Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem worldwide and the most common presenting illness among people living with HIV, including those who are taking antiretroviral treatment. TB is also the leading cause of death among people living with HIV, accounting for one in four HIV/AIDS-associated deaths. The development of shorter and simpler drug regimens that are well tolerated, effective against multiple and extensively drug-resistant (MDR/XDR) Mtb and appropriate for joint TB/HIV treatment is urgently needed. The return of interest in whole cell-based screens to identify novel anti-TB compound has led to the identification of a number of promising inhibitors. Intriguingly, several of them including the TB drug candidate SQ109 have been reported to kill Mtb through their inhibitory activity on the mycolic acid transporter, MmpL3. Mycolic acids are abundant long chain (C60-C90) fatty acids synthesized in the cytoplasm that populate both the inner and the outer leaflets of the outer membrane of all mycobacteria; by preventing mycolic acids from being exported to the periplasmic space, the inhibition of MmpL3 prevents the formation of the outer membrane of Mtb. The fact that multiple chemical scaffolds block the growth of Mtb through the apparent inhibition of MmpL3 may reflect the unusual vulnerability and druggability of this transporter. Alternatively, our preliminary results have led us to question the direct mechanism of inhibition of MmpL3 by some of these compounds. Thus, in spite of the growing interest in MmpL3 as a novel therapeutic target and owing to the technical challenges associated with the development of transport assays for this large membrane protein, MmpL3 has not yet been validated as the direct target of any Mtb inhibitor. The critical need for novel therapeutic approaches to treat MDR/XDR-Mtb infections and the importance of understanding the mode of action of small molecule inhibitors to drive their optimization process make the validation of MmpL3 as a novel drug target and the development of inhibition assays for this transporter a high priority. This proposal is to establish the therapeutic potential of MmpL3 by determining its vulnerability (i.e., how much inhibition of the target's activity is required to block bacterial growth) in vitro and in vivo (Aim 1), to develop the whole cell-based and cell-free assays required for the screening, validation and optimization of inhibitors of this transporter (Aim 2) and to provide proof-of-concept of its druggability by small molecule inhibitors (Aim 3).