Glucose analog inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes

Abstract

The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analogue inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for additional atoms attached at the C1-beta position of glucose and some scope for additional atoms at the C1-alpha position. Kinetic parameters were determined for alpha-D-glucose: Ki = 1.7 mM, Hill coefficient n = 1.5, and alpha (synergism with caffeine) = 0.2. For beta-D-glucose, Ki = 7.4 mM, n = 1.5, and alpha = 0.4. More than 20 glucose analogues have been synthesized and tested in kinetic experiments. Most were less effective inhibitors than glucose itself and the best inhibitor was alpha-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, alpha = 0.4). The binding of 14 glucose analogues to glycogen phosphorylase b in the crystal has been studied at 2.4-A resolution and the structure have been refined to crystallographic R values of less than 0.20. The kinetic and crystallographic studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogues. The results show the discrimination against beta-D-glucose in favor of alpha-D-glucose is achieved by an additional hydrogen bond made in the alpha-glucose complex through water to a protein group and an unfavorable environment for a polar group in the beta pocket. The compound alpha-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogues with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl beta-D-glucoside) with those whose substituent atoms are more rigid (e.g., beta-azidomethyl-1-deoxyglucose or beta-cyanomethyl-1-deoxyglucose) indicates that although all three compounds make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, alpha-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compound made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a number of compounds (beta-aminoethyl, beta-mesylate, and beta-azidomethyl analogues). The beta pocket was shown to bind gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.

N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose

“…The molecule makes a total of 12 hydrogen bonds and 63 van der Waals interactions, the most important of which are to side-chains of Leu 136, His 377, and Thr 378. In the a-D-glucose complex, there are, in total, 12 hydrogen bonds and 56 van der Waals contacts with a-D-glucose, but there is very little complementarity between the nonpolar groups of the sugar and nonpolar groups on the enzyme (Martin et al, 1991). The crystallographic analysis showed that the glucosyl moiety of I-GlcNAc made one less hydrogen bond to the protein than in the a-D-glucose complex.…”

“…At the allosteric site, apparent features of the difference map indicated ( I ) a shift of the side chain of Arg 309 in order to optimize contacts with the nucleotide phosphate group, (2) a small change in the position of Tyr 75, and (3) displacement of a few water molecules near the nucleotide base. At the catalytic site, the main features of the difference map included: ( I ) the presence of negative den- 14 86 17 83 16 84 18 82 14 86 sity over His 377, and associated positive density nearby, indicating a shift of the side-chain of histidine away from the I-GlcNAc molecule to optimize contact with the glucosyl0-6 hydroxyl; this movement is seen for all complexes with glucose-like compounds (Barford et al, 1988;Oikonomakos et al, 1988;Martin et al, 1990Martin et al, , 1991Papageorgiou et al, 1991), and (2) the presence of negative density near the 0 -2 hydroxyl indicating displacement of a water molecule (Wat 9.904).…”

“…Glucose also exerts its effects through conversion to Glc-6-P, and it appears that only those 2469 analogues that are converted can cause activation of glycogen synthase, presumably as a result of hexose-6-P binding to synthase b (Carabaza et al, 1992;Massillon et al, 1995). It has been postulated that glucose analogues with greater inhibition of glycogen phosphorylase may result in more effective regulatory agents than glucose (Martin et al, 1991). There is the expectation that if a sufficiently powerful and specific inhibitor of glycogen phosphorylase could be achieved, then such an agent should shift the balance between glycogen breakdown and glycogen synthesis in favor of the latter and may be of therapeutic benefit in the treatment of the noninsulin-independent form of diabetes mellitus (Type I1 diabetes), where failure to suppress hepatic output of glucose is one of the major manifestations of the disease (DeFronzo, 1988).…”

“…1-GlcNAc acts as a competitive inhibitor with respect to Glc-I-P with a K j value of 32 pM, determined from the in- Figure 2. Data from Martin et al (1991); the parameters refer to a-D-glucose.…”

Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal

“…The difference electron density maps for PL-GPb-phosphate-Glc-IMP and PL-GPb-fluorophosphateGlc-IMP complexes against PL-GPb-phosphite-Glc-IMP complex, in the vicinity of the catalytic site, are shown in Figure 3. In the X-PLOR refined structures of the three complexes, the conformational changes in the enzyme are small and similar to those observed when Glc (Martin et ai., 1990) or a Glc-like inhibitor (Martin et al, 1991;Oikonomakos et al, 1995) binds. We describe in some detail mainly the PL-GPb-phosphate-b-GIc-IMP complex.…”