Defining the Mechanism of Action and Resistance of New <i>Mycobacterium abscessus</i> MmpL3 Inhibitors

<i>Mycobacterium abscessus</i> (Mab) is difficult to treat due to intrinsic and acquired resistance to diverse antibiotics. Among the intrinsic resistance factors is the mycomembrane, a complex structure that limits permeability to several classes of antibiotics. Here, we report new inhibitors of MmpL3, an essential transporter required to build the mycomembrane. Several of the MmpL3 inhibitors have comparable activity in vitro to standard-of-care treatments, exhibit both time- and dose-dependent bactericidal activity, have low eukaryotic cytotoxicity, and are efficacious against Mab growing in macrophages or in biofilms. The inhibitors had varying activities against a panel of 30 different multidrug-resistant clinical isolates and are additive or synergistic with standard-of-care antibiotics, suggesting they could be included in combination therapy. The inhibitors also exhibit a low frequency of resistance, with some of the isolated mutants displaying differential patterns of sensitivity and resistance to the different MmpL3 inhibitors and putative fitness defects. Cross-resistance profiles of 15 structurally related inhibitors against 16 different MmpL3 resistant mutants demonstrate structure-driven clustering patterns of the inhibitors, where those carrying a similar scaffold cluster together and different MmpL3 amino-acid substitutions account for these differences. Cross-resistance profiles were also simulated computationally, showing significant correlation between the computationally calculated parameters and the biological patterns of cross-resistance and emphasizing specific structural-functional associations driving resistance or susceptibility. These inhibitors and their analogs hold promise for clinical translation, and the established structural-functional associations provide mechanistic insights into the function of MmpL3, resistance and susceptibility of MmpL3 inhibitors, and fitness costs associated with MmpL3 resistance.