Exploring the role of chemical reactions in the selectivity of tyrosine kinase inhibitors*

A variety of diseases are associated with tyrosine kinase enzymes that activate many proteins via signal transduction cascades. The similar ATP-binding pockets of these tyrosine kinases make it extremely difficult to design selective covalent inhibitors. The present study explores the contribution of the chemical reaction steps to the selectivity of the commercialized inhibitor acalabrutinib over Bruton’s tyrosine kinase (BTK) and the interleukin-2- inducible T-cell kinase (ITK). Ab initio and empirical valence bond (EVB)

simulations of the two kinases indicate that the most favorable reaction path involves a water-assisted mechanism of the 2-butynamide reactive group of acalabrutinib. BTK reacts with acalabrutinib with a substantially lower barrier than ITK, according to our calculated free-energy profile and kinetic simulations. Such a difference is due to the microenvironment of the

active site, as further supported by a sequence-based analysis of specificity determinants for several commercialized inhibitors. Our study involves a new approach of simulating directly the IC50 and inactivation efficiency k_eff, instead of using the standard formulas. This new strategy is particularly important in studies of covalent inhibitors with a very exothermic bonding step. Overall, our results demonstrate the importance of understanding the chemical reaction steps in designing selective covalent inhibitors for tyrosine kinases.