Unusual Targeting of the human Thymidylate synthase interface: a tethering approach with mass-spectrometric detection.

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