Currently this project focuses on four key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb), (2) the lead development of an oxazolidinone with optimized activity against MTb, (3) the synthesis and evaluation of inhibitors of the inosine 5'-monophosphate dehydrogenase and (4) structure-based design and screening of inhibitors of NAD synthetase and fumarase. Most of our effort currently is devoted to Project (1) in which we are screening large compounds libraries obtained from global collaborators including several large pharmaceutical companies to identify inhibitors of MTb growth under in vivo relevant conditions, performing dose-titration follow-up of hits and synthesizing or purchasing chemically similar compounds. These series are evaluated using secondary screens with 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. Since September 2015 we have completed primary screening as well as hit confirmation screens by dose titration screens of the resultant primary hits from a new 250,000 compound deck from the Medicines for Malaria Venture (MMV) under two carbon source including a beta-oxidation substrate and glucose, a 60,000 new compound deck from the Dundee Drug Discovery Unit under 2 different growth condition and a 69,000 compound deck the Global Chemical Diversity Library under two growth conditions. Reconfirmed hits with IC50 values less than 10uM are then assayed for their ability to completely inhibit growth of the organism under a panel of in vitro growth conditions reflecting all carbon sources thought to be in vivo relevant by determination of the minimum inhibitory concentration (MIC) and parallel determination of their cytotoxicity to HepG2 cells. Every attempt is 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 are prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in Lead Optimization. In contrast, series with structural alerts suggesting toxicophores are deprioritized. To rapidly expand the SAR for the prioritized chemotypes, commercially available analogs are purchased and tested in MIC assays. Target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, microarray and metabolomics analyses. In addition, kinetic and thermodynamic solubility determinations and microsomal stability assays are done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. A major reason for the low success rates in TB drug development is that the leads ultimately fail to reduce bacterial burdens in TB patients. Failure of a drug to reach sufficient levels at the site of disease is a leading cause of treatment failure. We are currently performing human alveolar epithelial transport assays in an attempt to develop an assay that will prioritize those compounds that we predict will accumulate in the epithelial lining fluid providing a reservoir of drug that will penetrate into adjacent granulomas. In addition, we are attempting to establish a primary alveolar epithelial cell line from marmoset lungs since this is our animal model of choice for the ultimate validation of a drugs efficacy before consideration of human clinical trials and thus ensuring that our compounds of interest are similarly transported when comparing marmoset to human epithelial transport assays would be a critical consideration. We have determined that the majority of series that progressed into hit-to-lead chemistry, were discontinued due to failing biological target validation and have expended considerable effort in attempting to optimize the process of biological triage. Target identification for compounds with potent anti-tubercular activity has shown that certain pathways are apparently susceptible to a diversity of chemical scaffolds. These apparently promiscuous targets include several enzymes involved in mycobacterial cell wall mycolyl-arabinogalactan biosynthesis as well as the respiratory bc1 complex. Neither of these targets are thought to have significant promise for treating TB since cell wall targets are already well hit by existing TB drugs and show little promise for radically changing the duration of therapy. TB bacteria are largely only slowly replicating in human infections and appear relatively insensitive to inhibitors of cell wall enzymes. The development of additional drugs which target aspects of cell wall biosynthesis would thus be expected to have little effect on the duration of chemotherapy and would only be useful as new drugs to treat drug-resistant disease. We have found that inhibitors of the respiratory bc1 complex show strain dependence as well as medium dependence in their ability to inhibit growth. Metabolic and transcriptional analyses of MTb growing under hypoxia as well as of MTb recovered from rabbit and monkey granulomas, have indicated major alterations in utilization of components of respiratory pathways. These results suggest that careful target validation is essential before progressing any inhibitors of respiration and coupled energy generation into lead optimization. To optimize our chances of identifying series with the potential to improve the speed of cure of TB, we have now applied our 4-tiered approach to accelerating the process of drug development through early application of counter-screens and other informative assays that will increase early lead candidate attrition rates while decreasing attrition rates in the expensive later stages of drug development. This hit triage process ensures that compounds get binned into groups based on the first tier of counter-screens with each bin subsequently progressing through a logical cascade of mechanism of action assays that will ensure that only relevant assays are run on each compound class. Using this hit triage approach we identified a scaffold with novel mechanism of action in tiers 1-2 assays that targeted the inosine monophosphate dehydrogenase as shown by macromolecular incorporation assays, metabolomics, transcriptomics and mutant generation followed by genoe resquencing. Enzyme kinetic studies showed that the inhibitor was an uncompetitive inhibitor of the enzyme with crystallographic studies further defining the binding mode of the compound. In project 2, we are working on developing an oxazolidinone with an increased potency against Mtb combined with a lower ability to inhibit mitochondrial protein synthesis in order to decrease the toxicities associated with linezolid chemotherapy. We are developing SAR based on anti-tubercular potency and lack of mitochondrial toxicity using mitobiogenesis assays and screening for antibacterial spectrum using B. subtilis as an indicator strain tested in parallel. Compounds with optimized properties including good PK values are progressed to evaluation in marmosets. In Projects 3-4 we are taking a variety of approaches to develop inhibitors and potential lead series against specific enzyme targets that are considered well validated as druggable in TB.
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