Design, synthesis and screening of a 2-aminothiazole library for inhibition of nucleotide-diphospho-sugar utilizing enzymes

This thesis describes the synthesis of a 2-aminothiazole library as potential inhibitors of nucleotide-diphospho-sugar (NDP-sugar) utilizing enzymes and the design of a high throughput assay for screening chemical libraries to such inhibitors. The results are important because selective inhibitors of NDP-sugar utilizing enzymes would be invaluable tools for studying cellular signaling, and the physiological responses that result (e.g., immunity or development). Furthermore, small molecule inhibitors would offer temporal control of the different NDP-sugar utilizing enzymes. Thus, we sought to develop strategies that can be used to generate and identify such compounds.; To design small molecule inhibitors with selectivity between NDP-sugar utilizing enzymes, computational methods were used to examine recently published structures of glycosyltransferases, a type of NDP-sugar utilizing enzyme. A scaffold was designed using GrowMol, a computer program for de novo molecular growth. The scaffold incorporated a 2-aminothiazole moiety and is hypothesized to occupy the nucleotide-diphospho-sugar binding pocket of NDP-sugar utilizing enzymes based on GrowMol calculations. We used the 2-aminiothiazole scaffold to design a library from which potential inhibitors could be identified. Specifically, a 1950-member library of 2-aminothiazoles was assembled using solid-phase synthesis and each compound was arrayed into 384-well microtiter plates for high throughput screening.; To rapidly screen libraries of compounds with various NDP-sugar utilizing enzymes, a fluorescent probe was synthesized to be used in a fluorescent polarization assay. This probe was found to be a competitive binder with several NDP-sugar utilizing enzymes: UDP-galactopyranose mutase (UGM), alpha-1,3-galactosyltransferase and beta-1,4-galactosyltransferase. Using the fluorescent polarization assay, several 2-aminothiazoles from the computational designed library were found to bind competitively to UGM. Finally, a screen of a commercial library against UGM using the newly developed fluorescent polarization assay identified several inhibitors of UGM with IC50 values in the 1-10 muM range. The chemical structures of theses inhibitors were similar to designed 2-aminothiazole scaffold. Indeed, some members of the 2-aminothiazole library also appear to inhibit UGM. These studies highlight the value of interweaving molecular design, combinatorial chemistry and high throughput screening to identify inhibitors when few lead candidates are known.