Currently this project focuses on seven key areas: (1) the chemical synthesis of analogs of nitroimidazoles such as PA-824, (2) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb), (3) the synthesis and evaluation of inhibitors of synthesis of the mycobacterial acyl adenylating enzymes, (4) the synthesis of inhibitors of biotin protein ligase, (5) the synthesis and evaluation of inhibitors of the nonmevalonate biosynthetic pathway and (6) the synthesis and evaluation of inhibitors of the inosine 5'-monophosphate dehydrogenase (IMPDH). In Project (1) we are synthesizing analogs of nitroimidazooxazines related to PA-824, a drug that is currently in Phase II studies in humans for the treatment of tuberculosis. Several PA-824 analogs have progressed to efficacy studies in a mouse model of granuloma formation in order to determine the compound with the best ability to kill the slowly replicating Mtb found in hypoxic environments. In addition, the analogs have progressed to pharmacokinetic analyses in relevant animal models to determine the optimal dose for non-human primate efficacy studies. We have completed our mechanistic analyses of the nitroreduction of PA-824 by the deazaflavin dependent nitroreductase (Ddn) encoded by Rv3547 with the ultimate goal of designing an optimized analog with enhanced activity against non-replicating Mtb. Studies on Ddn were performed with deuterated substrate (PA-824), cofactor (F420H2), buffer and LC_MS solvent system in order to understand the mechanism of hydride transfer and determination of the rate determining step for the enzymatic bio-reduction. The key to understanding the mechanism was to establish a structure of the intermediate which is the source of all the metabolites. Since the intermediate was inherently unstable to various isolation techniques and due to the complex NMR spectra of the reaction mixture, it was difficult to assign a structure to the intermediate. UV-visible difference spectra of the reaction mixture along with calculations done with the time-dependent density functional theory for the intermediate, helped in establishing its structure. A solvent isotope effect was seen suggesting the participation of a water molecule during catalysis. Incorporating water molecules in quantum chemical calculations for constructing putative transitions states of reduced intermediates helped in understanding the product distribution ratio. In Project (2) we are synthesizing analogs from clusters of hits identified in whole cells screens for inhibitors of mycobacterial survival under a variety of in vivo relevant conditions. Since September 2011, we have completed our biological evaluation based on SAR studies coupled to biological evaluation of this identified in a screen performed in collaboration with the NCGC. For the salicylanilides, more than 200 analogs were synthesized and SAR studies coupled to cytotoxicity evaluation indicated that potency against Mtb tracked with cytotoxicity which, coupled to our inability to generate resistant mutants, suggested a non-specific mechanism of action. Two chemically different scaffolds that we previously had demonstrated to inhibit DprE1, an enzyme critical for mycobacterial cell wall arabinogalactan synthesis, and that we optimized for microsomal and pharmacokinetic properties, were tested in vivo and shown to be mildly bacteriostatic in a chronic model of murine tuberculosis. We have synthesized several inhibitors with reported antitubercular activities in the hope of progressing these to SAR studies for drug target identification. Unfortunately this only highlighted the inaccuracies of published results since several compounds were found to be completely inactive against Mtb. However, we are pursuing a few promising leads from the published literature including an isoxazole-based scaffold for which we have generated a compound with submicromolar potency against MTb and similarly a hydroxyindole scaffold for which we have achieved submicromolar potency. Metabolic stability both in culture as well as to microsomes have highlighted some liabilities that need to be addressed if these are pursued any further. Target studies of these are underway which will be critical in determining further medicinal chemistry efforts on these compounds. Several kinase scaffolds were identified in the screens and SAR studies on these have suggested independent targets for a few of these whereas at least two scaffolds, one being an imidazopyridine core, suggested the same target based on convergent SAR. For the imidazopyridine scaffold microsomal stability assays have suggested some points of CYP450-dependent metabolism that need to be addressed for future in vivo studies. Finally a scaffold which we identified as a potential fatty acid-CoA ligase inhibitor was explored for SAR analyses. Fifteen analogs were synthesized potencies of which suggested a very steep SAR. We have initiated analog searches of compounds libraries of several pharmaceutical companies (as part of the TB Drug Accelerator program of the Gates Foundation) in order to screen more analogs against Mtb to gain a better understanding of SAR of this scaffold. In Project (3) we are continuing our evaluation of approaches to the inhibition of enzymes that are acyl-adenylated by ATP. Several acyl-adenylating enzymes are essential for a variety of metabolic processes in Mtb including mycobactin biosynthesis. An enzyme that is essential for the initial steps in the biosynthesis of the iron-acquiring siderophore of MTb, mycobactin, is adenylated by ATP as are certain enzymes in lipid biosynthesis of this organism. These enzymes share a common mechanism and share some unique characteristics of their active sites. In collaboration with scientists at the University of Minnesota's Center for Drug Design we are continuing to test inhibitors of mycobactin and lipid biosynthesis. In Project (4) we are collaborating with the Aldrich laboratory in evaluating inhibitors of the mycobacterial biotin protein ligase (BPL) which links biotin to the biotin-dependent carboxylases that play essential roles in fatty acid biosynthesis and gluconeogenesis. A bisubstrate BPL inhibitor was designed which showed potent on-target inhibition of drug-sensitive as well as resistant strains of Mtb. In project (5) we are working with the Dowd laboratory that is developing inhibitors of the nonmevalonate biosynthetic pathway which is essential for isoprenoid biosynthesis. The first committed step in this pathway in some organisms is the reaction by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) , a known target of the antibiotic fosfidomycin. Fosfidomycin has no activity against Mtb due to its poor cell penetration. Inhibitors with improved cell penetration were designed and shown to inhibit growth of Mtb suggesting that Dxr may be an attractive target for drug development. In project (6) we are collaborating with researchers at Brandeis University who are developing inhibitors of bacterial inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH is essential for guanine nucleotide biosynthesis. This target has been extensively exploited for cancer and immunotherapies but poorly exploited for anti-infective drug development due to the need to develop selectivity for the bacterial enzyme relative to its human counterpart. Inhibitors have been developed with high selectivity for the bacterial enzyme which display on-target micromolar efficacy against Mtb.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2012
Total Cost
$635,835
Indirect Cost
City
State
Country
Zip Code
Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T et al. (2016) Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site. Proc Natl Acad Sci U S A 113:7503-8
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