Design of Inhibitors of Glycogen Phosphorylase: A Study of .alpha.- and .beta.-C-Glucosides and 1-Thio-.beta.-D-glucose Compounds

Abstract

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)

Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo

“…6B). Treatment of the primary human hepatocytes with CP-91149 also increased basal 14 C-glycogen content (44%, P Ͻ 0.001) compared with the control (Fig. 6A).…”

“…5B). A hormonally responsive primary human hepatocyte culture system was then developed for determining CP-91149's effects on glycogenolysis by using 14 C-glycogen prelabeled cells. Primary human hepatocytes displayed a 60% (P Ͻ 0.001) reduction in 14 C-glycogen content in response to both GGN and forskolin, whereas coincubation with 30 M CP-91149 inhibited the GGN and forskolin effect by Ͼ80% (P Ͻ 0.01) compared with untreated hepatocytes (Fig.…”

“…A sensitive method for measuring hepatic glycogenolysis in ob͞ob mice, based on 14 C-prelabeling of peripheral glucosyl residues of hepatic glycogen (36), was used to further assess glycogenolysis in vivo. Following prelabeling, the ob͞ob mice were treated with either vehicle or 50 mg͞kg CP-91149, and the residual 14 C-liver glycogen content and plasma glucose concentration were measured 3 h later. As shown in Table 2, mice treated with CP-91149 had 3-fold higher (P Ͻ 0.01) 14 C-liver glycogen content than the vehicle-treated control mice.…”

“…Assays were terminated 2 h later by washing with ice-cold PBS, solubilizing with 1 M NaOH, and quantitation of the 14 C-glycogen (35). Results are expressed as dpm 14 C-glycogen content per well.…”

“…Glucose production from the catalysis of glycogen to glucose-1-phosphate is rate-limited by phosphorylase a, a well-studied enzyme that is regulated by multiple covalent, substrate, and allosteric effectors (12). To date, two types of glycogen phosphorylase inhibitors have been reported: glucose analogs bearing multiple polar functionalities, which bind near the active site of the enzyme (13)(14)(15)(16)(17)(18), and caffeine and other heteroaromatic analogs, which bind at the purine inhibitory site, also known as the I-site (19)(20)(21)(22). Although some glucose and purine nucleoside phosphorylase inhibitors reportedly inhibit glycogenolysis in rodent cells and tissues (22)(23)(24), none of these are known to be orally active in vivo, possibly because of inadequate potency or poor pharmacokinetics, limiting their utility in determining the effect of glycogenolysis inhibition on HGP.…”