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Kinetic and crystallographic studies on 2-(-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

¹ Institute of Organic and Pharmaceutical Chemistry, and ² Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 11635 Athens, Greece³ Department of Organic Chemistry, Faculty of Science, and ⁴ Department of Medical Chemistry, Research Centre for Molecular Medicine, University of Debrecen, Debrecen, Hungary

(RECEIVED November 4, 2004; FINAL REVISION December 7, 2004; ACCEPTED December 10, 2004)

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of -D-glucopyranose, 2-(-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K_i values of 145.2 (±11.6), 76 (±4.8), and 8.6 (±0.7) µM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 Å; GPb-benzothiazole, 2.10 Å; GPb-benzimidazole, 1.93 Å). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (~ 32 Å) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.

Keywords: type 2 diabetes; glycogen phosphorylase; -D-glucopyranosyl analogs; inhibition; X-ray crystallography

Abbreviations: GPb, muscle glycogen phosphorylase b • PLP, pyridoxal 5'-phosphate • glucose, -D-glucose • Glc-1-P, -D-glucose 1-phosphate • methyl-oxadiazole, 2-(-D-glucopyranosyl)-5-methyl-1,3,4-oxadiazole • benzothiazole, 2-(-D-glucopyranosyl)-benzothiazole • benzimidazole, 2-(-D-glucopyranosyl)-benzimidazole • r.m.s. deviation, root-mean-square deviation.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041216105.

Reprint requests to: Nikos G. Oikonomakos, Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, 116 35 Athens, Greece; e-mail: ngo{at}eie.gr; fax: +30-210-7273-831.

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