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

Abstract

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 ␤-Dglucopyranose, 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) M, 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-methyloxadiazole 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 crystallographyGlycogen phosphorylase (GP), because of its central role in glycogen metabolism, has been exploited as a potential target for structure-based design of potent inhibitors, that may be relevant to the control of blood glucose concentrations in type 2 diabetes (Aiston et al. 2001(Aiston et al. , 2003Latsis et al. 2002). Several regulatory binding sites, identified in GP, have been used as molecular targets (for review, see Oikonomakos 2002). These are the allosteric site that binds the activator AMP and the inhibitor glucose-6-P, the catalytic site that binds substrates glucose-1-P and glycogen, and the inhibitor Article published online ahead of print. Article and publication date are at

4(5)-Aryl-2-C-glucopyranosyl-imidazoles as New Nanomolar Glucose Analogue Inhibitors of Glycogen Phosphorylase

“…The significant difference in the binding strength of these compounds was ascribed to the H-bond forming capacity of the NH moiety in 3 to the main chain CO of His377 next to the catalytic site of the protein as revealed by X-ray crystallography of the RMGPb-3 complex. 22 Further studies with each isomer of C-glucosyl oxadiazoles 21,23,24 4−6 indicated that the constitution of the heterocycle significantly influenced the inhibition. The 1,2,4-triazoles 7, with the heteroatoms in the same relative position as in the oxadiazoles, proved even stronger inhibitors.…”

Carbohydrate Derivatives and Glycomimetic Compounds in Established and Investigational Therapies of Type 2 Diabetes Mellitus

“…For the inhibitory glucose derivatives (Gimisis, 2010;Praly & Vidal, 2010;Somsák, 2011;Somsák et al, 2003;Somsák et al, 2005) protein crystallography showed primary binding to the catalytic site of the enzyme. Some glucose analogues can also occupy the new allosteric site , and the benzimidazole-site was evidenced by a 2-(-Dglucopyranosyl)-benzimidazole (Chrysina et al, 2005). The studied O-, and S-glucopyranosides proved very weak inhibitors with K i values in the 2000-25000 M and 650-21100 M, respectively (Somsák et al, 2003;Somsák et al, 2005).…”

“…Protein crystallographic studies (Chrysina, 2010;Oikonomakos, 2002) revealed the existence of six binding sites in GP (Fig. 1): the catalytic, the inhibitor, the allosteric, the glycogen storage, and the new allosteric sites, as well as the newly discovered benzimidazole site (Chrysina et al, 2005). Each binding site can be targeted by small molecules, and a large variety of inhibitors were tested as described in recent reviews (Loughlin, 2010;Somsák et al, 2008).…”

C-(β-d-Glucopyranosyl)formamidrazones, formic acid hydrazides and their transformations into 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazoles: a synthetic and computational study

“…The corresponding thioamide remained also intact on treatment with benzhydrazide even at elevated temperature (conditions adapted from reports for non-sugar 29,30 or ribofuranose 19 based compounds) as well as under microwave irradiation. Therefore, application of more reactive precursors such as Operbenzoylated C-(b-D-glucopyranosyl)formimidate, 9 (1), -formamidrazones (3,4), -formamidine (5) and C-(b-D-glucopyranosyl) formyl chloride, 31 (13), was envisaged for the construction of the target triazoles. Synthesis of formamidrazone 3 was carried out by the treatment of ethyl C-(b-D-glucopyranosyl)formimidate 9 (1) with hydrazine hydrate in EtOH (Scheme 1).…”