Establishment Of The Recombinant Yeast System To Study CYP2C9, 2C19 And Their SNPs In Drug-drug Interaction

As a member of the cytochrome P-450 enzyme superfamily, CYP2C9 and CYP2C19 play the important role in the metabolism of drugs and xenobiotics, about 20% drugs are metabolited by CYP2C in the clinical therapy, because of their gene polymorphism, the phenomenons of adverse drug reaction(ADR) and drug drug interaction (DDI) always happen, therefore, the research on genetic polymorphism become more important.The research cloned CYP2C9,CYP2C19 wild types and their mutations (CYP2C9-R144C,CYP2C9-I359L,CYP2C19-S51G,CYP2C19-R329H,CYP2C19-R442C) by recombinant method in vitro, expressed their proteins in yeast Saccharomyces cerevisiaee, then using these recombinant enzymes to develop a broadly applicable assay system for studying human CYP2C19 polymorphic enzymes, the result may help us to know the genetic polymorphism effects on drug drug interaction.Firstly, the mutant sites were introduced to wild type through site-directed mutagenesis PCR, and cloned into pYES2/CT vector for galactose-inducible expression in yeast which was already integreted with CYP450 Oxidoreductase (POR), the expressed proteins were validated by western blot and detected the P450 content using reduced CO difference spectrum, the results were as follows: CYP2C9-WT 30pmol/mg,CYP2C9-R144C 60 pmol/mg,CYP2C9-I359L 8 pmol/mg ,CYP2C19-WT 30pmol/mg ,CYP2C19-R329H 53 pmol/mg, CYP2C19-R442C 27pmol/mg, but CYP2C19-S51G did not get the content, that's to say the yeast system can express the P450 protein.Secondly, the system was verified by HPLC which was FDA recommended , next, to make sure the experiments, the enzymes' characters were detected which include the comparison with human liver microsomes, the enzymes' stability. And the outcomings were good for experiments.Thirdly, do the enzyme analysis using the enzymes we made.1. the enzyme activity variation made by site mutations was checked through marker substrate and fluorescent substrate.(the marker substrate of CYP2C9 is diclofenac , fluorescent substrate is BOMCC; the marker substrate of CYP2C19 is S-mephenytion, fluorescent substrate is CEC) ,As a result, the CYP enzyme activity was declined by every mutant site, besides, as to CYP2C9-I359L and CYP2C19-R442C, the declined extent was different between marker substrate and fluorescent substrate.2. The parameters of enzyme kinetics (Km,Vmax,Clint)were also detected by marker substrate and fluorescent substrate, the results were as follows: every mutation led to increscent Km, depressed Clint and variational Vmax.3. Used fluorogenic high throughput technique to detect the inhibition of 22 drugs. The substrate concentration was constant, made the drugs dilute several times (128-0.125μM), the inhibition parameters(IC₅₀) about different enzymes were showed differently, besides, Drugs had similar structure or therapy had different inhibition on CYP2C9,CYP2C19, The inhibitory degree of one drug on different genotype varied a lot. Meanwhile, CYP2C9 was strongly inhibited by its known inhibitor sulfaphenazole, IC₅₀ was about 0.7μM, CYP2C19 was also strongly inhibited by its known inhibitor tranylcypromine, IC₅₀ was about 7.1μM, both of them were close to FDA recommended.Consequently, The yeast expression system of CYP2C9, CYP2C19 and their genetic polymorphism were constructed successfully , besides, the detectable method of enzyme kinetics and drug inhibion for CYP2C9,CYP2C19 and their SNPs in vitro were applied initially. The result was good to instruct logical drug using , avoid the ADR and DDI at best , also provide theoretical basis on new drug research.