Vanoxerine kills mycobacteria through membrane depolarization and efflux inhibition.

is a deadly pathogen, currently the leading cause of death worldwide from a single infectious agent through tuberculosis infections. If the End TB 2030 strategy is to be achieved, additional drugs need to be identified and made available to supplement the current treatment regimen. In addition, drug resistance is a growing issue, leading to significantly lower treatment success rates, necessitating further drug development. Vanoxerine (GBR12909), a dopamine re-uptake inhibitor, was recently identified as having anti-mycobacterial activity during a drug repurposing screening effort. However, its effects on mycobacteria were not well characterized. Herein, we report vanoxerine as a disruptor of the membrane electric potential, inhibiting mycobacterial efflux and growth. Vanoxerine had an undetectable level of resistance, highlighting the lack of a protein target. This study suggests a mechanism of action for vanoxerine, which will allow for its continued development or use as a tool compound.