人类 P450 氧化还原酶遗传变异对 CYP2D6 体外催化作用的影响。
Effects of genetic variants of human P450 oxidoreductase on catalysis by CYP2D6 in vitro.
机构信息
Department of Pediatrics, University of California, San Francisco, California 94143-0978, USA.
出版信息
Pharmacogenet Genomics. 2010 Nov;20(11):677-86. doi: 10.1097/FPC.0b013e32833f4f9b.
OBJECTIVES
Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6.
METHODS
N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (K(m)) and maximum velocity (V(max)) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as V(max)/K(m).
RESULTS
Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol.
CONCLUSION
POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40-50%, potentially explaining some genetic variation in drug metabolism.
目的
细胞色素 P450（P450）氧化还原酶（POR）将电子捐赠给所有微粒体细胞色素 P450，包括药物代谢和甾体生成酶。严重的 POR 突变会导致骨骼畸形和类固醇生成紊乱。POR 多态性 A503V 约存在于人类等位基因的 28%，降低 CYP3A4 和甾体生成 CYP17 的活性，但不降低甾体生成 CYP21 或药物代谢 CYP1A2 和 CYP2C19 的活性。CYP2D6 代谢约 25%的临床用药；我们评估了 POR 变体支持人类 CYP2D6 活性的能力。
方法
野生型（WT）、Q153R、A287P、R457H 和 A503V POR 的 N-27 形式以及 WT CYP2D6 在大肠杆菌中表达。用纯化的 CYP2D6 重建细菌膜中的 POR 蛋白。通过 EOMCC（2H-1-苯并吡喃-3-甲腈，7-（乙氧基-甲氧基）-2-氧-（9Cl））、右美沙芬和布非洛尔的代谢来测量 CYP2D6 的支持。在每个反应的三个重复实验中确定米氏常数（K(m)）和最大速度（V(max)）；催化效率表示为 V(max)/K(m)。
结果
与 WT POR 相比，致病 POR 突变体 A287P 和 R457H 对 EOMCC 无检测到的 CYP2D6 活性，但 A287P 对右美沙芬和布非洛尔的活性约为 25%。Q153R 对 CYP2D6 的功能增加（EOMCC 为 128%，右美沙芬为 198%，布非洛尔为 153%）。A503V 支持 CYP2D6 活性降低：EOMCC 为 85%，右美沙芬为 62%，布非洛尔为 53%。
结论
POR 变体的影响取决于代谢的底物。致病 POR 突变体 R457H 和 A287P 活性较差，表明受影响的患者应考虑药物代谢减少。常见的 A503V 多态性使两种常用药物的 CYP2D6 活性降低 40-50%，可能解释了药物代谢的一些遗传变异。
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