Rigorous Absolute Binding Free Energy Calculations Elucidate Mutation–Driven Resistance Mechanisms of Non–Covalent Bruton’s Tyrosine Kinase (BTK) Inhibitors

Abstract

Abstract Drug resistance driven by acquired mutations in Bruton’s tyrosine kinase (BTK) threatens the long–term clinical efficacy of non–covalent BTK inhibitors (NCIs). While clinically observed variants such as A428D, C481S, and the double mutant C481S/T474I display distinct resistance profiles, their differential impact on inhibitor recognition remains poorly characterized at the atomic level. In this study, we employed rigorous alchemical absolute binding free energy calculations to quantify the thermodynamic consequences of these mutations across five chemically diverse NCIs. Benchmarking against wild–type experimental binding free energy values demonstrates that the Binding Free Energy Estimator 2 (BFEE2) framework yields highly accurate and robust predictions, significantly outperforming end–point methods like MM–PBSA. Subsequent structural and interaction fingerprint analyses reveal a distinct hierarchy of resistance mechanisms. The A428D mutation triggers widespread electrostatic and hydrophobic disruption near the hinge region, driving severe, broad–spectrum affinity loss. In contrast, the C481S substitution largely preserves the active site architecture, allowing NCIs to maintain full native potency. Finally, the C481S/T474I double mutant introduces localized steric clashes at the gatekeeper position, resulting in moderate, inhibitor–specific binding penalties. Together, these findings establish a robust thermodynamic framework for predicting mutation–induced resistance and offer actionable structural principles for the rational design of next–generation, mutation–resilient NCIs.