Mechanism-based Inhibition of P450 2D6 by a Piperazine-containing Compound

描述（由申请人提供）：人类细胞色素P450酶是一个含血红素酶家族，参与类固醇、维生素、类维生素素和药物、复杂植物生物碱、致癌物和其他小分子等异种生物质的代谢。随着人类基因组的测序，已经确定了57个家庭成员。p450在几乎所有药物的代谢中的作用使这一酶家族在医学和人类健康方面引起了相当大的兴趣。虽然只有大约12个p450参与药物代谢，但它们能够代谢数十万种化合物。在药物代谢P450酶中如此广泛的底物特异性的一个警告是，这些酶也容易受到自身反应产物的抑制。这种类型的抑制被称为基于机制的抑制，并导致酶的不可逆失活。作为机制抑制剂的化合物是了解这些酶的结构/功能的重要工具。由于它们与酶靶共价结合，基于机制的抑制剂可以帮助鉴定酶机制中重要的氨基酸侧链。此外，了解机制抑制的途径和药物官能团有助于未来药物的合理设计。本研究的总体目标是了解不同化合物对P450不可逆抑制的机制。一个重要的药物代谢酶是P450 2D6。虽然该酶只占肝脏总P450的不到5%，但它负责约25%的药物化合物的代谢。该酶还显示出多种多态性形式，这有助于对P450 2D6代谢的药物反应的个体差异。这项工作的目的是确定在P450 2D6和药物Schering 66712之间形成的加合物的类型，Schering 66712是已知的第一个基于机制的2D6抑制剂和含有哌嗪的化合物，在结构上代表了许多药物。多种实验方法，包括14c标记化合物凝胶电泳，HPLC，分子模型和质谱，将被用来了解Schering 66712和P450 2D6之间相互作用的性质。最后，我们将鉴定可能对其激活很重要的先灵66712代谢物。我们相信，从这些实验中获得的知识将有助于理解含哌嗪的化合物如何与P450 2D6相互作用，并有助于未来的药物设计，以寻求避免这种药物诱导的抑制，可能导致不利的药物-药物相互作用。重要的是，这里提出的项目将有助于理解基于机制的P450 2D6的抑制- P450 2D6是一种高度多态性，在药物代谢中重要的酶，其基于机制的抑制知之甚少。公共卫生相关性：不良的药物-药物相互作用在服用多种药物（非处方药和处方药）的个人中很常见，特别是在老年人中。药物-药物相互作用的一个重要原因是药物失活的酶负责代谢其他共同给药的药物。这里提出的研究将通过增加我们对某些类别的药物如何在个体中相互作用并导致药物引起的不利医疗事件的理解而有益于人类健康。

DESCRIPTION (provided by applicant): Cytochrome P450 enzymes 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. With the sequencing of the human genome, fifty-seven family members have been identified. The role of P450s in metabolism of nearly all drugs has made this family of enzymes of considerable interest in medicine and for human health. While only about a dozen P450s are involved in drug metabolism, they are able to metabolize hundreds of thousands of compounds. One caveat of such broad substrate specificity among drug metabolizing P450 enzymes is that the enzymes are also susceptible to inhibition by products of their own reactions. This type of inhibition is called mechanism-based inhibition and leads to irreversible inactivation of the enzyme. Compounds that act as mechanism-based inhibitors are important tools for understanding structure/function of these enzymes. Because they become covalently attached to their enzyme targets, mechanism-based inhibitors can aid in identification of important amino acid side chains in the enzyme mechanism. Also, understanding of pathways and drug functional groups involved in mechanism-based inhibition can aid in rational design of future drugs. The overall goal of this research is to understand mechanisms of P450 irreversible inhibition by different compounds. One important drug-metabolizing enzyme is P450 2D6. While constituting less than 5% of the total liver P450, the enzyme is responsible for metabolism of ~25% of pharmaceutical compounds. The enzyme also displays multiple polymorphic forms that contribute to inter-individual difference in responses to drugs metabolized by P450 2D6. The objectives of this work are to identify the type of adduct formed between P450 2D6 and the drug Schering 66712 - the first known mechanism based-inhibitor of 2D6 and a piperazine- containing compound structurally representative of numerous drugs. Multiple experimental approaches including gel electrophoresis with 14C-labeled compound, HPLC, molecular modeling, and mass spectrometry, will be used to understand the nature of the interaction between Schering 66712 and P450 2D6. Finally, we will identify metabolites of Schering 66712 that may be important for its activation. We believe that the knowledge gained from these experiments will contribute to the understanding of how piperazine-containing compounds interact with P450 2D6 and aid in future drug design that seeks to avoid this type of drug induced inhibition that can lead to unfavorable drug-drug interactions. Importantly, the project proposed here will contribute to the understanding of mechanism-based inhibition of P450 2D6 - a highly polymorphic, important enzyme in drug metabolism for which little is known of its mechanism-based inhibition. PUBLIC HEALTH RELEVANCE: Adverse drug-drug interactions are common among individuals who take multiple drugs (both over the counter and prescribed), particularly among older persons. A significant cause of drug-drug interactions is inactivation by drugs of the enzymes responsible for metabolism of other co-administered drugs. The research proposed here will benefit human health by adding to our understanding of how certain classes of drugs may interact in individuals and cause drug-drug induced unfavorable medical events.