The long-term objective of the proposed research is to elucidate the mechanisms of drug- and xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. The inactivation of NOS by drugs, such as guanabenz and aminoguanidine, involves the metabolic formation of reactive drug and oxygen intermediates. Our approach is to first characterize the disposition, in particular, the metabolism of the drug and related xenobiotics by NOS with the use of analytical techniques such as LC-MS/MS. We will then study how the metabolites, especially those bound to heme and protein, enhance the proteasomal degradation of the drug-altered enzyme. We have also discovered that cellular factors facilitate the insertion of heme into heme-deficient apo-NOS and we postulate that this may be a repair mechanism for metabolite- inactivated NOS. Thus, in order to understand how drug metabolism by NOS can cause adverse drug effects we propose the following specific aims: (1) To characterize the mechanism of drug-mediated inactivation of NOS. (2) To characterize what forms of inactivated and dysfunctional NOS are recognized for ubiquitylation and proteasomal degradation. (3) To characterize the heme insertion process into heme- deficient apo-NOS that is facilitated by cellular protein factors. The completion of these studies will define the chemistry of how drugs are metabolized by NOS and lead to NOS inactivation. We will show how these molecular interactions lead to defined and predictable biological responses that ultimately define the pharmacological and toxicological profiles of certain drugs. This will aid in the design of safe and effective drugs to control NOS as well as strategies to decrease adverse drug effects related to NOS. We also address the fundamental biological processes of how the heme prosthetic group is inserted into P450 NOS and how cells maintain NOS protein quality control. Overall, this work furthers our understanding of how the metabolism of drugs, especially those used chronically, can alter the normal biological processes to give rise to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics.
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