Studies On The Inhibition Of Flavonoids To Dihydrofolate Reductase

Dihydrofolate reductase(DHFR)is an enzyme,which can reduce dihydrofolate to tetrahydrofolate(THF).THF involves the de novo synthesis of DNA,RNA and some amino acids.Thus,this enzyme is for biological cell proliferation.Therefore,DHFR is considered as an important drug target for anti-malarial,anti-viral,anti-bacterial and anti-cancer.Flavonoids are secondary metabolites of plants which are widely present in nature.They are capable of inhibiting DHFR’s activity.Besides,flavonoids have great potential to be medicine due to low side effects.We study DHFR from Escherichia coli,Rhizoctonia solani and human with the following research.First part of this thesis is the study on the optimal conditions of expressing of DHFR.And the optimal conditions of expressing of Escherichia coli DHFR are 0.1 mM IPTG,22℃,200 rpm for 19 h;the optimal conditions of expressing of Rhizoctonia solani DHFR are 0.1 mM IPTG,24℃,200 rpm for 20 h;the optimal conditions of expressing of human DHFR are 0.1 mM IPTG,26℃,200 rpm for 14 h.Secondly,the study on the effect of NusA on DHFR.It turns out that NusA will weaken the enzyme activity and thermostability of Rhizoctonia solani and human DHFR.Thirdly,three pairs of glycine and serine were added between the TEV restriction site and human DHFR.The digestion efficiency was increased from 58.6%to 91.4%.Second part of this thesis,the study on the inhibition of flavonoids to DHFR.Firstly,combing the AutoDock program and inhibition experiments,three flavonoids with low IC50 values within 200 μM are discovered.IC50 value of a-naphthoflavone is 131.1 μM,IC50 value of geraniol is 158.1 μM and IC50 value of liquiritigenin is 43.5 μM.Secondly,the docking experiments show that:Liquiritigenin has two H-bonds(3.12 A,2.76 A)with Asp21 and Glu30 respectively.The compound has hydrophobic interactions with Asp21,Glu30,Phe34,Phe31,Ser60,Pro61,Leu22,Ile60 of human DHFR and NADPH.Diosmetin has three H-bonds(2.75 ?,3.01 A,2.94 ?)with Val115,Ile7,Glu30 respectively.The compound has hydrophobic interactions with Val115,Ile7,Glu30,Phe34,Phe31,Pro61,Leu22,Asn64,Ala9,Val8 and NADPH of human DHFR.α-naphthoflavone has hydrophobic interactions with Phe31,Leu22,Leu27,Ile60,Val15,Ile7,Phe34,Val8,Ala9,Glu30 of human DHFR.In this thesis,we obtained the purified DHFR.Besides,we discovered a few flavonoids,which can inhibit human DHFR using virutual screening and confirmed their inhibition with IC50 values.The results of the thesis can be used the as the basis for the design of new drugs.