Investigating the unbinding mechanisms and kinetics of MmpL3 inhibitors: A computational study

Abstract Mycobacterial membrane protein Large 3 (MmpL3) is responsible for transporting trehalose monomycolates across the inner membrane for cell wall biosynthesis, a process driven by the proton motive force and essential for the survival of Mycobacterium tuberculosis . As a result, MmpL3 has become a promising target for anti‐tuberculosis drugs. Although many inhibitors targeting MmpL3 have been discovered, their unbinding mechanisms and kinetics remain poorly understood. In this study, the τ‐random acceleration molecular dynamics (τRAMD) and steered molecular dynamics (SMD) methods were employed to investigate the unbinding mechanisms and kinetics of four representative MmpL3 inhibitors: SQ109, AU1235, NITD349, and BM212. Analysis of 320 RAMD dissociation trajectories revealed considerable diversity in the dissociation pathways for these inhibitors, dissociating into intracellular, extracellular, or transmembrane regions. Notably, the H4H5H10 pathway, dissociating to the intracellular region, was the primary route. Also, τRAMD results demonstrated a strong correlation between the computed relative residence times and experimental data. Furthermore, SMD simulations along the H4H5H10 pathway indicated that SQ109, AU1235, and NITD349 disrupted hydrogen bonding with MmpL3 prior to dissociation. Meanwhile, inhibitor BM212 underwent conformational adjustments within the binding pocket. All these inhibitors must traverse the channel formed by Phe255 and Phe644 via the H4H5H10 pathway, necessitating the overcoming of significant energy barriers. Based on these findings, we suggest that enhancing inhibitor interactions with MmpL3, such as through hydrogen bonding or increasing inhibitor size to create larger physical barriers (e.g., interactions with Phe255 and Phe644), may prolong the inhibitors' residence times.