Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis

Abstract

dMmpL3, 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.

Antiinfectives targeting enzymes and the proton motive force

“…The detailed molecular mechanism of action of the neutral uncouplers is not known but has been proposed to be related to either conformational changes in membrane proteins or changes in phospholipid bilayer organization (57). We find that AU1235 (9) collapses the pH gradient in the EcIMV assay, consistent with the NMR observations (20). In addition, we find that N 5 ,N 6 -bis(2-fluorophenyl)- [1,2,5]oxadiazolo [3,4-b]pyrazine-5,6-diamine (BAM15; 32), a mitochondrial uncoupler that does not depolarize the plasma membrane (59), also acts as a (neutral) uncoupler in the EcIMV assay.…”

“…It thus seems likely that clofazimine (17), as well as TMC207 (1), can act, at least in part, in a similar manner to the potent anionic protonophores, such as carbonyl cyanide m-chlorophenyl hydrazone Table 1. Uncoupling effects, computed molecular properties, and M. tuberculosis cell growth inhibition for compounds investigated (CCCP) (20), which has an EC 50 of ∼400 nM in this assay, about the same as clofazimine (17). We found similar results with both Escherichia coli IMVs (EcIMVs) and Mycobacterium smegmatis IMVs (MsIMVs) ( Table 1), so it is unlikely that there are specific drug-membrane interactions with the mycobacterial inner membrane lipids.…”

“…More recently, the analog 1-(2-adamantyl)-3-(2,3,4-trifluorophenyl)urea [AU1235 (9)] has been found to kill replicating but not nonreplicating M. tuberculosis (58) and to collapse the pH gradient in M. smegmatis (20). The detailed molecular mechanism of action of the neutral uncouplers is not known but has been proposed to be related to either conformational changes in membrane proteins or changes in phospholipid bilayer organization (57).…”

“…Amiodarone (4) and dronedarone (5) had little uncoupling activity against host cells. In related work, Li et al (20) found that other TB drug leads, BM212 (6), THPP-2 (7), Ro 48-8071 (8), the urea AU1235 (9), and the indolecarboxamide 2418 (10), most of which had been proposed to target MmpL3, likewise had activity as uncouplers, collapsing pH gradients, and in some cases were active against the Significance Uncoupling agents might be expected to be generally cytotoxic, but many US Food and Drug Administration (FDA)-approved drugs do have activity as uncouplers, in addition to targeting enzymes. There is therefore interest in the discovery of antibiotics that have such multitarget activity.…”

“…Several of these compounds also have enzyme targets. For example, SQ109 (2), ethanolamine (3), and Ro 48-8071 (8) have been found (13,20) to inhibit enzymes involved in menaquinone biosynthesis, particularly the prenyl transferase 1,4-dihydroxy-2-naphthoate octaprenyltransferase (MenA) and human oxidosqualene cyclase (OSC) (21), and bedaquiline (1) is a potent ATP synthase inhibitor, indicating the possibility of multitarget activity for such compounds. These results are of interest because they show that several recently discovered M. tuberculosis drug leads can act as uncouplers in addition to targeting one or more enzymes that are essential for bacterial cell growth, with membrane targeting being of particular interest because it might be expected to be less susceptible to the development of resistance than is purely enzyme targeting, and SQ109 (2) does indeed have a low frequency of resistance in M. tuberculosis (∼2.55 × 10 −11 ) (22).…”