chrysin and quercetagetin

Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 μM) than flavopiridol (Ki=1.24 μM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding.

Topics: Adenosine Monophosphate; Animals; Binding Sites; Binding, Competitive; Chromones; Crystallography, X-Ray; Enzyme Inhibitors; Flavones; Flavonoids; Glycogen Phosphorylase; Kinetics; Models, Molecular; Molecular Docking Simulation; Piperidines; Protein Conformation; Rabbits; Structure-Activity Relationship

The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3,3',4',5,6,7-hydroxyflavone) was identified as a moderately potent, ATP-competitive inhibitor (IC(50), 0.34 micromol/L). Resolution of the crystal structure of PIM1 in complex with quercetagetin or two other flavonoids revealed a spectrum of binding poses and hydrogen-bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared with PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED(50), 5.5 micromol/L). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked PIM1 kinase activity. Furthermore, the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.

Topics: Chromones; Crystallography, X-Ray; Flavones; Flavonoids; Humans; Male; Phenotype; Prostatic Neoplasms; Protein Kinase Inhibitors; Protein Structure, Secondary; Proto-Oncogene Proteins c-pim-1; Sensitivity and Specificity; Substrate Specificity