Abstract 3892: Anti-cooperative allosteric inhibitors of thymidylate synthase as novel antineoplastic agents
Authors: Kulemina Lidia V. , Morgan Kevin , Shabashvili Daniil , Allegra Carmen , Chen Min , Zajac-Kaye Maria 
Published: August 2012

Abstract
Thymidylate synthase (TS) inhibitors are a class of antineoplastic agents widely used for the treatment of gastrointestinal, breast, ovarian and other cancers. TS catalyzes reductive methylation of dUMP to dTMP and serves as the sole de novo source of thymidylate inside the cell. While TS is essential for DNA biosynthesis and normal cell proliferation, TS overexpression has been associated with resistant tumor phenotype and poor prognosis. Although TS inhibitor 5-flourouracil (5-FU) continues to be one of the most frequently used chemo-therapeutic agents, 5-FU and its metabolites can cause significant liver, cardiac and bone marrow toxicity and are associated with development of drug resistance. In this study, we developed a new strategy that targets TS subunit cooperativity by freezing isoform interconversions and structural dynamics of the enzyme. In TS catalytic mechanism, conserted motion of amino acid residues 181-197 brings the catalytic Cys195 in and out of the active site. These small scale motions that translate across the dimer interface upon substrate binding and product release are integral to TS enzymatic function and can be taken advantage of in structure-based design of its inhibitors. For this purpose, we selected TS crystal structure with high asymmetry between the subunits (PDBID:1HVY) and used it as a template for molecular docking. The structure was prepared by removing heteroatoms and liganded molecules, followed by energy minimization in UCSF Chimera (1.5.2) and testing resulting geometries in Coot (0.6.2). We identified 5 potential docking sites and selected the site at the dimer interface specific to inactive conformation of TS. National Cancer Institute (NCI) small molecule library of 139,735 compounds was extracted from ZINC and pruned for redundant structures and molecules with molecular weight less than 200 Da. Resulting compound library was docked (DOCK6.4) into the selected site in 1000 orientations with flexible bond parameters enabled. The top 1000 hits were re-scored using AMBER and visualized in PyMOL to check for consistency and geometric fit. 837 out of 1000 molecules were further selected and re-ranked based on predicted physicochemical characteristics (cLogP, pKa, solubility, etc.). The twenty-seven highest ranked compounds were requested from the NCI and tested for their effects on viability in HeLa, K562 and A549 human cancer cell lines. Five out of 27 compounds reduced viability by more than 50% at a concentration of 10uM; one of the compounds was effective below 1uM. In conclusion, by using this new approach that targets TS subunit cooperativity, we identified small molecules with potential anti-cancer activity.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3892. doi:1538-7445.AM2012-3892