The role of the A428D mutation in conferring resistance to pirtobrutinib in Bruton's tyrosine kinase: insights from molecular dynamics simulations

Acquired resistance to non-covalent, reversible Bruton's tyrosine kinase (BTK) inhibitors poses a major therapeutic challenge in B-cell malignancies, yet the underlying biophysical mechanisms remain poorly understood. In this study, we employed comprehensive molecular dynamics (MD) simulations and free energy landscape (FEL) analysis to elucidate how the BTK A428D mutation confers resistance to pirtobrutinib. Our results demonstrate that the A428D mutation destabilizes the kinase domain and increases conformational heterogeneity by driving an inward displacement of the activation loop (A-loop) toward the catalytic cleft, which disrupts the shape complementarity of the binding pocket and elevates the energetic barrier for inhibitor retention. Integrated network analyses including dynamic cross-correlation matrix (DCCM), community coupling, and residue-wise centrality reveal that the A428D mutation impairs the synergistic motions and topological connectivity of the BTK scaffold, thereby disrupting residue-level communication and preventing stable inhibitor-induced conformational convergence. At the atomic level, the introduction of the negatively charged Asp428 disrupts the hydrophobic-polar balance and destabilizes critical hinge-region interactions involving M477 and E475, leading to a significant reduction in binding affinity and a lower energy barrier for drug dissociation, as confirmed by molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and adaptive steered MD (ASMD) simulations. Collectively, our findings establish a mechanistic link between A428D-induced conformational rewiring and clinical resistance, providing a molecular rationale for disease relapse and the design of next-generation BTK inhibitors. • Microsecond simulations show that A428D destabilizes the BTK kinase domain. • The A428D mutation drives A-loop inward displacement to disrupt pocket complementarity. • Integrated MM/PBSA and ASMD reveal reduced binding affinity and lower dissociation barriers. • Network analysis identifies impaired topological connectivity and lost conformational convergence. • Our findings link A428D induced conformational rewiring to clinical pirtobrutinib resistance.