bm-212 and trehalose-monomycolate

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of

Topics: Bacterial Proteins; Cord Factors; Lipid Metabolism; Membrane Proteins; Mycobacterium smegmatis; Mycolic Acids; Piperazines; Pyrroles; Spheroplasts

MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.

Topics: Adamantane; Anti-Bacterial Agents; Antitubercular Agents; Bacterial Proteins; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Cell Membrane; Cord Factors; Drug Resistance, Multiple, Bacterial; Ethylenediamines; Membrane Proteins; Membrane Transport Proteins; Microbial Sensitivity Tests; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acids; Phenylurea Compounds; Piperazines; Proton Ionophores; Pyrroles; Tuberculosis, Multidrug-Resistant; Valinomycin; Vitamin K 2