pelà M, Guerrini R, Amoroso MR, Esposito F, Martello A, Ponterini G, D'Arca D,Costi MP. Mass spectrometric/bioinformatic identification of a protein subset that characterizes the cellular activity of anticancer peptides. J Proteome Res. 2014 Nov 7;13(11):5250-61. doi: 10.1021/pr500510v. Epub 2014 Sep 29. Erratum in: J Proteome Res. 2016 Oct 7;15(10 ):3944. PubMed PMID: 25196676.
Cannazza G, Cazzato AS, Marraccini C, Pavesi G, Pirondi S, Guerrini R, Pelà M, Frassineti C, Ferrari S, Marverti G, Ponterini G, Costi MP. Internalizationand stability of a thymidylate synthase Peptide inhibitor in ovarian cancer cells. J Med Chem. 2014 Dec 26;57(24):10551-6. doi: 10.1021/jm501397h. Epub 2014 Dec 3. PubMed PMID: 25353379.