MmpL3 as a target for novel anti-TB agents

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Summary Mycolic acids are essential building blocks of the outer membrane of all mycobacteria and the interest in drugs targeting their biosynthesis and export is clearly illustrated by the therapeutic efficacy of several past and present anti-mycobacterial agents including isoniazid, ethionamide, thiacetazone and isoxyl, and a number of recently discovered small molecule inhibitors reported to inhibit the integral membrane mycolic acid transporter, MmpL3. Joint efforts by our Consortium prior to and during the previous funding period have led to the discovery of MmpL3 and to the validation of this transporter as a target of therapeutic interest. These studies further allowed us to gain the first insights into its structure, energetics and mechanisms of susceptibility and resistance to inhibitors. Despite these advances, important gaps still remain in our understanding of the translocation of mycolic acids from the cytoplasm to the periplasm and outer membrane, the precise role played by MmpL3 in this process, and the molecular mechanisms involved. Several lines of evidence indicate that the export of mycolic acids to the cell surface involves more components than the only MmpL3 protein, and that MmpL3 may act as a scaffold for a multiprotein complex coupling synthesis and export. Our preliminary evidence further indicates that MmpL3 may play a role in coordinating, at the level of the plasma membrane, the focal deposition of cell wall core constituents during cell elongation and cell division. Aim 1 of this basic research proposal is to define the nature of the MmpL3-associated machinery responsible for the efficient synthesis and export of mycolic acids in Mycobacterium tuberculosis, and to probe the involvement of MmpL3 in the spatiotemporal coordination of new cell wall deposition during cell elongation and division. Under Aim 2, a detailed biochemical examination of MmpL3 will be conducted to delineate the precise extent of mycolic acid transport mediated by MmpL3 in mycobacterial cells and to gain insight in the molecular mechanisms involved. The assays developed under this aim will further be used to probe the precise mechanism of action of MmpL3 inhibitors and associated mechanisms of resistance. In addition to providing fundamental knowledge about essential physiological processes of all mycobacteria, the results from these studies will facilitate current drug development efforts targeted to MmpL3 and could further lead to innovative therapeutic strategies targeting key metabolic processes at the intersection of cell envelope biosynthesis and cell elongation and division.