Delineating the Mechanism of Action of the Repurposed Drug Benzatropine against Mycobacterium tuberculosis

In this study, we have performed luminescence-based screening assays and identified benzatropine, an FDA-approved drug that inhibits the intracellular growth of drug-sensitive and drug-resistant M. tuberculosis strains. We demonstrate that oral administration of benzatropine inhibits the growth of M. tuberculosis in aerosol-infected mice. Furthermore, we show that benzatropine enhances the activity of rifampicin and pretomanid against M. tuberculosis in THP-1 macrophages. Detailed analysis of the proteomics data revealed that, in comparison to the untreated and infected macrophages, the relative expression of several autophagy-related proteins was altered in benzatropine-treated and M. tuberculosis-infected macrophages. We demonstrate that exposure to benzatropine reduced phosphorylation of JAK2 and STAT3, increased intracellular ROS and calcium levels, and induced autophagy in macrophages. We further report that preincubation with histamine, wortmannin, and N-acetylcysteine reduced benzatropine’s ability to induce autophagy and inhibit the growth of intracellular bacteria. Also, activation of STAT3 signaling with colivelin and inhibition of AMPK signalling with compound C reduced benzatropine-induced autophagy. Taken together, we have shown that benzatropine, an FDA-approved drug, induces autophagy and inhibits the growth of intracellular drug-sensitive and drug-resistant M. tuberculosis.