Abstract
ABSTRACTMycobacterium tuberculosis 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 antituberculosis (anti-TB) drugs in vitro and in vivo. This potentiation is widely attributed to inhibition of the efflux pumps of M. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with its in vitro potentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activities in vitro using inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating “persister” M. tuberculosis that was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin 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 anti-TB drugs in vitro and in vivo and highlight a previously unrecognized role for the membrane of M. tuberculosis as a pharmacologic target.
Highlights
Mycobacterium tuberculosis kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance
We found a dose-proportional increase in the levels of both sigE and clgR transcripts following exposure of growing cultures to verapamil for 1 h at concentrations similar to those required for M. tuberculosis growth inhibition and killing of nonreplicating M. tuberculosis bacteria
Similar results were obtained in two independent assays, excluding the possible presence of technical artifacts in the absence of a suitable positive control with demonstrated and direct efflux pump inhibitory activity in M. tuberculosis
Summary
Mycobacterium tuberculosis kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. An FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugs in vitro and in vivo This potentiation is widely attributed to inhibition of the efflux pumps of M. tuberculosis, resulting in intrabacterial drug accumulation. Consistent with its in vitro potentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. Addition of verapamil to the first-line drug regimen accelerated the sterilization of infected lungs [19] These observations suggested that verapamil could potentiate anti-TB drugs in the clinic [15], leading to the initiation of at least one clinical trial (Annual Report of the National Institute for Research in Tuberculosis; http://www .nirt.res.in)
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