The long-range objective of our research program is to develop mechanism based irreversible inactivators (suicide inhibitors) that will be selective for particular P450 isozymes and that can therefore be used to investigate the mechanism of chemical carcinogenesis. The three broad objectives of this proposed research are subdivided into a set of specific aims. I. The synthesis and investigation of ethynyl compounds as suicide inhibitors of P450 isozymes potentially involved in chemical precarcinogen activation in humans. 1. 2-Ethynyl and 4-ethynylpyrene (2EP, 4EP) will be tested as suicide inhibitors of P4501A1. 2. Ethynyl derivatives of adamantane will be tested as suicide inhibitors capable of differentiating P4501A2, 2B1, and 2E1 dependent activities. 3. 4- Ethynylacetanilide will be investigated as a potential selective suicide inhibitor of P4501A2 from human liver. II. The effects of ethynyl suicide inhibitors of P450 isozymes will be examined in model biological systems selected to demonstrate the potential use of such reagents in studies of chemical carcinogenesis. 1. 2EP and 4EP will be tested as selective in vivo inhibitors of benzo[a]pyrene and of 7,12-dimethylbenz[a]anthracene (DMBA) induced skin tumors in the two-stage mouse model 2. Experiments will be carried out to establish why co-application of 2-ethynylnaphthalene (2EN) changes the mutational spectrum produced by DMBA in mouse skin and causes both H-ras and K-ras mutations in the resulting tumors. 3. The effects of selective ethynyl inhibitors of P450 isozymes upon the metabolism of the tobacco specific nitrosamine, 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone in bronochiolar Clara cells and aveolar type II cells from mouse lung will be determined. III. Selective ethynyl inhibitors will be used as active site probes to label and identify features of the substrate binding sites of P450 isozymes. 1. Samples of (3)H-labeled ethynyl compounds will be used to label the active site regions of P4501A1, 1A2, 2B1, and 2E1 and the sites of the labeling established by proteolysis of the labeled P450 proteins and separation and identification of the resulting labeled peptides. 2. Fluorine-labeled 2EN will be used to label the active site region of P4502B1 and fluorine-NMR then used to establish the relative orientation of the fluorine label on the bound ethynyl inhibitor to the P450 heme iron. Appropriately designed ethynyl compounds, because of their special biological activities, can be used to make significant contributions to investigations of fundamental aspects of P450 involvement in chemical carcinogenesis.
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