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 and evaluation of substrates for, and inhibitors of, transpeptidases that are responsible for remodeling the TB cell envelope, (5) the synthesis of inhibitors of adenosylmethionine-8-amino-7-oxononanoate aminotransferase (BioA), (6) synthesis of analogs of SQ109, and (7) the synthesis and evaluation of isoprenoid biosynthetic inhibitors. 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. Two 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. In collaboration with NITD, computational (3D QSAR pharmacophore) modeling was used to predict the activity of derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail of the 6-amino derivative of PA-824. These were tested for whole-cell activity against both replicating and non-replicating Mtb, as well as determination of their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from MTb that reductively activates this pro-drug. Neither whole cell activity nor enzymatic kinetic parameters were significantly different suggesting that this site is not involved in important enzyme contacts. Substitution on both unoccupied positions of the trifluoromethoxyphenyl ring yielded multiple compounds with 40nM aerobic whole cell activity and 1.6M anaerobic whole cell activity tenfold improvement over the parent molecules. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism and are undergoing in vivo testing. 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. An anaerobic screen performed at GNF in collaboration with researchers at NITD identified 230 molecules that killed MTb during anaerobic survival. Cheminformatic analysis clustered these into 83 scaffolds of which 9 were prioritized based on chemical sensibility and published biological data. More than 50 analogs of 3 of these top priority clusters were synthesized and evaluated for cidal effect against anaerobically persisting MTb. Currently, a series of molecules with a defined structure-activity relationship against whole cells have been synthesized that have sub-micromolar potency against non-replicating MTb. The target of these molecules is under investigation. In addition, we have synthesized and biologically evaluated more than 200 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 of molecules within a hit cluster. In Project (3) we are evaluating 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 exploring the biological role of the transpeptidases that are essential for cell wall peptidoglycan biosynthesis in MTb. We have synthesized the native peptide substrates as tools to explore the function of the unique L,D-transpeptidases. In addition, we have synthesized a panel of beta-lactam analogs for biological evaluation against MTb and other bacterial pathogens. 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. 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 synthesizing analogs of the diamine drug SQ109 initially developed by TRS researchers which is currently in clinical development for TB chemotherapy. The target of SQ109 is unknown and synthesis of analogs has allowed us to explore the target pathway of this drug. In project (7) we are collaborating with researchers at George Washington University who are developing inhibitors of the deoxyxylulose pathway of isoprenoid synthesis to test the vulnerability of this metabolic pathway to inhibition during growth of MTb.
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