Calculating the Full Free Energy Profile for Covalent Modification of a Druggable Cysteine in Bruton’s Tyrosine Kinase

Targeted Covalent Inhibitors bind to their targets both covalent and non-covalent 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 (7), 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. The specific mechanism of the binding and the relative importance of the covalent and non-covalent interactions is difficult to determine experimentally, but 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. The mechanism is ionic, where 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 non covalently bound ligand. This model predicts that the formation of the covalent linkage makes binding 19.3 kcal/mol more exergonic than the non-covalent binding alone. Nevertheless, non-covalent 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 that 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, non-covalent interactions, and the formation of covalent linkage are all significant factors in the ultimate

binding affinity of a covalent drug to its target.