sq-109 and trehalose-monomycolate

The mycolic acid transporter MmpL3 is driven by proton motive forces (PMF) and functions via an antiport mechanism. Although the crystal structures of the

Topics: Biological Transport; Membrane Transport Proteins; Mycobacterium tuberculosis; Protons

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

SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of Mycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates. In vitro assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential mmpL3 gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.

Topics: Acyltransferases; Adamantane; Aerobiosis; Antigens, Bacterial; Antitubercular Agents; Bacterial Proteins; Cell Wall; Chromatography, Thin Layer; Cord Factors; Drug Resistance, Bacterial; Ethylenediamines; Lipid Metabolism; Membrane Transport Proteins; Microbial Sensitivity Tests; Microscopy, Electron; Mutation; Mycobacterium tuberculosis; Mycolic Acids