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) under in vivo relevant conditions, (2) the lead development of an oxazolidinone with optimized activity against MTb, (3) identification of environmental microbes that produce anti-tubercular secondary metabolites, (4) unraveling the mechanism of action of hits of interest as well as the mechanisms by which the pathogen adapts to the xenobiotic stress either through modulation of compound uptake, compound metabolism or mutations in the target pathway and in (5) we are exploring the physiological function of important mycobacterial enzymes and microbial biochemistry underlying host pathogenesis. 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 2018, we have completed formal hit assessment (FHA) of inhibitors validated in our HTS. The FHA entails our hit prioritization screens and counter-screens using our 4-tiered hit prioritization approach that bins compounds into major mechanistic classes, in particular highlighting those compounds that hit well-known targets in cell wall synthesis or respiration and excluding generally cytotoxic compounds. 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 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. In addition, selected compounds are synthesized to explore preliminary SAR. Kinetic and thermodynamic solubility determinations and microsomal stability assays are also done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. 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. WE have identified two lead compound that are 10-fold less toxic in mitochondrial biogenesis assays and 5-10-fold more potent against the pathogen than linezolid. Human dose prediction assay have indicated favorable dosing and PK/PD analysis is underway. Compounds with optimized properties including good PK values have been progressed to evaluation in marmosets. In project 3, we have identified environmental reservoirs that are rich in mycobacteria that compete with other environmental bacteria for limited nutrients. Specifically, sphagnum peat bogs have been reported to support diverse bacterial communities including slow-growing mycobacteria closely related to Mtb that compete for nutrients under conditions that recapitulate some of the defining characteristics of human granulomas including an acidic pH, hypoxia as well as nutrient limitation. We have screened microbes from these peat bogs and identified novel bacteria that produce secondary metabolites capable of inhibiting the growth of Mtb. Genome sequencing of some of these bacteria has allowed us to identify several genome clusters that encode enzymes known to be involved in secondary metabolite production and the purification and characterization of these metabolites is currently in progress. We have expanded our collection of microbes from these peat bogs to expand the repertoire of antibiotic-producing bog isolates with activity not only against Mtb but also other important bacterial pathogens and will be using genome-guided hit deconvolution to identify potential encoded metabolites. Currently we have the largest global collection of acidobacteria which we are characterizing for their spectrum of antitubercular antibiotics. In project 4, target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, microarray and metabolomics analyses. In addition, for top hits of interest where SAR indicates that certain positions on the molecule can be modified while retaining anti-tubercular activity, we have chemically modified the compounds by addition of a linker that can be UV-crosslinked onto the putative targets, as well as a linker moiety that provides a handle allowing purification of the resultant ligand-protein complexes. This chemical biology approach is guiding our efforts in target identification. To further expand our arsenal of molecular and chemical biology tools available to explore the role of enzymes or pathways of interest, we have developed a panel of fluorescent proteins with a variety of regulated promoters that are used to report on the role or regulation of particular pathways during drug treatment. Whole genome sequencing data combined with compound metabolite analyses have also highlighted the extensive repertoire of xenobiotic metabolizing enzymes that Mtb possesses that either inactivate or in some cases activate the hit of interest. An understanding of these metabolic processes will help us exploit or circumvent the activity of these enzymes in our drug development process. On a similar vein, we have identified the important role of cell wall and cell membrane associated proteins that facilitate the uptake of compounds including both nutrients and drugs. We have now uncovered the role of a large family of proteins that facilitate substrate transport through the outer mycomembrane of the cell wall. In project 5 we are continuing work to explore the importance of the biosynthesis of various cofactors, isoprenoids as well as Mtb-specific metabolites such as trehalose in maintaining viability under replicating as well as non-replicating conditions that reflect host-relevant conditions important during pathogenesis of the human host.
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