This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-AI-102*: Develop diagnostics and drugs for multiple or extensively drug-resistant tuberculosis (MDR/XDR TB). Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB) in humans, currently infects one-third of the world's population in its latent form. The emergence of multi-drug- and extremely drug- resistant (MDR/XDR) strains has highlighted the need for new drugs with bactericidal mechanisms different from those of presently available agents and capable of reducing treatment duration. We have identified a series of urea derivatives showing potent bactericidal activity on M. tb in vitro with MICs in the 0.1 1/4M range. These inhibitors appear to be specific to mycobacteria and to show best activity against M. tb, including MDR strains. Preliminary studies on their mode of action indicate that they inhibit in M. tb the synthesis of oxygenated mycolates, trehalose dimycolates (TDM) and phosphatidylinositol mannosides (PIM), all essential components of the mycobacterial cell envelope. These various effects seem to relate, at least in part, to their ability to inhibit in vitro multiple structurally-related ?/?-hydrolase fold enzymes. Their unique effects on the synthesis of mycolic acids and derived lipids suggest that their targets in these pathways differ from those of other anti-TB agents such as isoniazid, ethionamide, thiacetazone and Isoxyl. Likewise, although the importance of PIM in the immunopathogenesis of TB and as essential structural components of the mycobacterial cell envelope has been well documented, to our knowledge, no specific inhibitors of this biosynthetic pathway have yet been identified. Given the important impact of PIM content on the permeability of the cell envelope and susceptibility of M. tb to drugs, we expect inhibitors targeting PIM synthesis to not only be bactericidal to M. tb but also potentiate the effect of other drugs used concomitantly in the treatment of the disease, facilitating their entry inside mycobacterial cells. This could in turn result in the shortening of treatment duration. Based on their potent antimycobacterial activity, novelty of their mode of action and potential to reduce treatment duration, we propose to further develop this family of compounds with the explicit goals of identifying inhibitors efficacious in mice and delineating which aspect of their mode of action accounts for their bactericidal activity. Derivatives of existing products will be synthesized and their MIC/MBC values, toxicity, and bioavailability parameters determined. The most promising compounds, including already synthesized ones, will be tested for efficacy in a mouse model of TB infection. Toward the elucidation of their mode of action, we will assess and compare the macromolecular effects of two prototype urea derivatives on wild-type strains and urea-resistant mutants of M. tb and M. smegmatis that we have isolated. The mutations responsible for conferring urea-resistance in these mutants will be identified and their direct involvement in drug resistance validated using genetic approaches. Finally, in vitro assays will be developed for the most relevant enzymatic targets of this family of inhibitors so as to provide strong bases for the future rational design of optimized compounds.
Novel drugs with bactericidal mechanisms different from those of presently available agents, capable of killing MDR/XDR Mycobacterium tuberculosis and reducing treatment duration are urgently needed. We propose to develop a series of urea-based inhibitors efficacious in mice and to determine the fundamentals of their mode of action.
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