Currently this project focuses on five key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb), (2) the synthesis and evaluation of inhibitors of synthesis of the mycobacterial acyl adenylating enzymes, (3) the synthesis and evaluation of inhibitors of the nonmevalonate biosynthetic pathway (4) the synthesis and evaluation of inhibitors of the inosine 5'-monophosphate dehydrogenase (IMPDH) and (5) design, synthesis and evaluation of inhibitors of peptidoglycan biosynthesis. In Project (1) we are screening large compounds decks to identify inhibitors of MTb growth under in vivo relevant conditions, dose-titration follow-up of hits and chemically similar compounds, and secondary screening using a battery of conditions that are thought to be relevant during in vivo growth of MTb. We selected carbon source as an in vivo relevant variable since numerous studies have shown the metabolism of glucose, cholesterol and/ or other lipids are critical for growth and survival of this pathogen in host tissues. We have focused on a much larger source of quality compound diversity to effectively interrogate bacterial metabolism and since September 2012 have screened a 250,000 compound library from Merck under 2 carbon sources, a 100,000 compound deck from Bayer under 3 carbon sources, a 500,000 deck from the Medicines for Malaria Venture under 2 conditions and a 5000 compound library enriched for anti-infectives from Pfizer under three screening conditions. Hits from primary screens were confirmed in dose titration screens followed by determination of the minimum inhibitory concentration (MIC) of reconfirmed hits to determine the concentration of compound required that results in full growth arrest. Those compounds that inhibited growth of MTb in vitro were subsequently prioritized. Every attempt was made to progress as many chemo-types as possible to increase the likelihood of hitting a diversity of targets. Hit series with multiple members showing activity for the scaffold with low-complexity, acceptable solubility and promising physicochemical properties for profiling were prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in the Lead Optimisation phase. In contrast, series with structural alerts suggesting toxicophores were deprioritized. For the prioritized chemo-types, additional profiling was performed by cross-screening against MTb mutants containing resistance mutations in promiscuous drug targets (MmpL3, DprE1), a katG mutant of MTb that is resistant to all compounds activated by an isoniazid-like mechanism, testing against a reporter strain that highlights compounds that act against targets in cell wall synthesis, a panel of other mutants with known polymorphisms in specific drug targets as well as hepG2 cytotoxicity screening to eliminate generally cytotoxic compounds. We hypothesize that drugs that target cell wall biosynthesis would not lead to a significant shortening of the duration of chemotherapy since current first-line TB treatment contains two drugs (isoniazid and ethambutol) that target aspects of cell wall biosynthesis with a 6-9 month treatment time required for success and clinical data additionally suggest that these are mainly active against a small population of rapidly growing MTb in airways and cavities that have erupted into airways. To rapidly expand the structure-activity relationships (SAR) for a series, commercially available analogs were purchased and tested in the MIC assays. Based on the preliminary SAR and results from the additional profiling assays, series were further prioritized to expand SAR by rational design and chemical synthesis of the hits and close analogs. In parallel, target identification was initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, microarray analysis and metabolomics analyses. In addition, kinetic and thermodynamic solubility determinations and microsomal stability assays were done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. Our mutant generation studies have also indicated that single nucleotide polymorphisms in actual target genes are rare. The majority of mutations identified in resistant mutants map to transcriptional regulators, efflux or compound modification systems, or genes suspected to affect cell wall permeability. This has been particularly true for low to moderate level resistant mutants. Transcriptional profiling analyses have been useful in binning compounds into groups based major mechanism of action and we are currently attempting to combine transcriptional regulation data with metabolomics data to generate a systems level overview of major pathways affected. To date, we have identified several compounds that block the function of MmpL3, the trehalose monomycolate transporter essential for cell wall synthesis. In addition, three scaffolds were identified that target the DprE1 epimerase involved in cell wall arabinan biosynthesis. The apparent promiscuity of these and potentially other enzymes involved in cell wall assembly suggested that cell wall synthesis is either a particularly vulnerable chokepoint during in vitro growth of this pathogen or that the localization as supramolecular cell wall assembly complexes at the bacterial poles facilitates access to variety of particularly hydrophobic compounds that directly or indirectly disrupt function or assembly of these multiprotein cell wall assembly factories. 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. Transcriptional profiling analyses had indicated that these imidazopyridines affected cellular respiration. This was subsequently corroborated in mutant generation studies which demonstrated that mutations in qcrB, a component of the cytochrome C oxidase complex involved in aerobic respiration, conferred high-level resistance to this scaffold. 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. In Project (2) we are continuing our evaluation of approaches to the inhibition of enzymes that are acyl-adenylated by ATP. 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 (3) 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 analogs 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 (4) we are collaborating with researchers at Brandeis University who are developing inhibitors of bacterial inosine 5'-monophosphate dehydrogenase which is essential for guanine nucleotide biosynthesis. Inhibitors have been developed with high selectivity for the bacterial enzyme which display on-target micromolar efficacy against Mtb. In Project (5) we are collaborating with Dr. Monika Konakleivas laboratory who are synthesizing azetinones (monocyclic -lactams) that target peptidoglycan biosynthetic enzyme(s). We have found several with activity against MTb in vitro.
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