Application of Mechanistic Enzymology in Identifying Inhibitors of Critical Enzymes from Human Pathogens

The single-celled pathogens Mycobacterium tuberculosis and Trypanosoma cruzi are the causative agents of the human diseases tuberculosis and Chagas disease, respectively. In an effort to develop potentially new treatments for both of these diseases we have utilized an understanding of enzyme mechanisms to guide the identification and development of potential inhibitors for critical enzymes of these two pathogens. In chapter II the work towards developing/discovering inhibitors of the D-alanine:D-alanine ligase enzyme from M. tuberculosis (MtDdl) is detailed. MtDdl is a target for the antibiotic D-cycloserine (DCS), the use of which is limited, in part, due to side-effects associated with the drug. The goal of this study was to determine if alternative D-alanine-like molecules could inhibit the function of this enzyme in vitro. The inhibitory effect of a small library of D-alanine analogs was evaluated. MtDdl was found to have a high degree of ligand selectivity; however two compounds that mimic the catalytic intermediates were characterized by kinetic and biochemical methods. The results of this study showed that both of these compounds exhibit similar inhibitory potency to DCS. Additionally, evidence for DCS phosphorylation was provided by positional isotope exchange, supporting a mechanism of inhibition which contradicts previous studies. In chapter III the study of a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from T. cruzi is discussed. Due to the role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatments for Chagas disease. In this study we have found that T. cruzi CL Brener strain possesses a pair of functionally identical genes encoding enzymes with HGPRT activity in vitro. One of these enzymes was further characterized and was found to be rate-limited by product release, which prevented accurate measurement of kinetic isotope effects. Potential transition-state analogs were tested against the enzyme and the most potent of which were found to bind with low nanomolar affinity. Analysis of the structure-activity relationship of the putative transition-state analog inhibitors provided convincing evidence for a chemical mechanism involving an SN1-like transition-state

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine–Guanine Phosphoribosyltransferases

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine–guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved