Computation of the binding affinities of catechol‐O‐methyltransferase inhibitors: Multisubstate relative free energy calculations

Alchemical free energy simulations are amongst the most accurate techniques for the computation of the free energy changes associated with noncovalent protein–ligand interactions. A procedure is presented to estimate the relative binding free energies of several ligands to the same protein target where multiple, low‐energy configurational substates might coexist, as opposed to one unique structure. The contributions of all individual substates were estimated, explicitly, with the free energy perturbation method, and combined in a rigorous fashion to compute the overall relative binding free energies and dissociation constants. It is shown that, unless the most stable bound forms are known a priori, inaccurate results may be obtained if the contributions of multiple substates are ignored. The method was applied to study the complex formed between human catechol‐O‐methyltransferase and BIA 9‐1067, a newly developed tight‐binding inhibitor that is currently under clinical evaluation for the therapy of Parkinson's disease. Our results reveal an exceptionally high‐binding affinity (K_d in subpicomolar range) and provide insightful clues on the interactions and mechanism of inhibition. The inhibitor is, itself, a slowly reacting substrate of the target enzyme and is released from the complex in the form of O‐methylated product. By comparing the experimental catalytic rate (k_cat) and the estimated dissociation rate (k_off) constants of the enzyme‐inhibitor complex, one can conclude that the observed inhibition potency (K_i) is primarily dependent on the catalytic rate constant of the inhibitor's O‐methylation, rather than the rate constant of dissociation of the complex. © 2012 Wiley Periodicals, Inc.