Potency Increase of Spiroketal Analogs of Membrane Inserting Indolyl Mannich Base Antimycobacterials Is Due to Acquisition of MmpL3 Inhibition

Chemistry campaigns identified amphiphilic indolyl Mannich bases as novel membrane-permeabilizing antimycobacterials. Spiroketal analogs of this series showed increased potency, and the lead compound displayed efficacy in a mouse model of tuberculosis. Yet the mechanism by which the spiroketal moiety accomplished the potency "jump" remained unknown. Consistent with its membrane-permeabilizing mechanism, no resistant mutants could be isolated against indolyl Mannich base lacking the spiroketal moiety. In contrast, mutations resistant against spiroketal analog were obtained in mycobacterial membrane protein large 3 (MmpL3), a proton motive force (PMF)-dependent mycolate transporter. Thus, we hypothesized that the potency jump observed for may be due to MmpL3 inhibition acquired by the addition of the spiroketal moiety. Here we showed that inhibited MmpL3 flippase activity without loss of the PMF, colocalized with MmpL3tb-GFP in intact organisms, and yielded a consistent docking pose within the "common inhibitor binding pocket" of MmpL3. The presence of the spiroketal motif in ostensibly augmented its interaction with MmpL3, an outcome not observed in the nonspiroketal analog , which displayed no cross-resistance to mutants, dissipated the PMF, and docked poorly in the MmpL3 binding pocket. Surprisingly, inhibited MmpL3 flippase activity, which may be an epiphenomenon arising from its wider membrane disruptive effects. Hence, we conclude that the potency increase associated with the spiroketal analog is linked to the acquisition of a second mechanism, MmpL3 inhibition. In contrast, the nonspiroketal analog acts pleiotropically, affecting several cell membrane-embedded targets, including MmpL3, through its membrane permeabilizing and depolarizing effects.