MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212

Abstract

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 that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed 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 was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [(14)C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

Fig 1

Chemical structure of BM212. Me, methyl.

Fig 2

Predicted topology of MmpL3 from M. smegmatis (MSMEG_0250) (A) and M. bovis BCG (BCG0243c) (B). The arrows indicate the amino acid substitutions observed for M. smegmatis (A) (see also Table 2) and M. bovis (B) (see also Table 3) mutants resistant to BM212. The amino acid substitution observed for the only resistant M. tuberculosis mutant is highlighted in gray. The MEMSAT2 program was used to predict topology (11).

Fig 3

[¹⁴C]BM212 accumulation by M. smegmatis wild-type and M. smegmatis MSMEG-GR12.5 cells. [¹⁴C]BM212 was added to the cells at time zero. The results are the averages of data from three experiments, and error bars indicate standard deviations.