Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance
The Mycobacterium tuberculosis (Mtb) mycolic acid flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole genome sequencing of isolated resistant mutants. Here we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel Mtb growth inhibitors for differential growth inhibition of wild type Mtb as compared to a pool of twenty-four unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular Mtb. Mtb cells treated with these compounds were shown to accumulate trehalose monomycolate and have reduced levels of trehalose dimycolate, supporting MmpL3 as the target. The inhibitors were mycobacteria specific with several also showing activity against the non-tuberculosis mycobacterial species M. abscessus. Cluster analysis of cross resistance profiles generated by dose response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small molecule inhibitors.