Capillary Electrophoresis Screening Of Dihydrofolate Reductase Inhibitors And Their Inhibition Kinetics Studies

Dihydrofolate reductase (DHFR) which is the essential enzymes for the biosynthesis of DNA, has long been recognized as a drug target of anticancer,antimicrobial,antifungal,antiviral and antimalarial drugs. In this paper, the separations of DHFR and the application of High Performance Capillary Electrophoresis(HPCE)method in the research of kinetics of enzyme reactions of DHFR were introduced, including the measurement of kinetic parameters of enzyme reactions,screening the inhibitors of DHFR and the research of kinetics of inhibitory activities.Through the established analysis model for screening of Dihydrofolate reductase inhibitors(DHFRIs) by HPCE, the inhibitions of DHFR activity by Luteolin, Epicatechin, Ursolic Acid, Paclitaxel, Quercetin, Podophyllotoxin, Oxymatrine, Baicalin,Vincristine ,Apigenin ,ECG and EGCG were studied. It was found Epicatechin,Luteolin,Paclitaxel,ECG,EGCG and Quercetin could markedly inhibit DHFR. The inhibition rate to DHFR is Luteolinï¹¥ECGï¹¥EGCG Paclitaxolï¹¥Epicatechinï¹¥Quercetin.Among these twelve compounds, luteolin was found to inhibit the activity of DHFR apparently. The inhibition mechanism of luteolin and inhibitory kinetics were studied by the difference of peak areas between reactants and products . According to the experiment results, luteolin is a noncompetitive inhibitor with respect to FH2 and a counter-competitive inhibitor with respect to NADPH..The Ki of Luteolin was calculated. It was demonstrated that the proposed CE method could be used to screen the inhibitors of DHFR and study the inhibitory mechanism of inhibitors.DHFR was separated from chicken liver by Sephadex G-75.