Proton transfer activity of the reconstituted Mycobacterium tuberculosis MmpL3 is modulated by substrate mimics and inhibitors.

Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.

Direct Inhibition of MmpL3 by Novel Antitubercular Compounds.

MmpL3, an essential transporter involved in the export of mycolic acids, is the proposed target of a number of antimycobacterial inhibitors under development. Whether MmpL3 serves as the direct target of these compounds, however, has been called into question after the discovery that some of them dissipated the proton motive force from which MmpL transporters derive their energy. Using a combination of in vitro and whole-cell-based approaches, we here provide evidence that five structurally distinct MmpL3 inhibitor series, three of which impact proton motive force in Mycobacterium tuberculosis, directly interact with MmpL3. Medium- to high-throughput assays based on these approaches were developed to facilitate the future screening and optimization of MmpL3 inhibitors. The promiscuity of MmpL3 as a drug target and the mechanisms through which missense mutations located in a transmembrane region of this transporter may confer cross-resistance to a variety of chemical scaffolds are discussed in light of the exquisite vulnerability of MmpL3, its apparent mechanisms of interaction with inhibitors, and evidence of conformational changes induced both by the inhibitors and one of the most commonly identified resistance mutations in MmpL3.

MmpL3 as a Target for the Treatment of Drug-Resistant Nontuberculous Mycobacterial Infections.

Nontuberculous mycobacterial (NTM) pulmonary infections are emerging as a global health problem and pose a threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis. Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) species account for 70-95% of the pulmonary NTM infections worldwide. Treatment options for these pathogens are limited, involve lengthy multidrug regimens of 12-18 months with parenteral and oral drugs, and their outcome is often suboptimal. Development of new drugs and improved regimens to treat NTM infections are thus greatly needed. In the last 2 years, the screening of compound libraries against M. abscessus in culture has led to the discovery of a number of different chemotypes that target MmpL3, an essential inner membrane transporter involved in the export of the building blocks of the outer membrane of all mycobacteria known as the mycolic acids. This perspective reflects on the therapeutic potential of MmpL3 in Mycobacterium tuberculosis and NTM and the possible reasons underlying the outstanding promiscuity of this target. It further analyzes the physiological and structural factors that may account for the apparent looser structure-activity relationship of some of these compound series against M. tuberculosis compared to NTM.