Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: I. Assessment of inhibitor structure-activity relationship and cholesterol biosynthesis inhibition properties.

A series of 15-, 32-, and 15,32-substituted lanost-8-en-3 beta-ols is described which function as inhibitors of cholesterol biosynthesis. These agents inhibit lanosterol 14 alpha-methyl demethylase activity as well as suppress HMG-CoA reduction activity in cultured cells. Several of these agents are extremely potent as both demethylase inhibitors and reductase suppressors, while others are more selective in their activities. Selected regio double bond isomers show preference for demethylase inhibition with the following order: delta 8 > delta 7 > delta 6 = unsaturated sterols. Comparisons also show that 4,4-dimethyl sterols are always more potent demethylase inhibitors and reductase suppressors than their 4,4-bisnomethyl counterparts. However, evaluation of an extensive oxylanosterol series leads us to conclude that demethylase inhibition and reductase suppression are not parallel in the same molecule. In addition, the oxylanosterols, but not the oxycholesterols, are able to disrupt coordinate regulation of HMG-CoA reductase from the LDL receptor. Thus, oxylanosterol treatment at levels which suppress reductase activity enhances LDL receptor activity. These results demonstrate that compounds can be made which (1) are selective reductase suppressors enabling dissection of the dual inhibitor nature of these compounds and (2) maximize reductase suppression and LDL receptor induction without demethylase inhibition which could lead to novel agents for serum cholesterol lowering.

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme.

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.

In situ accumulation of 3 beta-hydroxylanost-8-en-32-aldehyde in hepatocyte cultures. A putative regulator of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity.

Biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) has been demonstrated in primary hepatocyte cultures treated with the lanosterol 14 alpha-methyl demethylase inhibitor miconazole. At concentrations of the drug which lead to suppressed levels of reductase activity, the appearance of a polar, mevalonate-derived sterol is noted. Cochromatography of the identified sterol with 3 beta-hydroxylanost-8-en-32-aldehyde tentatively identified the metabolite as a lanosterol 14 alpha-methyl demethylation intermediate. Subsequent isolation and characterization of the metabolite by gas chromatography/mass spectroscopy confirmed this structural assignment. When the lanosterol 14 alpha-methyl demethylase-deficient mutant, AR45, was treated with authentic metabolite, a suppression of HMG-CoA reductase was observed. These results demonstrate that metabolism of the oxygenated biosynthetic intermediate is not required to suppress reductase activity. The results also strongly support the hypothesis that oxygenated 14 alpha-methyl demethylase intermediates are endogenously generated modulators of HMG-CoA reductase activity.