Microsecond simulations to investigate the structural mechanism of super-resistant double mutations in BTK to the covalent inhibitor ibrutinib in multiple leukemia.

Bruton’s Tyrosine Kinase (BTK) is an anchor in B-cell receptor signaling and plays an important role in chronic lymphocytic leukemia (CLL). The use of covalent inhibitors of BTK, such as ibrutinib, enhances the survival of patients with CLL. However, mutations at the C481 residue cause resistance to ibrutinib and diminish its clinical efficacy. Recently, super-resistant mutants, i.e., T474M-C481S and T474I-C481S, were reported to cause manifold resistance to the BTK-targeting drug, ibrutinib; however, the mechanism of this resistance is still elusive. Structure-based approaches proved to be effective in deciphering drug resistance mechanisms (s) that could guide the development of novel, effective therapeutics. Therefore, we used molecular modeling combined with biophysical simulation approaches to determine the impact of T474M-C481S and T474I-C481S mutations on the binding of ibrutinib. Our results revealed that essential hydrogen bonds and a covalent interaction with C481 are lost due to these mutations. Using µs simulations, our results revealed that the regions 432–439 and 545–559 demonstrated dynamically unstable behavior with the transition of secondary structure, where a helix to loop and loop to helix transition could be observed. Structural compactness, residue flexibility, and average hydrogen bonds in each trajectory reported significant variations. The binding free energy calculation using MM-GBSA (Molecular Mechanics Generalized Born Surface Area) and MM-PBSA (Molecular Mechanics Poisson–Boltzmann Surface Area) approaches revealed that both the vdW and electrostatic energies are reduced in mutants. Using the MM-PBSA approach, the wild type demonstrated a total binding free energy (TBE) of -42.65 ± 0.08 kcal/mol, while T474M-C481S and T474I-C481S had TBE values of -38.81 ± 0.18 kcal/mol, and − 33.04 ± 0.13 kcal/mol, respectively. The MM-GBSA results revealed that the wild type had a TBE of -60.33 ± 0.06 kcal/mol, while the TBE values for T474M-C481S and T474M-C481S mutants were − 53.18 ± 0.12 kcal/mol and − 49.12 ± 0.10 kcal/mol, respectively. PCA and FEL results further revealed the dynamic variations caused by these mutations. These findings underline the significant impact of mutations T474M and C481S on the binding free energy, highlighting the importance of these residues in ibrutinib-BTK interactions.