title: Capillary electrophoresis as a novel technique for screening natural flavonoids as kinase inhibitors.
abstract: Capillary electrophoresis (CE) was used for the first time to evaluate the inhibition activity of aglycone flavonoids (such as quercetin and isorhamnetin) and some of their glycosylated derivatives toward human kinases. The cyclin-dependant kinase 5 (CDK5/p25) and the glycogen synthase kinase 3β (GSK3β) were chosen since they are very promising biological targets for developing treatments against neurodegenerative diseases and cancer. In a previous work, we developed an in-capillary kinase CE assay where the capillary was used as an enzymatic nanoreactor in which the kinase, its substrate, adenosine 5'-triphosphate (ATP) and its potential inhibitor were mixed by using transverse diffusion of laminar flow profiles (TDLFP). The product adenosine 5'-diphosphate (ADP) was then detected at 254nm and quantified. In this work, this assay was improved to reduce, for the first time, the dilution effect commonly observed with the TDLFP approach. Under the new conditions established herein, IC50 values for quercetin, kaempferol and flavopiridol were successfully obtained and were in the same order of magnitude of those reported in the literature using the conventional assay using radioactive (33)P-ATP. It was shown that aglycone flavonoids have an inhibition activity more important than their glycosylated derivatives. CE was also proved to be very efficient for evaluating inhibition activity of complex samples such as crude extracts of sea buckthorn (SBT) berries obtained by solvent-free microwave extraction (SFME). This novel approach to combine SFME technique to a CE-based enzymatic assay is very interesting for evaluating the biological activity of natural material in a fast, simple, economic (no use of neither fluorescent nor radiometric labels) and green (no organic solvents) manner.
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans - CNRS FR 2708, UMR 7311, Orléans, France. Electronic address: reine.nehme@univ-orleans.fr.
authors.forename: Reine
authors.initials: R
authors.lastname: Nehmé
authors.forename: Hala
authors.initials: H
authors.lastname: Nehmé
authors.forename: Grégory
authors.initials: G
authors.lastname: Roux
authors.forename: Emilie
authors.initials: E
authors.lastname: Destandau
authors.forename: Bérengère
authors.initials: B
authors.lastname: Claude
authors.forename: Philippe
authors.initials: P
authors.lastname: Morin
mesh_terms: D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005419:Flavonoids / Q000032:analysis*; D005638:Fruit / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D031281:Hippophae / Q000737:chemistry*; D006801:Humans; D010936:Plant Extracts / Q000032:analysis*; D047428:Protein Kinase Inhibitors / Q000032:analysis*
chemical_list: D005419:Flavonoids; D010936:Plant Extracts; D047428:Protein Kinase Inhibitors; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3
keywords: Capillary electrophoresis; Enzymatic inhibition; Flavonoids; Hippophaë rhamnoides; Kinases; Transverse diffusion of laminar flow profile