Recent clinical trials have confirmed the value of lowered dietary cholesterol intake on the incidence of atherosclerotic heart disease. Esterification is considered an essential step in the overall absorption of cholesterol in the intestine. There is considerable evidence that this is catalyzed by cholesterol esterase. We have shown that depletion of intestinal cholesterol esterase, by specific immunoprecipitation or diversion of pancreatic juice or inactivation of the enzyme by deprivation of bile acid cofactors, markedly impairs cholesterol absorption. This occurs despite normal levels of intestinal ACAT activity. The principal objective of this project is to develop specific inhibitors of intestinal cholesterol esterase and to examine their influence on the activity of the enzyme and on cholesterol absorption. Studies on the nature of the catalytic site using the purified enzyme have confirmed the suitability of the system for development of enzyme-activated antagonists (suicide inhibitors). These will be of two main types: those in which the sterol molecule is replaced by a ligand which is activated when the ester linkage is cleaved, and those in which the fatty acid ligand is replaced by a poor 'leaving group', resulting in permanent acylation of the enzyme. Based on initial tests of potential inhibitors and the effectiveness of 2-halocyclohexanones, the halo-enol lactone system of Katzenellenbogen has been selected for further in-depth study. Substrate analogs will be synthesized in which the esterified ligand contains a latent haloketone or halohydrin function which is activated upon cleavage of the ester linkage by the enzyme. The inhibitory potenties of the synthetic analogs will be evaluated using purified intestinal cholesterol esterase and established enzyme assay procedures with C14-labelled cholesterol as substrate. The short-term physiological effectiveness will be tested by administration to lymph-fistula rats and measurement of changes in absorption of test doses of C14-cholesterol into lymph. Long-term effectiveness of the analogs will be evaluated in the cholesterol-fed rabbit model. Suicide inhibitors, by their nature, are chemically unreactive until activated by the target enzyme. They thus offer the dual advantages of low toxicity and high specificity. Since sterol esters must be cleaved to be absorbed, the analogs offer the additional pharmacological advantage that they will not enter the circulation and should be excreted unchanged.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Physiological Chemistry Study Section (PC)
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George Washington University
Schools of Medicine
United States
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