No abstract

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 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 N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

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)

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to a-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of P-D-glucopyranosylamine, N-acetyl-P-D-glucopyranosylamine (I-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K, = 32 pM) and the a ( K , = 35 PM) forms of the enzyme with respect to glucose I-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-I-GlcNAc-IMP complex were grown in space group P432,2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1 %, for data between 8 and 2.3 A resolution. I-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of a-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-I-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-a-D-glucose complex structure. The structure of the I-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

alpha-D-glucose is a weak inhibitor (Ki = 1.7 mM) of glycogen phosphorylase (GP) and acts as physiological regulator of hepatic glycogen metabolism; it binds to GP at the catalytic site and stabilizes the inactive T state of the enzyme promoting the action of protein phosphatase 1 and stimulating glycogen synthase. The three-dimensional structures of T state rabbit muscle GPb and the GPb-alpha-D-glucose complex have been exploited in the design of better regulators of GP that could shift the balance between glycogen synthesis and glycogen degradation in favour of the former. Close examination of the catalytic site with alpha-D-glucose bound shows that there is an empty pocket adjacent to the beta-1-C position. beta-D-glucose is a poorer inhibitor (Ki = 7.4 mM) than alpha-D-glucose, but mutarotation has prevented the binding of beta-D-glucose in T state GP crystals. A series of beta-D-glucose analogues has been designed and tested in kinetic and crystallographic experiments. Several compounds have been discovered that have an increased affinity for GP than the parent compound.