Investigation of the Flavin Dependent Thymidylate Synthase enzyme as a potential antimicrobial target - PROJECT SUMMARY With the increase in antibiotic resistance bacterial infections there is an urgent need for novel antimicrobial agents that target alternative cellular pathways. One attractive strategy for the development of new antibiotics is to target DNA biosynthesis by inhibiting thymine biosynthesis. It has been shown that several severe human pathogens, such as Mycobacterium tuberculosis (M. tb) and Clostridioides difficile (C. diff) (and several other severe human pathogens), produce the DNA base thymine using an alternative enzymatic pathway that relies on a unique flavin- dependent thymidylate synthase (FDTS) that is coded for by the thyX gene. In eukaryotes and humans, the thyA or TYMS gene respectively code for “classical TSase” enzymes, which are well-characterized and the target of several chemotherapeutic drugs. Both the FDTS and classical TSase enzymes catalyze the same net reaction of uridylate (dUMP), which forms the product thymidylate, dTMP; however, the chemical mechanism, catalytic intermediates, and protein/active stie structure have been shown to be very different. Since these two proteins have distinct structures and mechanism of catalysis, it should be feasible to inhibit FDTS selectively over human TSase thereby interrupting DNA production in the pathogen but not in humans, providing antibiotics with low toxicity. In the proposed research plan our main goals are i) to study the molecular mechanism of MtbFDTS and CdiffFDTS catalysis and understand the nature of enzyme inhibition using biochemical, biophysical, and structural techniques to develop a consensus mechanism for this putative antibiotic drug target, ii) Develop biochemical methodologies to understand the nature of ligand binding to the MtbFDTS enzyme and relate this to assays that evaluate inhibition, such that these methods can be used for testing the efficacy and potency of newly synthesized molecules at inhibiting FDTS selectively over classical TSase., iii) to synthesize new compounds that we hypothesize to display selective inhibition of FDTS over human TSase, and to create a structure-function relationship for rational design of antibiotic compounds that may be effective against several human pathogens. By completing these experimental goals, we expect to discover unique mechanistic features of the MtbFDTS and CdiffFDTS enzymes, solve novel enzyme structures, and uncover physical parameters for selective inhibition of thymidylate production within pathogenic bacteria, without interrupting human TSase enzymes. This approach will help to define a structure-function relationship for selective enzyme inhibition. This will ultimately increase the overall chances of future antibiotic drug discovery through the rational design of compounds using fragment-based docking models using the crystal structures that we’ll solve for the MtbFDTS and CdiffFDTS enzymes, and through future high-throughput inhibitor screening using the assays we develop. Our studies are expected to discover underlying principles that will allow us to design and test new molecules that will not interfere with human DNA biosynthetic enzymes, which will potentially give rise to antibiotic drugs with low human toxicity.
