Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate

The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlor ophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose ac tivity is primarily controlled by reversible phosphorylation of Ser14 of th e dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carrie s the Ser14(P), changes its conformation into a distorted 3(10) helix and i ts contacts from intrasubunit to intersubunit. This alteration causes a ser ies of tertiary and quaternary conformational changes that lead to activati on of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regul ation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has kla found as the most potent inhibitor of GPb (K-i = 1.6 n M) that binds at the allosteric site of T-state GPb and producer further co nformational changes, characteristic of a T'-like state. Kinetics show W180 7 is a potent competitive inhibitor of GPa (-AMP) (K-i = 10.8 nM) and of GP a (+1 mM AMP) (K-i = 19.4 mu M) with respect to glucose 1-phosphate and act s in synergism with glucose. To elucidate the structural features that cont ribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 Angstrom and 2.1 Angstrom resolution, and refine d to crystallographic R-values of 0.179 (R-free = 0.230) and 0.189 (R-free = 0.263), respectively. W1807 binds tightly at the allosteric site and indu ces substantial conformational changes both in the vicinity of the alloster ic site and the subunit interface. A disordering of the N-terminal tail occ urs, while the loop of chain containing residues 192-196 and residues 43'-3 9' shift to accommodate the ligand. Structural comparisons show that the T- state GPa-glucose-W1807 structure is overall more similar to the T-state GP b-W1807 complex structure than to the GPa-glucose complex structure, indica ting that W1807 is able to transform GPa to the T'-like state already obser ved with GPb. The structures provide a rational for the potency of the inhi bitor and explain GPa allosteric inhibition of activity upon W1807 binding.