Discovery of novel aldose reductase inhibitors using a protein structure-based approach: 3D-database search followed by design and synthesis.

Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out structure-based drug design and synthesis in an attempt to find new types of AR inhibitors. With the ADAM&EVE program, a three-dimensional database (ACD3D) was searched using the ligand binding site of the AR crystal structure. Out of 179 compounds selected through this search followed by visual inspection, 36 compounds were purchased and subjected to a biological assay. Ten compounds showed more than 40% inhibition of AR at a 15 microg/mL concentration. In a subsequent lead optimization, a series of analogues of the most active compound were synthesized based on the docking mode derived by ADAM&EVE. Many of these congeners exhibited higher activities compared to the mother compound. Indeed, the most potent, synthesized compound showed an approximately 20-fold increase in inhibitory activity (IC(50) = 0.21 vs 4.3 microM). Furthermore, a hydrophobic subsite was newly inferred, which would be useful for the design of inhibitors with improved affinity for AR.

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation.

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.

Rational automatic search method for stable docking models of protein and ligand.

An efficient automatic method has been developed for docking a ligand molecule to a protein molecule. The method can construct energetically favorable docking models, considering specific interactions between the two molecules and conformational flexibility in the ligand. In the first stage of docking, likely binding modes are searched and estimated effectively in terms of hydrogen bonds, together with conformations in part of the ligand structure that includes hydrogen bonding groups. After that part is placed in the protein cavity and is optimized, conformations in the remaining part are also examined systematically. Finally, several stable docking models are obtained after optimization of the position, orientation and conformation of the whole ligand molecule. In all the screening processes, the total potential energy including intra- and intermolecular interaction energy, consisting of van der Waals, electrostatic and hydrogen bonding energies, is used as the index. The characteristics of our docking method are high accuracy of the results, fully automatic generation of models and short computational time. The efficiency of the method was confirmed by four docking trials using two enzyme systems. In two attempts to dock methotrexate to dihydrofolate reductase and 2'-GMP to ribonuclease T1, the exact structures of complexes in crystals were reproduced as the most stable docking models, without any assumptions concerning the binding modes and ligand conformations. The most stable docking models of dihydrofolate and trimethoprim, respectively, to dihydrofolate reductase were also in good agreement with those suggested by experiment. In all test cases, it was shown that our method can accurately predict the correct docking structures, discriminating the correct model from incorrect ones. The efficiency of our method was further tested from the viewpoint of ability to predict the relative stability of the docking structures of two triazine derivatives to dihydrofolate reductase. Our docking method provides a useful tool for rational drug design and investigations of biochemical reaction mechanisms.