Citrus flavonoids luteolin, apigenin, and quercetin inhibit glycogen synthase kinase-3β enzymatic activity by lowering the interaction energy within the binding cavity

Abstract

Pancreatic cancer studies have shown that inhibition of glycogen synthase kinase-3β (GSK-3β) leads to decreased cancer cell proliferation and survival by abrogating nuclear factor κB (NFκB) activity. In this investigation, various citrus compounds, including flavonoids, phenolic acids, and limonoids, were individually investigated for their inhibitory effects on GSK-3β by using a luminescence assay. Of the 22 citrus compounds tested, the flavonoids luteolin, apigenin, and quercetin had the highest inhibitory effects on GSK-3β, with 50% inhibitory values of 1.5, 1.9, and 2.0 μM, respectively. Molecular dockings were then performed to determine the potential interactions of each citrus flavonoid with GSK-3β. Luteolin, apigenin, and quercetin were predicted to fit within the binding pocket of GSK-3β with low interaction energies (-76.4, -76.1, and -84.6 kcal·mol(-1), respectively) and low complex energies (-718.1, -688.1, and -719.7 kcal·mol(-1), respectively). Our results indicate that several citrus flavonoids inhibit GSK-3β activity and suggest that these have potential to suppress the growth of pancreatic tumors.

FIG. 1.

Sequence alignments. Amino acid sequences of the glycogen synthase kinase-3β (GSK-3β) structures reported in PDB 1H8F and 1I09 and the complete 420 amino acid coding sequence of GSK-3β are aligned. Both 1H8F and 1I09 are missing residues from their N- and C-termini; 1I09 is missing 2 loop regions between residues 120-126 and 286-300.

FIG. 2.

Flavonoids and known glycogen synthase kinase-3β (GSK-3β) inhibitor structures. (A) Basic flavonoid structure. The A-ring is commonly hydroxylated at positions 5 and 7, and the B-ring is hydroxylated at positions 4′, 3′4′ or 3′4′5′. (B) Structures of flavonoids presented in Table 1. (C) Some of the flavonoid structures and the 2 known inhibitor structures presented in Table 2.

FIG. 3.

Predicted structure of glycogen synthase kinase-3β (GSK-3β) complexed with I-5. The predicted protein backbone of GSK-3β is shown in ribbon format, with α-helices shown in red, β-sheets in yellow, and loops in blue. The active site is located between the β-rich N-terminus and the α-rich C-terminus, and the predicted binding mode of I-5 is shown in space-filling format.

FIG. 4.

Predicted docking mode for luteolin in the binding cavity of glycogen synthase kinase-3β (GSK-3β). (A) Surface representation of the binding cavity of GSK-3β is shown with the predicted mode for luteolin binding and the interacting amino acid residues. Binding cavity residues are shown with acidic residues in red, basic residues in dark blue, hydrophobic residues in green, and hydrophilic residues in light blue. (B) Two-dimensional representation of the luteolin interacting residues following the method of Clark and Labute. Binding cavity residues that are shown with green circles indicate residues with no polar or charged side chains. Residues with light mauve circles indicate polar side chains that can be acidic, indicated by a red ring, or basic, indicated by a blue ring. The arrows indicate hydrogen bonds to side chain residues in green and backbone residues in blue.

FIG. 5.

Predicted docking modes for rutin and narirutin in the binding cavity of glycogen synthase kinase-3β (GSK-3β). Surface representation of the binding cavity of GSK-3β is shown with the predicted modes for rutin in blue and for narirutin in orange. The binding cavity residues are shown with acidic residues in red, basic residues in dark blue, hydrophobic residues in green, and hydrophilic residues in light blue.