The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor

Abstract

The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.

Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.

“…Catalysis by phosphorylase b in this system depends on the presence of lyotropic salt, organic solvent, and AMP. BAY W1807, the active (-) S-enantiomer in BAY U6751, is known to be a potent inhibitor of muscle phosphorylase b (assayed in the synthetic direction) and to act as a competitive inhibitor with respect to the stimulation of the enzyme by AMP (28). We confirmed this conclusion (results not shown).…”

“…3). The binding of the inhibitor to muscle phosphorylase b induces a T-state, reminiscent of the binding of G6P (28). We could not test for cooperative inhibition of phosphorylase a by G6P and BAY U6751 because detection of phosphorylase activity in our current assay relies on the coupled oxidation of G6P.…”

“…The active R-state is induced by substrate and allosteric activators, including covalent phosphorylation, whereas the less active T-state is promoted by dephosphorylation and by a different set of allosteric inhibitors. The 3 main classes of phosphorylase inhibitors that have been tested in intact systems are as follows: 1) glucose analogs that bind near the active site (11,26), 2) heteroaromatic analogs of caffeine (2) that possibly bind at the purine inhibitory site, and 3) 1-alkyl-1,4-dihydropyridine-2,3-dicarboxylic acids (27) that bind at the AMP site and induce a so-called T-state, which is also adopted in the presence of the natural inhibitor G6P (28).…”

“…1). Only the active (-) S-enantiomer fits in the binding pocket on phosphorylase (28,29). The (+)-enantiomer is completely inactive.…”

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

“…GPb-flavopiridol and GPa-glucose-flavopiridol complexes were co-crystallized as previously (21,23) in a medium consisting of 25 mg/ml enzyme, 1 mM flavopiridol, 3 mM DTT, 10 mM Bes, 0.1 mM EDTA, 0.02% sodium azide, pH 6.7, and 1 mM spermine (GPb) or 10 mM magnesium acetate and 50 mM glucose (GPa); as flavopiridol is insoluble under FIG. 1.…”