The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants resistant to BM212 were isolated. By screening of genomic libraries and by whole genome sequencing we found that all the characterized mutants show mutations in the mmpL3 gene allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (Mycobacterial membrane protein, Large) family. Susceptibility is unaffected by the efﬂux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrate that resistance is not driven by efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.
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