In this competing continuation, mycobacterial cell wall metabolism is targeted as a route to new tuberculosis drugs. In one approach, essential cell wall biosynthetic enzymes are targetted. It is proposed to prepare M. tuberculosis luciferase reporter strains that respond specifically to inhibitors of the following essential enzymes: rhamnosyl transferase (WbbL), galactofuranosyl transferase (GlfT), dTDP-Rha formation enzymes (RmIB-D), and the UDP-Galf formation enzyme (Glf). In addition, direct enzyme assays amenable to screening for inhibitors will be developed for WbbL and GlfT (such assays are already in place for RmIB-D and Glf). It is further proposed to identify the genes encoding the three enzymes that form the arabinosyl donor, decaprenylphosphoryl-D-arabinose, show that their corresponding enzymes are essential, and prepare M. tuberculosis luciferase reporter strains and enzyme assays for use in finding inhibitors of them. In the second approach it is hypothesized that compounds that activate or deregulate the cell wall degrading arabinases and peptidoglycan hydrolyases can act more quickly than inhibition based drugs and thereby shorten tuberculosis therapy. Thus newly discovered M. tuberculosis endogenous arabinanases (of unknown function) that cleave cell wall arabinan will be purified and their genes identified. The effect of overexpressing these enzymes in M. tuberculosis will be tested; it is hoped the effect will be lethal. Reporter strains of M. tuberculosis to detect small molecules that up regulate the expression of the arabinanases will be prepared. The presence of a cell wall metabolic enzyme complex containing these arabinases (which can potentially be disrupted for therapy) will be searched for using a novel cross-linking reagent, cyanogen complexes of peptidoglycan synthetic and degradative enzymes, after covalent crosslinking with cyanogen, will be isolated. The identity of all proteins present in the complex will be determined by LC/MS and how individual proteins interact in the complex will be determined for the long-term goal of disrupting the complex to release the peptidoglycan cleaving enzymes. We will test M. tuberculosis for increased arabinase activity after exposure to novel highly active ethambutol like compounds (the presence of endogenous arabinase was first recognized during ethambutol treatment). Finally, large numbers of novel Betalactams will be tested for their action against M. tuberculosis with and without ethambutol like compounds anticipating that some will strongly stimulate cell wail degradation and bacterial death.
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