Targeted covalent inhibitors (TCIs) bind to their targets in both covalent and noncovalent modes, providing exceptionally high affinity and selectivity. These inhibitors have been effectively employed as inhibitors of protein kinases, with Taunton and coworkers (Nat. Chem. Biol.2015,11, 525–531) reporting a notable example of a TCI with a cyanoacrylamide warhead that forms a covalent thioether linkage to an active-site cysteine (Cys481) of Bruton’s tyrosine kinase (BTK). The specific mechanism of the binding and the relative importance of the covalent and noncovalent interactions is difficult to determine experimentally, and established simulation methods for calculating the absolute binding affinity of an inhibitor cannot describe the covalent bond-forming steps. Here, an integrated approach using alchemical free-energy perturbation and QM/MM molecular dynamics methods was employed to model the complete Gibbs energy profile for the covalent inhibition of BTK by a cyanoacrylamide TCI. These calculations provide a rigorous and complete absolute Gibbs energy profile of the covalent modification binding process. Following a classic thiol-Michael addition mechanism, the target cysteine is deprotonated to form a nucleophilic thiolate, which then undergoes a facile conjugate addition to the electrophilic functional group to form a bond with the noncovalently bound ligand. This model predicts that the formation of the covalent linkage is highly exergonic relative to the noncovalent binding alone. Nevertheless, noncovalent interactions between the ligand and individual amino acid residues in the binding pocket of the enzyme are also essential for ligand binding, particularly van der Waals dispersion forces, which have a larger contribution to the binding energy than the covalent component in absolute terms. This model also shows that the mechanism of covalent modification of a protein occurs through a complex series of steps and that entropy, conformational flexibility, noncovalent interactions, and the formation of covalent linkage are all significant factors in the ultimate binding affinity of a covalent drug to its target.
