The Establishment Of Polymorphic Human Cytochrome P450 2D6 Heterogenous Expression System And Its Primary Application In Drug Screen

Cytochromes P450 is a superfamily of hemethiolate-containing proteins involved in the oxidative metabolism of a large variety of xenobiotics and endobiotics. Many P450 enzymes participate in the conversion of carcinogens, environmental pollutants, and drugs to more polar metabolites, thereby facilitating their excretion and preventing the accumulation of these potentially harmful compounds. Predominantly expressed in liver, members of the CYP1-3 families exhibit broad substrate specificity and metabolize the majority of administered drugs. Human CYP2D6 is responsible for the metabolism of large number of prescription drugs. Pharmacogenetic analysis has shown that single nucleotide polymorphisms (SNPs) in this gene can cause altered trajectory of drug metabolism. In order to study the effects of single amino acid change on CYP2D6 biochemical properties, eight crude non-synonymous SNPs have been chosen.A yeast expression vector pYES2/CT was used to clone the expression constructs. The SNPs were introduced into the prototype CYP2D6 gene by PCR mutagenesis method. Following sequencing, the recombinants were co-expressed with POR in yeast by galactose induction. The S9 microsome fractions containing recombinant CYP2D6 enzymes were isolated by differential centrifugation and were used to test the enzymatic activities in real-time kinetic assays using a CYP2D6-specific fluorogenic substrate AMMC. The kinetic data was fit to nonlinear regression analysis for determination of Km and Vmax values. The inhibition assay was applied to screen for the comparative inhibitory potential of 12 clinically important drugs with the polymorphic variants of CYP2D6 isozyme.Eight non-synonymous SNPs of CYP2D6 (R26H, P34S, A85V, N166D,V136M, V338M, L213P, and I369T) were expressed to high levels in yeast by galactose induction, which was identified by Western blot. Fluorogenic assays using isolated microsomes showed robust catalytic activity of the prototype CYP2D6 enzymes with Km and Vmax of 2.9μmol/L and 17.1RFU/min/mg microsome protein. Of the eight SNP enzymes, L213P and I369T were completely inactive whereas the activity of P34S enzyme was markedly reduced with 3-fold higher Km than the prototype CYP2D6 enzyme. A85V had a half lower Km than the prototype CYP2D6 enzyme, which indicated that its activity increased slightly. Other SNP enzymes retained comparable levels of Km as the prototype enzyme. The IC50 values showed that all of the CYP2D6 prototype and variants were completely inhibited by CYP2D6-specific inhibitor quinidine. Generally, drugs we tested had the same inhibitory trend to 8 SNPs, but for a certain drug, the inhibition potential varied a lot; while for a specific variant enzyme, different drugs had different inhibition range.Human CYP2D6 enzymes containing 8 naturally occurring non-synonymous SNPs have been produced and biochemically characterized. The comparison of these enzymes to the prototype enzyme shows significant differences in their kinetic properties. 12 drugs have been assessed with the polymorphic variants of CYP2D6 isozyme. The inhibition potential differs from drugs and SNPs. Establishing polymorphic CYP2D6 heterogenous expression system and screening drugs that are substrates of CYP2D6 will facilitate drug development and customized pharmacotherapy.