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Research on the Human CYP2D6 Polymorphism and the Relationship with Drug Metabolism

Author: WangLingLing
Tutor: ChenChao
School: Northwestern University
Course: Microbiology
Keywords: Human Cytochrome 2D6 Saccharomyces cerevisiae BJ5628 Geng Clone Protein Expression Fluorogenic Assays Drug inhibition assay
CLC: R96
Type: Master's thesis
Year: 2007
Downloads: 303
Quote: 0
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Abstract


Drug is majorly metabolized in hepatics, depending on many enzymes, especially the CYP450.The polymorphism of CYP450 is the main reason to cause drug adverse reaction that raises the death rate nowdays.In the passed years, people use animal models in the drug screening research. However, this method has huge disadvantages; there are many differences between animals and human, such as: different kinds of enzyme, different kinds of reaction, different contribution extent, and different kinds of production. So, it is inaccurate, low-throughput, and high cost if we get the human information from the animals in the drug metabolism screening. With the develop of the Molecular Biology, Genetics, Proteins, Bio-information, the upstream basic technology of the drug research get huge breakup. If using humanized organization or humanized enzyme to do research in vitro, we can extinct the influence of the species differences, and it’s low cost,short cycle.In this paper,I built CYP450 screening system in vitro,and prospect the metabolism data and properity in vivo,which is high-throughput, high- efficiency,and high-exact.The concretely Methods include: The CYP2D6 prototype gene was sub-cloned from a commercial cDNA into a yeast expression vector pYES2/CT. The prototype construct was then used as a template to separately introduce 8 single nucleic acid changes using two-step PCR mutagenesis method. Following sequencing, the CYP genes were expressed in yeast BJ5628 integrated POR by galactose induction, and S9 microsome fractions containing CYP enzymes were isolated by differential centrifugation. Biochemical assays were performed with the microsomes using CYP2D6-specific fluorogenic probe AMMC, and the CYP2D6 activity was validated by enzyme inhibition with a known inhibitor drug quinidine. For kinetic constants, the enzyme velocity was measured at six different substrate concentrations, and the data were treated by nonlinear regression analysis; And then we choose 12 kinds clinical drugs to do inhibition experiment and get IC50. Results suggest that the cloning of 8 non-synonymous SNPs (28C, P34S, L91M, R173C, R201H, H324P, E410K, R440H) of CYP2D6 was confirmed by nucleotide sequencing of the entire gene. The expression of 2D6 enzyme in yeast following galactose induction was confirmed by Western analysis using antibody specific for the V5 immunotag present at the C-termini of recombinant enzymes. Fluorogenic assays using isolated microsomes with AMMC showed robust catalytic activity of the prototype 2D6 enzymes, which was inhibited by a known inhibitor quinidine in a dose-dependent manner. The enzymes containing SNPs showed variable activities as determined by Km and Vmax. Of the 8 SNPs, two SNPs (R440H and H324P) were catalytically inactive, whereas P34S showed markedly reduced activity. The drug inhibition assay differs among different drug and SNPs: five drugs don’t inhibit CYP2D6 and 8 SNPs; some drugs depend on the polymorphism of CYP2D6; quinidine has the strongest inhibition.Then we can get the Conclusion that we have developed a comprehensive technology for evaluating a large number of non-synonymous SNPs in CYP2D6 gene. The expression system produces high levels of enzymes, and the isolated microsomes produce reliable 2D6-specific enzyme activities. Assays with fluorogenic substrate and the drug inhibition assay allow sensitive analysis of enzyme activities and high-throughput drug screening. The application of this technology for drug screening promises to produce valuable pharmacogenetic information.

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