Abstract
Understanding competitive inhibition at the molecular level is essential for unraveling the dynamics of enzyme-inhibitor interactions and predicting the evolutionary outcomes of resistance mutations. In this study, we present a framework linking competitive inhibition to alchemical free energy perturbation (FEP) calculations, focusing on Escherichia coli dihydrofolate reductase (DHFR) and its inhibition by trimethoprim (TMP). Using thermodynamic cycles, we relate experimentally measured binding constants (K(i) and K(m)) to free energy differences associated with wild-type and mutant forms of DHFR with a mean error of 0.9 kcal/mol, providing insight into the molecular underpinnings of TMP resistance. Our findings highlight the importance of local conformational dynamics in competitive inhibition. Mutations in DHFR affect substrate and inhibitor binding affinities differently, influencing the fitness landscape under selective pressure from TMP. Our FEP simulations reveal that resistance mutations stabilize inhibitor-bound or substrate-bound states through specific structural and/or dynamical effects. The interplay of these effects showcases significant molecular-level epistasis in certain cases. The ability to separately assess substrate and inhibitor binding provides valuable insights, allowing for a more precise interpretation of mutation effects and epistatic interactions. Furthermore, we identify key challenges in FEP simulations, including convergence issues arising from charge-changing mutations and long-range allosteric effects. By integrating computational and experimental data, we provide an effective approach for predicting the functional impact of resistance mutations and their contributions to evolutionary fitness landscapes. These insights pave the way for constructing robust mutational scanning protocols and designing more effective therapeutic strategies against resistant bacterial strains.