title: Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.
abstract: Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; College of Pharmacy, South Central University for Nationalities, Wuhan 430074, China.
authors.forename: Yunfang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Xu
authors.affiliation: College of Chemistry and Material Sciences, South Central University for Nationalities, Wuhan 430074, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: College of Pharmacy, South Central University for Nationalities, Wuhan 430074, China.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Mei
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China. Electronic address: yuxiuxiao2011@whu.edu.cn.
authors.forename: Yuxiu
authors.initials: Y
authors.lastname: Xiao
mesh_terms: D044946:Biflavonoids / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D004800:Enzymes, Immobilized; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D007700:Kinetics; D060328:Magnetic Phenomena; D062105:Molecular Docking Simulation; D010455:Peptides / Q000302:isolation & purification; D010766:Phosphorylation
chemical_list: D044946:Biflavonoids; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D010455:Peptides; C105620:morelloflavone; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3
keywords: GSK-3β; HPLC; Inhibitor screening; Magnetic bead; Non-ATP competitive inhibitor; Traditional Chinese medicine