Identification And Functional Characterization Of A New CYP2C9 Variant CYP2C9*62 And The Drug Metabolic Activity Analysis Of The Defective CYP2C9 Variant CYP2C9*18

Objective To investigate the distribution pattern of CYP2C9 gene in Chinese Han population and to characterize the in vitro drug metabolism activity of CYP2C9 variants.Methods In this experiment,genetic polymorphism of CYP2C9 and VKORC1 genes was screened in 162 patients taking warfarin.The CYP2C9 plasmid vector containing the mutation site was constructed by the overlap extension PCR amplification method.Recombinant baculovirus expression vector systems(BEVS)were used to get recombinant bacmid and recombinantly express the target proteins in insect microsomes.Microsomes were extracted and then the target proteins were quantified with immunoblotting method.In vitro enzymatic activity of the target proteins was determined using losartan,tolbutamide and diclofenac as the probe substrates.Protein expression,quantitative characterization and metabolic activity analysis were performed using the same methods for another variant of CYP2C9,CYP2C9*18(1190A>C,1075A>C).The crystal structure of CYP2C9.18 variant was predicted by homology modeling with the deposited crystal structures of two human CYP2C9 proteins bound to losartan.Functional alterations of the proteins were then analyzed by examining their structural changes.Results Six CYP2C9 genotypes were obtained after screening of 162 subjects with the frequencies as following: 88.89% for *1/*1,0.62% for *1/*2,8.64% for *1/*3,0.62% for *1/*13,0.62% for *1/*29 and 0.62% for *1/*33.A new CYP2C9 variant,named CYP2C9*62(8576C>T),was identified from one of the patients,and the genotype of VKORC1 gene in this patient was detected as GA heterozygous.The recombinant plasmid and bacmid containing CYP2C9*62 were successfully constructed and the corresponding proteins were also highly expressed in insect cells.Compared with the wild-type,the protein expression level of variant CYP2C9.62 was significantly reduced.In vitro metabolic activity of the enzyme revealed that the drug clearance of this new CYP2C9 variant toward losartan,tolbutamide and diclofenac was only 0.83%,7.48% and 32.68% of that of the wild-type,respectively.Expression and function of variants D397 A and CYP2C9.18 were investigated using the same method to that of variant CYP2C9.62.Compared to the wild-type,the variant D397 A exhibited similar expression level in insect cells,accompanied by reduced drug metabolism activity.Whereas the protein expression level of CYP2C9.18 was significantly lower as compared with that of wild-type,and its drug metabolism activity was also decreased by more than 80%.By exploring the spatial crystal structure of CYP2C9.18,we found that the amino acid substitution of D397 A altered the spatial orientation of amino acid residues at the substrate binding site,which in turn reduced the overall protein structure and substrate binding capacity.Conclusion In this study,we identified a new CYP2C9 allelic variant CYP2C9*62.With the insect baculovirus expression vector system(BEVS),variants CYP2C9.62,CYP2C9.18 and D397 A were successfully expressed in insect microsomes.Using three drugs as the substrate probes,the in vitro pharmacokinetic activities of CYP2C9 variants were systematically investigated.Our data and previous studies clearly indicated that CYP2C9*62 and CYP2C9*18 are rare allelic variants of CYP2C9 and carriers with these alleles should be regarded as the poor metabolizers.In order to prevent adverse drug reactions,patients with these alleles might be rigorously monitored and thoroughly evaluated when taking CYP2C9 metabolizing drugs in clinic.