Design of inhibitors of glycogen phosphorylase: a study of alpha- and beta-C-glucosides and 1-thio-beta-D-glucose compounds.

alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.