| Thymidylate synthase (TS) is an essential enzyme in ail living organisms. It has long been the focus of therapeutic studies for cancer, and more recently proposed as a target for treating infectious diseases. A fragment-based strategy was used to screen and identify small molecule fragments which bind to human TS using ligand-based nuclear magnetic resonance (NMR). This approach was also taken to analyze a dissimilar nucleotide binding protein from Trypanosoma brucei to determine ligand selectivity. A test for fragment pairs binding the folate site of human TS was devised and validated, using a pair of ligands which recapitulate a known potent inhibitor. These tests and other NMR-based studies were conducted to establish specificity and proximal binding information for a variety of fragment hits, which were employed in the design of novel lead compounds. Commercially available analogs of these leads were purchased and tested for potency by in vitro activity assay, and their affinities determined by quantitative NMR titrations. Two of these compounds were observed to have low micromolar affinity, moderate potency, and excellent binding efficiency against human TS. Relative binding orientations for both leads were modeled using AutoDock, and the complexes confirmed using data from NMR binding epitope experiments. Both lead compounds represent original starting points to generate potent and selective TS inhibitors. |
