MmpL3 (Mycobacterium membrane protein Large 3) is a common target of inhibitors of mycobacterial growth identified by whole cell, phenotypic, high throughput screens. MmpL3 is a mycolate flippase that moves trehalose monomycolate (TMM) to the pseudoperiplasmic space, from where TMM is modified to trehalose dimycolate (TDM) and incorporated into the mycomembrane. Mycobacterium tuberculosis (Mtb) and M. smegmatis mmpL3 knockdown strains show that mmpL3 is essential for survival both in vitro and in mice. This phenotype makes MmpL3 an attractive therapeutic target and supports efforts to characterize molecules targeting MmpL3. Multiple MmpL3 inhibitors exhibit synergistic interactions with TB drugs, further supporting interest in this target. Using an innovative combination of untargeted and targeted mutant screens, we have identified ten new and distinct scaffolds that inhibit MmpL3 function. These compounds are bactericidal both in vitro and against intracellular Mtb in primary murine macrophages. The inhibitors are mycobacteria specific with several showing activity against the non-tuberculous mycobacterial (NTM) species M. abscessus (Mab), including the HC2099 and HC2091 series. Pilot structure activity relationship (SAR) studies involving the synthesis of over 100 analogs of HC2099 and HC2091 have identified analogs with whole cell Mtb half-maximal efficacies of ~80 nM and ~280 nM, respectively. Several analogs exhibited high solubility, stability in microsomes and no cytotoxicity in macrophages, supporting their further development. For example, MSU-43085, an analog of HC2099, is orally bioavailable, active against Mtb in an acute murine model of infection and has activity comparable to standard of care drugs against Mab in vitro and in macrophages. Therefore, these series will be valuable tools to understand inhibitor-MmpL3 structure-function interactions and as leads for new TB drug development. Our library of MmpL3 inhibitors and mutants also will enable our team to define mechanisms of resistance in MmpL3. Cluster analysis of cross resistance profiles, generated by dose response experiments for each combination of 13 MmpL3 inhibitors against 24 different mmpL3 mutants, defined two clades of inhibitors and two clades of resistant mutants. Pairwise combination studies of the inhibitors revealed antagonistic, synergistic and additive interactions that were specific to the identified clades. Modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3. These findings support our hypothesis that combinations of MmpL3 inhibitors or rationally designed molecules can be employed to reduce the frequency of resistance. The overall goals of this study are to: 1) optimize new MmpL3 inhibitors to define inhibitor MmpL3 interactions and generate proof-of-concept data showing efficacy in vivo (Aim 1); and 2) define mechanisms of resistance in MmpL3 to devise strategi...