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 proteinligand 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 (Kd 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 (kcat) and the estimated dissociation rate (koff) constants of the enzyme-inhibitor complex, one can conclude that the observed inhibition potency (Ki) is primarily dependent on the catalytic rate constant of the inhibitor's O-methylation, rather than the rate constant of dissociation of the complex. (c) 2012 Wiley Periodicals, Inc.