Binding of oxalyl derivatives of β-d-glucopyranosylamine to muscle glycogen phosphorylase b

Abstract

Five oxalyl derivatives of β-d-glucopyranosylamine were synthesized as potential inhibitors of glycogen phosphorylase (GP). The compounds 1–4 were competitive inhibitors of rabbit muscle GPb (with respect to α-d-glucose-1-phosphate) with K_i values of 0.2–1.4 mM, while compound 5 was not effective up to a concentration of 10 mM. In order to elucidate the structural basis of their inhibition, we analysed the structures of compounds 1–4 in complex with GPb at 1.93–1.96 Å resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as α-d-glucose and stabilize the T-state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. Comparison with the lead compound N-acetyl-β-d-glucopyranosylamine (6) shows that the hydrogen bonding interaction of the amide nitrogen with the main-chain carbonyl oxygen of His377 is not present in these complexes. The differences observed in the K_i values of the four analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interactions, conformational entropy and desolvation effects.

Introduction

Understanding the fundamental mechanisms that regulate glycogen metabolism provides a basis for therapy of type 2 diabetes. High blood glucose concentration in type 2 diabetes is, in part, due to abnormal production of glucose by the liver. Glycogen is an important source of hepatic glucose production, therefore, inhibition of hepatic glycogen phosphorylase (GP), which catalyses the first step in glycogen breakdown, is a potential therapeutic strategy.1, 2, 3, 4, 5 GP inhibitors may bind predominantly to four distinct sites, the catalytic, the allosteric, the indole carboxamide and the inhibitor or caffeine site,6, 7, 8, 9, 10, 11, 12, 13, 14 while a new binding site capable of accommodating benzimidazole has been recently discovered.¹⁵ Recently, the synthesis and in vitro and in vivo biological evaluation of corosolic acid and maslinic acid derivatives, inhibitors of GP, have been reported;42, 43 furthermore, FR258900, a novel GP inhibitor, isolated from fungal strain No. 138354, was shown to stimulate glycogen synthesis in primary hepatocytes via GP inhibition.44, 45 The efficacy of such inhibitors on control of blood glucose and hepatic glycogen balance has been confirmed from animal studies and in vitro cell biology experiments.3, 16

The catalytic site of GP has been extensively exploited for development of potent inhibitors of the enzyme.15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 Sometime ago N-acetyl-β-d-glucopyranosylamine (6) was found to be a potent inhibitor of muscle GPb with a K_i = 32 μM.²⁰ Structural information available from the crystal structure of the inhibitor complex at 2.3 Å²⁰ and recently at 1.9 Å resolution³¹ showed that 6, on binding to GPb, promotes the less active T state enzyme through stabilization of the closed position of 280 s loop (residues 282–287), which blocks access of the substrate to the catalytic site. The inhibitor fits tightly into the so-called β-pocket, a side channel from the catalytic site with no access to the bulk solvent.¹⁷ In addition, there is a hydrogen bond between N1 of 6 and carbonyl O of His377, an interaction that since then has been observed in all β-d-glucopyranosylamine and spirohydantoin analogues of β-d-glucopyranose.15, 21, 26, 29, 30 Identification of the structural determinants contributing to inhibitor binding mode at the catalytic site should provide a better understanding of the mechanism of inhibition of GP and aid in the design of compounds with improved potency against GP.

A novel class of oxalyl derivatives of β-d-glucopyranosylamine (1–5, Scheme 1) were identified as competitive inhibitors of GPb with K_i values 0.2–1.4 mM. We report here on the crystallographic binding of derivatives 1–4 to GPb, in order to provide rationalizations for the kinetic properties of the compounds. The crystallographic data show that the compounds bind at the catalytic site and occupy a position similar but not identical to that of the lead compound 6. On binding of 1–4 to the enzyme there are subtle changes of the amino acid side chains and water structure in the vicinity of the catalytic site, while the characteristic hydrogen bond between the amide nitrogen N1 and main-chain O of His377 is not present in the crystal complex structures studied.