The Effect of Genetic Diversity on Mechanism-Based Inactivation of CYP2D6

描述(申请人提供)：人体细胞色素P450酶(Cyps)是一类含血红素的酶，参与类固醇、维生素、环烷类化合物和外源物质(如药物、复杂植物生物碱、致癌物和其他小分子)的代谢。Cyps在几乎所有药物的新陈代谢中的作用使这个酶家族在医学和人类健康方面引起了极大的兴趣。CYP2D6是一种重要的药物代谢酶，负责代谢~25%的药物化合物，但只占肝脏CYP含量的一小部分。该酶还表现出多种多态形式，导致对由CYP2D6代谢的药物的反应存在个体间的差异。药物代谢环磷酰胺的广泛底物专一性的一个警告是，这些酶很容易受到自身反应的副产物的抑制。这种类型的抑制称为基于机制的失活，并导致不可逆的 酶的失活。作为机理失活剂的化合物是了解这些酶的结构/功能的重要工具，目前只有少数几种化合物是已知的。该项目将有助于理解细胞色素P450 2 D6的失活机制，并通过两类失活剂--蛋白质内收剂和血红素修饰剂来筛选等位基因变体。这些互补的研究将通过两个目标来进行：目标1：确定基于机制的蛋白加合物失活剂靶向的CYP2D6的氨基酸(S)。在电子计算机和LC/MS中，将通过假设的亲核性氨基酸对非亲核性氨基酸的突变来检验与载脂蛋白加成有关的氨基酸的预测。表达的突变酶(S)将在正常和失活底物上进行动力学表征，底物包括两个蛋白加入子(SCH66712和EMTPP)和两个血红素修饰剂(帕罗西汀和MDMA)。目的2：确定细胞色素P4502D6基因最常见的多态性与灭活剂的相互作用。几乎20%的药物治疗受到基因多态的影响。基因多态性导致药物的血药浓度和半衰期不同，从而导致药物不良反应。CYP2D6是高度多态的，多态在失活中的作用将被表征。总之，这些研究将提供对In激活剂如何与CYP2D6相互作用的理解，并在合理的药物设计和评估个性化药物时代药物代谢的个体差异方面带来新的机会。

DESCRIPTION (provided by applicant): Cytochrome P450 enzymes (CYPs) in humans are a family of heme-containing enzymes involved in metabolism of steroids, vitamins, eiconsanoids, and xenobiotics such as drugs, complex plant alkaloids, carcinogens, and other small molecules. The role of CYPs in metabolism of nearly all drugs has made this family of enzymes of considerable interest in medicine and for human health. CYP2D6 is one of the significant drug metabolizing enzymes responsible for metabolism of ~25% of pharmaceutical compounds while only accounting for a fraction of liver CYP content. The enzyme also displays multiple polymorphic forms that contribute to inter-individual difference in responses to drugs metabolized by CYP2D6. One caveat of such broad substrate specificity among drug metabolizing CYPs is that the enzymes are susceptible to inhibition by products of their own reactions. This type of inhibition is called mechanism-based inactivation and leads to irreversible inactivation of the enzyme. Compounds that act as mechanism-based inactivators are important tools for understanding structure/function of these enzymes and there are only a few known for CYP2D6. The project proposed here will contribute to understanding mechanisms inactivation of CYP2D6 and select allelic variants by two classes of inactivators - protein adductors and heme modifiers. These complimentary lines of investigation will be pursued through two aims: Aim 1: To determine the amino acid(s) of CYP2D6 targeted by mechanism-based protein adduct inactivators. In silico and LC/MS predictions of amino acids involved in apoprotein adduction will be tested by mutagenesis of postulated nucleophiles to non-nucleophilic amino acids. The expressed mutant enzyme(s) will be characterized in terms of kinetics with normal and inactivator substrates including two protein adductors (SCH66712 and EMTPP) and two heme modifiers (paroxetine and MDMA). Aim 2: To determine the interaction of the most common polymorphisms of CYP2D6 with inactivators. Almost 20% of drug therapies are affected by polymorphisms. Polymorphisms lead to different plasma concentrations and half-life of drugs that lead to adverse drug reactions. CYP2D6 is highly polymorphic and the role of polymorphisms in inactivation will be characterized. Together, these studies will provide an understanding of how in activators interact with CYP2D6 and lead to new opportunities in rational drug design and evaluation of inter-individual differences in drug metabolism in the age of personalized medicine.