Do Mutations in Mycobacterial ATP Synthase Undermine Bedaquiline Binding? Insights from Multiscale Molecular Simulations

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (MTB), remains a major global health problem. Current antitubercular drugs, used for the treatment of TB, pose a significant challenge in treating Multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. The recently developed drug Bedaquiline (BDQ) is used for MDR-TB and works by inhibiting the rotation of the ATP synthase enzyme, disrupting energy production, ultimately leading to bacterial cell death. BDQ binds to multiple sites within the ATP synthase, including the lagging, leading, and c-only sites; however, resistance has emerged due to mutations in the enzyme, and the underlying molecular mechanisms remain unclear. In this study, we performed all-atom molecular dynamics, umbrella sampling, and random accelerated molecular dynamics simulations to investigate the structural and energetic effects of clinically relevant mutations associated with BDQ resistance. The total binding free energies of BDQ estimated in wild-type (WT), E61D, and A63P mutants were -47.90, -38.90, and -37.12 kcal/mol, respectively, indicating that single-point mutations can reduce the total binding free energy by at least 9 kcal/mol, with the E61D and A63P mutants exhibiting the greatest loss of binding affinity for BDQ. Umbrella sampling further indicated that D28G and A63P mutants significantly lower the free-energy barrier compared to WT. Residence-time analyses further identify D28G, E61D, and A63P as key mutations that markedly reduce BDQ efficacy. Overall, these computational studies provide molecular-level insights into the mechanisms of BDQ resistance, which can aid in the design of novel inhibitors for MDR-TB treatment.