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

描述（由申请方提供）：人体中的细胞色素P450酶是一个含血红素的酶家族，参与类固醇、维生素、类花生烷和异生物质（如药物、复合植物生物碱、致癌物和其他小分子）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在几乎所有药物代谢中的作用使得这个酶家族在医学和人类健康方面具有相当大的兴趣。虽然只有十几个P450参与药物代谢，但它们能够代谢数十万种化合物。药物代谢P450酶中这种广泛的底物特异性的一个警告是，这些酶也容易受到其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。作为基于机制的抑制剂的化合物是理解这些酶的结构/功能的重要工具。由于它们与酶靶共价连接，基于机制的抑制剂可以帮助识别酶机制中重要的氨基酸侧链。此外，了解参与基于机制的抑制的途径和药物官能团可以帮助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的不可逆抑制机制。一种重要的药物代谢酶是P450 2D 6。虽然占总肝脏P450的不到5%，但该酶负责约25%的药物化合物的代谢。该酶还显示出多种多态性形式，这些多态性形式有助于对P450 2D 6代谢的药物的反应的个体间差异。这项工作的目的是确定P450 2D 6和药物Schering 66712之间形成的加合物的类型，Schering 66712是第一个已知的基于2D 6抑制剂的机制，是一种含有哌嗪的化合物，在结构上代表了许多药物。多种实验方法，包括凝胶电泳与14 C-标记的化合物，HPLC，分子建模和质谱，将被用来了解先灵66712和P450 2D 6之间的相互作用的性质。最后，我们将确定Schering 66712的代谢产物，这些代谢产物可能对其活化很重要。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 2D 6相互作用，并有助于未来的药物设计，以避免这种类型的药物诱导的抑制，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了解P450 2D 6的机制为基础的抑制-一个高度多态性，在药物代谢中的重要酶，其机制为基础的抑制知之甚少。公共卫生相关性：药物之间的不良相互作用在服用多种药物（非处方药和处方药）的人中很常见，特别是在老年人中。药物间相互作用的一个重要原因是药物使负责其他联合给药药物代谢的酶失活。这里提出的研究将有利于人类健康，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-药物诱导的不利医疗事件的理解。

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.