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

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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.