A Comprehensive Study on the Effect of Human Genetic Polymorphism on the Drug Metabolizing, Properties of CYP

本研究以人类药物代谢酶中占主导地位的细胞色素P450(CYP)为研究对象，通过对基于人类基因组单核苷酸多态性(SNPs)信息制备的突变体的药物结合和代谢活性的综合研究，旨在建立一种适用于定制化治疗的临床原则。我们根据SNP信息制备了细胞色素P450 1A2、细胞色素P450 2C9、细胞色素P450 2C19、细胞色素P450 D6和细胞色素P3A4，以及这些细胞色素P450的突变体，并测定了这些细胞色素P450的药物代谢特性。此外，我们还测量了它们的共振拉曼光谱，以阐明SNPs对结构因素的影响。此外，我们还测量了突变体的药物代谢特性，以考察SNPs对药物代谢的影响。我们可以建立一个参数Kd，它是药物结合亲和力的指数，与Km，一个代谢活性的衡量标准，是线性相关的。此外，还发现Vmax与5配位的血红素含量呈线性相关。因为Vmax/Km的值与药物清除量相关，现在很明显，Kd和5-配位的静态值反映了药物清除量。另一方面，通过添加底物药物来稳定蛋白质，并在纯化过程中对表面活性剂进行修饰，成功地制备了细胞色素P3A4和细胞色素P450 2D6。因此，我们最终确定可以利用这些细胞周期蛋白对药物代谢进行综合研究。

In this study, we focused on cytochrome P450 (CYP) which is a dominant group in the human drug metabolizing enzymes, and we aimed at the establishment of a clinical principle which is applicable to order-made treatment, through comprehensive studies on the drug binding and metabolizing activities of mutants which are prepared on the bases of the information on human genomic single-nucleotide polymorphisms (SNPs). We prepared CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, along with mutants of these CYPs based on the SNP information, and measured drug metabolizing properties of these CYPs. In addition, we measured resonance Raman spectra of them in order to clarify structural factors that are affected by SNPs. Furthermore, we measured drug metabolizing properties of the mutants in order to investigate the effect of SNPs. We could establish that a parameter Kd, which is an index of drug binding affinity, is linearly correlated with Km, a measure of metabolizing activity. In addition, Vmax was found to linearly correlate with the content of 5-coordinated heme. Because the value Vmax/Km is correlated with drug clearance, it is now clear that static values of Kd and 5-coordination reveals the drug clearance. On the other hand, we succeeded in the preparation of CYP3A4 and CYP2D6 by adding a substrate drug to stabilize the protein, and modifying surfactants during the purification step. Thus, we finally established that drug metabolism by CYPs can be conprehensively investigated using these CYPs.