THE BINDING MODE OF AN E-64 ANALOG TO THE ACTIVE-SITE OF CATHEPSIN-B

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhibitor to cathepsi n B have been proposed. Molecular docking using an empirical force fie ld was carried out to distinguish between the two modes. The search be gan with manual docking, followed by random perturbations of the docki ng conformation and cycles of Monte Carlo minimization. Finally, molec ular dynamics was carried out for the most favorable docking conformat ions. The present calculations predict that the isobutyl-NH-Eps-Leu-Pr o inhibitor preferentially binds to the S' rather than the S subsites' of cathepsin B. The S' binding mode prediction is supported by the X- ray crystal structure of cathepsin B bound to a closely related ethyl- O-Eps-Ile-Pro inhibitor, which was found to bind in the S' subsite wit h the C-terminal epoxy ring carbon making a covalent bond to the sulfu r atom of Cys29. This agreement, in turn, validates our docking strate gy. Furthermore, the calculations provide evidence that the dominant c ontribution to the total stabilization energy of the enzyme-inhibitor complex stems from the strong electrostatic interaction between the ne gatively charged C-terminal carboxylate group of the ligand and the po sitively charged imidazolium rings of His110 and His111. The latter ar e stabilized and held in an optimal orientation for interactions with the C-terminal end of the ligand through a salt bridge between the sid e chains of His110 and Asp22. By comparison with the crystal structure , some insight into the specificity of the epoxyldipeptide family towa rds cathepsin B inhibition has been extracted. Both the characteristic s of the enzyme (e.g. subsite size and hydrophobicity) as well as the nature of the inhibitor influence the selectivity of an inhibitor towa rds an enzyme.