Precision targeting of BTK in chronic lymphocytic leukemia: computational insights into structural dynamics and the influence of mutations on BTK inhibitors through QM and MM studies

Bruton's tyrosine kinase (BTK) is a central mediator of B-cell receptor signaling and a validated therapeutic target in chronic lymphocytic leukemia (CLL). However, the clinical efficacy of both covalent and non-covalent BTK inhibitors is increasingly undermined by resistance-conferring mutations, particularly at residues C481 and T474. These mutations, alone or in combination, pose a significant challenge to sustained therapeutic response. In this study, integrative quantum mechanical and molecular modeling approaches were employed to investigate the effects of clinically relevant BTK mutations on inhibitor binding. Ten FDA-approved covalent and non-covalent inhibitors were evaluated against four single mutants (C481S, A428D, V416L, and D539G) and four compound mutants (T474A/C481S, T474I/C481S, T474M/C481S, and T474S/C481S). Density functional theory-based local and global reactivity descriptors identified nucleophilic and electrophilic hotspots within the inhibitors, with nitrogen atoms in pirtobrutinib, zanubrutinib, and spebrutinib displaying pronounced nucleophilic potential, suggesting a key role in stabilizing interactions within the BTK active site. Molecular docking analyses revealed that these inhibitors maintained strong binding affinities across multiple BTK mutants, frequently exceeding that of ibrutinib. Molecular dynamics simulations confirmed the structural stability and compactness of selected inhibitor-BTK complexes. Binding free-energy calculations further supported these observations, with several mutant complexes demonstrating enhanced affinities relative to the wild type. Collectively, these findings highlight structurally resilient inhibitors capable of overcoming compound mutation-driven resistance and underscore the importance of BTK mutational profiling in guiding precision therapeutic strategies for BTK-driven malignancies.