Molecular Probes of the Mechanisms of Cytocrhome P450

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DESCRIPTION (provided by applicant): The central theme of this program involves studies of the mechanisms of action of the drug-metabolizing enzymes cytochrome P450 and the cell signaling protein nitric oxide synthase (NOS). Metalloporphyrin model compounds are also investigated to elucidate how these enzymatic processes occur. The principal approaches involve kinetic and mechanistic studies of enzyme-substrate interactions, the synthesis and characterization of reactive iron porphyrin species as models of putative enzymic intermediates and to relate the interconversions of these species toward a molecular understanding of these proteins. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while nitric oxide synthase is the source of the signal molecules nitric oxide and peroxynitrite. Synthetic metalloporphyrins can be employed as probes to intervene in these processes in diagnostic ways. Thus, these agents may prove to be significant tools for elaborating the biology of superoxide, peroxynitrite and NO. These same metalloporphyrins have shown impressive activity in animals suggesting their application as pharmaceutical agents. Our effort seeks to provide a foundation of mechanistic and kinetic information that can be applied to in vitro models, cell culture studies and whole animal models of specific disease states such as ischemia- reperfusion, sepsis and autoimmune diseases. Experiments are aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite and these other species, while studies of protein tyrosine nitration will elucidate how proteins are damaged under conditions of oxidative stress. The studies N-hydroxyarginine oxidation aims to illuminate the range of mechanisms for NOS and to seek out new oxidation processes that may help with the rational development of NOS inhibitors. Rapid kinetic techniques have been developed to study the reactivity of these species. A central question is how to modulate the chemistry of metalloporphyrin centers between nucleophilic and electrophilic pathways that are essential to understand the variety of P450 mediated processes. Synthetic and semi-synthetic phospholipid assemblies are used to model and understand the larger scale events in the role of cytochrome c in triggering lipid oxidation and programmed cell death (apoptosis).