Glycogen phosphorylase inhibitors: A free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-​1-​P. Because of its fundamental role in the metab. of glycogen, GP has been the target for a systematic structure-​assisted design of inhibitory compds., which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-​site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan)​. In this work, we employ MD free energy simulations to calc. the relative binding affinities for GP of hydan and two spirohydantoin analogs, methyl-​hydan and n-​hydan, in which a hydrogen atom is replaced by a methyl- or amino group, resp. The results are compared with the exptl. relative affinities of these ligands, estd. by kinetic measurements of the ligand inhibition consts. The calcd. binding affinity for methyl-​hydan (relative to hydan) is 3.75±1.4 kcal​/mol, in excellent agreement with the exptl. value (3.6±0.2 kcal​/mol)​. For n-​hydan, the calcd. value is 1.0±1.1 kcal​/mol, somewhat smaller than the exptl. result (2.3±0.1 kcal​/mol)​. A free energy decompn. anal. shows that hydan makes optimum interactions with protein residues and specific water mols. in the catalytic site. In the other two ligands, structural perturbations of the active site by the addnl. methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water mol. for two well-​defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water mols. in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in x-​ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to est. the order of relative binding strengths, in contrast to the rigorous free energy treatment.