a, Dose–response analysis of BRD-8000 on the growth of wild-type Mtb, the EfpA hypomorph, and a mutant overexpressing EfpA (pUV15::efpA), demonstrating hypersensitivity of the hypomorph. Independent biological replicates (n = 4) are shown as open circles; means are shown as filled circles; error bars denote 95% confidence intervals. b, BRD-8000.2 is bactericidal as demonstrated by reducing colony-forming units over time. Independent biological replicates (n = 8) are shown as open circles; means are shown as filled circles; error bars denote 95% confidence intervals. c, Toxicity and bioavailability measurements of BRD-8000.2 and BRD-8000.3. hERG, human ether a-go-go related gene. d, Cytochrome P450 (CYP) inhibition measurements of BRD-8000.2 and BRD-8000.3. e, Schematic of the EtBr efflux assay. Bacteria were loaded with EtBr and its efflux was monitored by change in fluorescence. f, Example kinetic time courses of EtBr fluorescence decay for Msm with BRD-8000.3. The concentration of EtBr used for pre-incubation is indicated in colour, with two different inhibitor concentrations shown. Numerically integrated Michaelis–Menten best-fit time courses are shown in red. Experiments were repeated independently once with similar results. g, Kinetic time course of EtBr fluorescence decay for Mtb. Numerically integrated Michaelis–Menten best-fit time courses are shown in red. The table shows best-fit Michaelis–Menten parameters and Fick’s diffusion constant for wild-type Mtb, an EfpA-overexpressor (pUV15::efpA), and the BRD-8000 resistant mutant (efpAV319F). Although the in vivo apparent maximal efflux rate (V max ) of the EfpA overexpressor is higher than the wild-type Mtb, that of the BRD-8000 resistant mutant is not, indicating that the resistant mutant is not hyperactive for efflux. Experiments were repeated independently once with similar results. h, Dose–response analysis of isoniazid against wild-type Mtb and the BRD-8000 resistant mutant (efpAV319F). Because isoniazid is a substrate of EfpA, no shift in the MIC 90 value for isoniazid with the BRD-8000 resistant mutant indicates that EfpA(V319F) is not hyperactive for efflux. Mean growth (n = 4 biologically independent replicates) is shown, with error bars indicating 95% confidence intervals. i, Results of an assay for intracellular accumulation of BRD-8000.3 in MsmΔefpA complemented with either Mtb wild-type efpA or efpAV319F. Fluorescence of BRD-8000.3 in bacterial lysates was measured, and lysate background fluorescence was subtracted. Although verapamil, a general efflux pump inhibitor, caused statistically significant intracellular accumulation of BRD-8000.3, there was no significant difference in accumulation between the different strains in the absence of verapamil, indicating that EfpA(V319F) is not hyperactive for efflux, and that BRD-8000.3 is not a substrate of EfpA. Independent biological replicates (n = 9 for wild-type control without verapamil; n = 3 for other conditions) are shown as open circles; means are shown as filled circles; error bars denote s.e.m. j, Additional compounds predicted and validated to be efflux inhibitors. k, Dose–response analysis of kinetic time courses of EtBr fluorescence decay for compounds in h. Increasing inhibitor concentration is denoted by colour. Experiments were repeated independently once with similar results. l, Example kinetic time courses of EtBr fluorescence decay for Msm at one concentration (6 µM) of BRD-9327. Numerically integrated Michaelis–Menten best-fit time courses are shown in red. The table shows global best-fit kinetic inhibition parameters across ten concentrations for this inhibitor. Experiments were repeated independently once with similar results. Source data