The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in NR-TB. These include one of the final steps in the biosynthesis of nicotinamide which is the amidation of nicotinic acid adenine denucleotide by the ATP-dependent enzyme NadE. Using genetic and biochemical approaches section scientists demonstrated that, contrary to contemporary wisdom, Mycobacterium tuberculosis is capable of scavenging nicotinamide from the human host but engages this system only during infection. This metabolic plasticity (the organism can both make NAD from scratch or scavenge it externally based on the environment it finds itself in) limits the possible choice of targets for impacting this pathway in NR-TB to the two final steps which are required for both pathways. Other pathways being explored include: the biosynthetic enzymes involved in biotin synthesis which have been shown genetically to contribute to NR-TB, the so-called suicide modules that contribute to shutdown of macromolecular processes in NR-TB, and the enzymes involved in cross-linking of peptidoglycan which introduce the unusual linkages found in NR-TB.? ? The second major focus area of this project starts from a different perspective and uses compounds that are in clinical development LPA-824 and SQ109) that are known to possess activity against NR-TB and attempts to understand how they work. Building on prior success in identifying Rv3547 as a critical enzyme for activation of nitroimidazoles we successfully reconstituted a two enzyme system that uses the deazaflavin cofactor F420 to reduce the drug in vitro. In doing so we showed that this unusual enzyme was a nitroreductase and discovered the biochemical function of an entire class of enzymes that is broadly distributed throughout many bacterial species. The mechanism of action of this agent involves release of nitric oxide and subsequent internal poisoning of cellular functions. This work suggests the possibility of structure-based optimization of NO-releasing prodrugs and represents an entirely new paradigm for antibacterial development. A manuscript describing this work has just been accepted for publication in Science.? ? The third major focus of this project involves global approaches to the elucidation of metabolic steps essential for NR-TB survival and includes techniques such as metabolomic analysis of fermertor-grown bacteria under non-replicating conditions as well as high-throughput screening of compounds under NR conditions to look for other lead or tool compounds from conventional chemical libraries. Such libraries have in the past proven to be poor sources for antibacterial lead molecules 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. Mycobacteria present in this environment face a very similar set of conditions to those within the diseased lung of a human TB patient. Competitor bacteria from the core of such bogs have been identified and characterized and some produce compounds of remarkable potency against NR-TB.
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