Abstract
The rise of antimicrobial resistance in Mycobacterium tuberculosis underscores the urgent need for novel therapeutic strategies. Mycobacterial membrane protein large 3 (MmpL3) has emerged as a promising drug target, given its essential role in trehalose monomycolate transport and cell-wall biosynthesis. We determined the crystal structure of M. smegmatis MmpL3 in complex with a potent indolecarboxamide inhibitor, UPAR-1109, at 2.15 Å resolution, the highest reported to date. This structure provided unprecedented insights into the binding mode of the inhibitor, highlighting strong polar interactions and extensive hydrophobic contacts, which disrupt proton translocation. Speculative analysis about the molecular mechanism of inhibition has been proposed based on this model. Computational studies, including docking, molecular-dynamics and enhanced sampling simulations, revealed the remarkable plasticity of the MmpL3 binding site and confirmed the crystallographic orientation of UPAR-1109 as the most stable and biologically relevant binding mode. Together, these findings advance our understanding of the function and inhibition of MmpL3, providing valuable information for the rational design of next-generation antituberculosis agents, also with potential applications against nontuberculous mycobacteria.