Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay

Abstract

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

An In Silico and an In Vitro Inhibition Analysis of Glycogen Phosphorylase by Flavonoids, Styrylchromones, and Pyrazoles

“…The GP activity was measured using a previously optimized and validated method [36]. In each assay, GP activity was measured by the direction of glycogen synthesis, which involved monitoring the colorimetric determination of inorganic phosphate deriving from glucose 1-phosphate.…”

“…The results are shown in Table 1. The selected panel of the compounds was distributed into five groups: 4-styrylpyrazoles (1-9), 5-styrylpyrazoles (10-15), 2-styrylchromones (16-29), 2-styrylchromone-related derivatives (30)(31)(32)(33)(34)(35), and flavonoids (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). In 4-styrylpyrazoles (1-9), all compounds contained a 2-hydroxyaryl substituent at C-3 of the pyrazole core.…”

“…Among the group 2-styrylchromone-related derivatives (30)(31)(32)(33)(34)(35), which display a 4-arylbuta-1,3-dien-1-yl at the C-2 of the chromone core, three compounds were hydroxylated (30)(31)(32), two were hydroxylated and methoxylated (33)(34), and one was only methoxylated (35). In the last group of compounds, the flavonoids (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52), almost all compounds were hydroxylated (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), one was hydroxylated and methoxylated (51) and only one was methoxylated (52). The hydroxylated flavonoid 49 also holds an O-glucuronide at C-7 of the A ring.…”

“…In the last group of compounds, the flavonoids (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52), almost all compounds were hydroxylated (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), one was hydroxylated and methoxylated (51) and only one was methoxylated (52). The hydroxylated flavonoid 49 also holds an O-glucuronide at C-7 of the A ring.…”

“…In the flavonoids family (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52), five compounds, namely 38, 46, 47, 49, and 51, showed remarkable inhibitory activities against GPa, with IC 50 values between 13.2 ± 1.4 and 23.5 ± 2.9 µM. Hydroxylated flavonoids at the A ring, together with hydroxylations at the C and/or B rings (37, 39-45, 48, and 50), showed inhibitory activities lower than 30%, with the exception of compound 42, which displayed an inhibitory activity of 35.7 ± 2.1%, at 50 µM.…”