A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3

Abstract

<i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against <i>M. tuberculosis</i> Herein, we show that PIPD1 and related analogs exert <i>in vitro</i> bactericidal activity against the <i>M. tuberculosis</i> strain mc²6230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of <i>M. tuberculosis</i>, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in <i>mmpL3</i>, encoding an inner membrane-associated mycolic acid flippase in <i>M. tuberculosis</i> Results from functional assays with spheroplasts derived from a <i>M. smegmatis</i> strain lacking the endogenous <i>mmpL3</i> gene but harboring the <i>M. tuberculosis mmpL3</i> homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from <i>M. tuberculosis</i>, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in <i>M. smegmatis</i> MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.