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

Abstract

Chemistry campaigns identified amphiphilic indolyl Mannich bases as novel membrane-permeabilizing antimycobacterials. Spiroketal analogs of this series showed increased potency, and the lead compound 1 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 2 lacking the spiroketal moiety. In contrast, mutations resistant against spiroketal analog 1 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 1 may be due to MmpL3 inhibition acquired by the addition of the spiroketal moiety. Here we showed that 1 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 1 ostensibly augmented its interaction with MmpL3, an outcome not observed in the nonspiroketal analog 2, which displayed no cross-resistance to mmpL3 mutants, dissipated the PMF, and docked poorly in the MmpL3 binding pocket. Surprisingly, 2 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 1 is linked to the acquisition of a second mechanism, MmpL3 inhibition. In contrast, the nonspiroketal analog 2 acts pleiotropically, affecting several cell membrane-embedded targets, including MmpL3, through its membrane permeabilizing and depolarizing effects.

Resistance against Membrane-Inserting MmpL3 Inhibitor through Upregulation of MmpL5 in Mycobacterium tuberculosis

“…Incorporation of a spiroketal moiety in the Mannich base caused a 10-fold increase in potency (4). Interestingly, mutants resistant to the spiroketal analogs could be isolated and mapped to the mycolic acid transporter MmpL3 (5). Biochemical, metabolic, computational and structure-activity-relationship analyses revealed that the potency improvement was caused by the acquisition of a second mechanism of action due to the inclusion of the spiroketal moiety (5).…”

“…Interestingly, mutants resistant to the spiroketal analogs could be isolated and mapped to the mycolic acid transporter MmpL3 (5). Biochemical, metabolic, computational and structure-activity-relationship analyses revealed that the potency improvement was caused by the acquisition of a second mechanism of action due to the inclusion of the spiroketal moiety (5). In addition to permeabilizing the membrane, spiroketal analogs of the indolyl Mannich bases (SIMBs) inhibit the flippase activity of the transmembrane MmpL3 protein and hence the transport of mycolic acids from the cytoplasm to the periplasmic space (5).…”

“…Biochemical, metabolic, computational and structure-activity-relationship analyses revealed that the potency improvement was caused by the acquisition of a second mechanism of action due to the inclusion of the spiroketal moiety (5). In addition to permeabilizing the membrane, spiroketal analogs of the indolyl Mannich bases (SIMBs) inhibit the flippase activity of the transmembrane MmpL3 protein and hence the transport of mycolic acids from the cytoplasm to the periplasmic space (5). Thus, SIMBs are novel dual-mechanism antibacterials, disrupting integrity of the bacterial cell membrane and blocking the transport of an essential cell wall component by inhibiting a transmembrane transporter (5).…”

“…In addition to permeabilizing the membrane, spiroketal analogs of the indolyl Mannich bases (SIMBs) inhibit the flippase activity of the transmembrane MmpL3 protein and hence the transport of mycolic acids from the cytoplasm to the periplasmic space (5). Thus, SIMBs are novel dual-mechanism antibacterials, disrupting integrity of the bacterial cell membrane and blocking the transport of an essential cell wall component by inhibiting a transmembrane transporter (5). Consistent with this dual mechanism, missense mutations at the binding site of SIMBs on MmpL3 reverted the 10-fold potency increase achieved by the addition of the spiroketal moiety (MIC 90 = 1 µM) back to that observed for non-spiroketal Mannich bases (MIC 90 = ~10 µM) which act only by disrupting membrane integrity (5).…”

“…Thus, SIMBs are novel dual-mechanism antibacterials, disrupting integrity of the bacterial cell membrane and blocking the transport of an essential cell wall component by inhibiting a transmembrane transporter (5). Consistent with this dual mechanism, missense mutations at the binding site of SIMBs on MmpL3 reverted the 10-fold potency increase achieved by the addition of the spiroketal moiety (MIC 90 = 1 µM) back to that observed for non-spiroketal Mannich bases (MIC 90 = ~10 µM) which act only by disrupting membrane integrity (5). Thus, the membranepermeabilizing mechanism endowed by the amphiphilic indolyl Mannich base scaffold of SIMBs ensures that these compounds retain appreciable activity even after bacteria have acquired resistance to the second, MmpL3-related mechanism (5).…”