Effects of novel synthetic sterol probes on enzymes of cholesterol metabolism in cell-free and cellular systems

A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7 alpha-hydroxylase. The compounds were 6-fluoro-5-cholesten-3 beta-ol (6-fluorocholesterol), 7-cholesten-3 beta-ol (7-cholestenol), 6 beta-fluorocholestan-3 beta-ol (6 beta-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3 beta-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3 beta-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3 beta-hydroxy group was essential for interaction with both enzymes since 3-acetoxy-6-fluorocholestanol did not affect any of the enzyme-catalysed reactions. 6-Fluorocholesterol and 7-cholestenol had no effect on cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6 beta-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around C-6 and C-7 of the sterol are important. These results show that it is possible to dissect components of cholesterol metabolism using simple, specifically substituted sterols and thus define a new approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.