Isolation of Mycobacterium tuberculosis mutants resistant to CWHM-728

Tuberculosis, caused by Mycobacterium tuberculosis, is a global health threat. The rise in antibiotic resistant M. tuberculosis isolates demonstrates a clear need for new antibiotics with novel mechanisms of action. To this end, we performed a screen for new compounds that inhibit M. tuberculosis, and identified a new series of anti-mycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines. To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine, named CWHM-728, and performed whole genome sequencing on four of these strains to identify the mutations that confer resistance. We found that each mutant harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex.