Mycobacterium tuberculosis (Mtb) kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several anti-tuberculosis (TB) drugs in vitro and in vivo. This potentiation is widely attributed to inhibition of Mtb's efflux pumps, resulting in intrabacterial drug accumulation. Here, we confirm and quantify verapamil's synergy with several anti-TB drugs, including bedaquiline and clofazimine, but find that this effect is not due to increased intrabacterial drug accumulation. Consistent with its in vitro potentiating effects on TB drugs that target or require oxidative phosphorylation, we show that the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response, similar to other membrane-active agents. We recapitulate these activities in vitro using inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. Consistent with such mechanism of action, we observe bactericidal activity against non-replicating 'persister' Mtb. In addition, we demonstrate a pharmacokinetic interaction whereby human-equivalent doses of verapamil cause a boost of rifampicin exposure in mice, providing a potential explanation for the observed treatment shortening effect of verapamil in mice receiving first line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of TB drugs in vitro and in vivo, and highlight a previously unrecognized role for Mtb's membrane as pharmacologic target.
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