The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in non-replicating (NR)-MTb. We are exploring the importance of biotin and pyridoxal biosynthesis, peptidoglycan turnover, the role of putative F420-binding and genetically annotated pyridoxal-generating enzymes and iron acquisition and validating these by chemical and genetic means in NR-MTb. We have been able to demonstrate that MTb peptidoglycan has interpeptide crosslinks which are proportionally different to those of bacteria such as E. coli and that these linkages and their relative amounts show subtle variations between different stages of growth and non-replicating persistence. The identity of the transpeptidases that play a role in crosslinking under the different conditions is being explored by analysis of genome sequences of clinical strains with differential beta-lactam susceptibilities, affinity pull-down and generation of mutants. We have shown that the beta-lactam meropenem results in very early release of cytosolic contents of MTb which is associated with ultrastructural changes in cell morphology as seen by scanning and transmission electron microscopy. We are exploring the mechanism of action of this drug by a combination of transcriptional profiling, metabolic labeling and protein tagging experiments to identify potential druggable targets in peptidoglycan biosynthesis. In addition, meropenem treatment of MTb is associated with a considerable fraction of phenotypically tolerant persisters. We are trying to understand the pathways that lead to a state of metabolic persistence as opposed to the pathways that are associated with cell death in order to identify targets of which inhibition would prevent the emergence of drug tolerant persisters or targets whose activation or inactivation could irreversibly set the course to a destiny of cell death. The second major focus area of this project starts from a different perspective and uses compounds that are in clinical development (PA-824 and SQ109) which are known to possess activity against replicating as well as NR-TB. We are attempting to dissect the catalytic mechanism of the reductase that activates PA-824 in the hope of developing PA-824 analogs that promote the formation of reactive nitrogen intermediates associated with cellular killing. In collaboration with researchers at GNF, we have solved the X-ray crystal structure of MTb Ddn without its cognate N-terminus, constructed dozens of site-directed mutants of Ddn as well as an active Nocardia farcina homolog of this protein, and solved crystal structures of the apo-enzyme as well as co-structures with its cognate oxidized F420 cofactor. The crystal structures have assisted us in mapping the F420 binding site but failed to divulge any information on the binding site of PA824. From our knowledge of the underlying chemistry and kinetic parameters from a huge amount of structure activity relationship (SAR) studies we identified a number of residues which might constitute the binding pocket of PA824. Site directed mutants of these putative active site residues were generated and specific activity data for these have helped in identifying the binding pocket for the drug PA824. Molecular dynamics were performed with various PA824 analogs to validate the SAR results. The results are in good agreement with the proposed binding site for the drug PA824. For SQ109 we have determined that it inhibits a unique aspect of cell wall biosynthesis which differs from other inhibitors of MTb mycolyl-arabinogalactan biosynthesis. This in combination with focused studies of enzymes that transfer mycolic acids to the cell wall has allowed us to start to unravel poorly understood aspects of key events in cell wall mycolyl-arabinogalactan synthesis. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. We have developed a chemostat model of MTb for growth of the organism under defined oxystatic conditions. We have determined the lower oxygen concentration limits at which measurable growth can still occur and are exploring metabolism by a combination of metabolomic, transcriptional and metabolic inhibitor sensitivity studies. Steady state isotope feeding experiments have provided a glimpse of how respiration is modulated during metabolic adaptation to hypoxia. In a second approach we are identifying inhibitors of metabolism by high-throughput screening approaches which would have as ultimate goal the mapping of metabolism under defined environmental growth or NR conditions. To date we have completed 9 high-throughput screens of MTb growing on defined carbon sources against a library of 15,000 compounds from the NCGC collection. We have also screened an 8,000 compound set of the BROAD Institutes Diversity Oriented Synthesis collection under 4 different carbon source conditions. In addition, a 35,000 compound library from BioFocus was screened under 3 different carbon source conditions. Understanding the differential susceptibility of MTb to inhibitors under these growth conditions will help define the relative role of the various metabolic pathways. Hits that are common to all or some screening conditions or unique to only one condition are now being followed up by chemoinformatic analyses of all available literature on these compounds and chemically similar analogs. Importantly, we have discovered that the carbon source has a dramatic effect on the types of the rank of hits identified during screening. Mutant selection with subsequent whole-genome re-sequencing and transcriptional profiling has allowed us to identify the target for 2 hits from the NCGC collection. We are continuing our studies to identify the target pathways of hits from the high-throughput screens by these approaches. Collaborative studies with researchers at Weill Cornell Medical College will allow us to further understand metabolic changes associated with hits of interest by metabolomic studies of MTb during treatment with these compounds. We are also studying the effect of changes in gene expression in cofactor metabolic pathways to identify how downregulation of cofactor biosynthesis affects target vulnerability. To date, we have completed a screen of MTb during restricted expression of panthotenate synthase which is essential for coenzyme A biosynthesis. A similar approach is being used to follow up on hits from a high-throughput screen of M. bovis BCG persisting under anaerobic conditions done in collaboration with NITD and GNF. The latter screen was designed to identify respiratory inhibitors since we have established that respiration is a bottleneck in survival of such non-replicating mycobacteria and in addition to the above approaches, we are also analyzing susceptibility of respiratory mutant strains to these hits and related analogs under a variety of conditions. We currently have sub-micromolar hits against anaerobic NR MTb for which target identification is in progress. Unfortunately 58,000 compounds do not provide sufficient chemical diversity to explore a significant fraction of metabolism. Moreover, no synthetic library would cover the chemical space required to map metabolism. So another aspect of this project has been to explore alternate sources for compounds that might be enriched in molecules with activity against NR-TB. One source that has been especially interesting is samples that have been collected from sphagnum peat bogs that harbor mycobacteria that compete for limited nutrients with other environmental bacteria under conditions which mimic aspects of human TB granulomas. Microarray analyses have proven to be a rapid method of eliminating those bog extracts containing an inhibitor that targets previously known metabolic targets such as protein synthesis, DNA integrity and iron acqui
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