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) identification of the most vulnerable targets of meropenem, (5) the synthesis of inhibitors of adenosylmethionine-8-amino-7-oxononanoate aminotransferase (BioA), (6) the synthesis and evaluation of proteasomal inhibitors, and (7) evaluation of novel translational inhibitors for the treatment of TB. 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. Eight PA-824 analogs synthesized in collaboration with the Novartis Institute for Tropical Diseases (NITD) are currently being tested in vivo for IND-enabling preclinical studies. The mechanism of catalysis of the deazaflavin dependent nitroreductase, Ddn, which is required for the reductive activation of PA-824 was investigated based on the hypothesis that a detailed mechanistic understanding of the enzymatic versus thermodynamic steps in pro-drug activation would enable development of a drug with enhanced activity against non-replicating Mtb. Quantum mechanical calculations were done to predict which steps would be rate-limiting during conversion of the substrate to its various intermediate and final metabolites and to identify which PA824 analogs would be most informative for unraveling each step in metabolite formation. These PA-824 analogs were synthesized and analyzed for their activity with whole cell assays as well as in enzyme assays with Ddn. Metabolite profiling of each analog was done and kinetic isotope effect studies done using deuterated PA824, deuterated water and/or deuterated F420 in order to understand what factors were determining in the observed metabolite ratios. In addition, in order to develop tools that would enable rapid progress for a backup candidate for PA824 for future clinical trials, methods were developed that would allow rapid identification of analogs with enhanced cell permeability as well as optimal pharmacokinetic properties. For this, a PA-824 permeability assay in Mtb and an analytical protocol for detection of PA-824 in tissue and blood samples for PK studies was developed. Work done in our lab showed that metronidazole was inactive against rapidly growing Mycobacterium abscessus, contrary to the published report in Journal of Antimicrobial Chemotherapy. 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 2010, we have synthesized and biologically evaluated more than 100 molecules that are derivatives of hit molecules obtained from quantitative high-throughput screening (qHTS) done in collaboration with NCGC which has allowed us to explore the structure-activity relationship (SAR) of molecules within a hit cluster. The scaffolds that gave us evidence of a specific target based on SAR studies were developed further to enable rapid proof of concept studies in animal models by performing cytotoxicity, solubility and rat and human microsomal stability assays. Several scaffolds that were hits in the screens were kinase scaffolds including scaffolds such as the imidazopyridines. The imidazopyridines have, for example, been developed by us in collaboration with collaborators at University of Notre Dame for their activity against MTb, including extensively drug resistant clinical strains of MTb. For 2 chemically different scaffolds inhibiting the same target in cell wall synthesis we identified molecules within these 2 scaffolds that had good microsomal stability, low cytotoxicity as well as acceptable oral bioavailability with sufficient serum concentrations and serum half-lives to warrant in vivo studies. Two molecules from each scaffold were taken into target validation studies in infected mice with results pending. In Project (3) we are continuing our evaluation of approaches to the inhibition of enzymes that are acyl-adenylated by ATP. 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 continuing our work on uncovering the mechanism of the beta-lactam, meropenem, which has remarkable cidal activity against both growing as well as anaerobically persisting MTb in the hope of uncovering the target which can be further exploited for drug development. We have synthesized labeled analogs of meropenem for identifying the mycobacterial target after labeling of whole cells and have used these in pull-down assays that resulted in identification of proteins involved in peptidoglycan assembly. In project (5) we are working with the Aldrich laboratory who are developing inhibitors of the biotin biosynthetic pathway. Based on the crystal structure of BioA and our previous success with synthetic analogs of a natural product inhibitor of BioA, the Aldrich laboratory have developed a series of inhibitors of BioA as well as BirA which biotinylates biotin biotin-accepting proteins which inhibit MTb growth with concomitant decreases in MTb protein biotinylation. In project (6) we are collaborating with researchers at George Washington University who are developing inhibitors of the proteasome to test the vulnerability of this metabolic pathway to inhibition during growth of MTb and have been able to identify a few modest inhibitors of MTb growth. In (7) we are testing the ability of a novel fluoroketolide antibiotic (solithromycin or CEM-101) which is currently in clinical development for other bacterial pathogens to inhibit growth of MTb in vitro under host relevant conditions, in infected macrophages as well as in infected animals. Our results have demonstrated that CEM-101 is active against clinical strains of MTb including XDR strains that are resistant to aminoglycosides, that it is active against MTb in macrophages and that it is equipotent to the second-line anti-tubercular streptomycin against acutely as well as chronically infected mice.
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