Squalene epoxide cyclase and microemulsion.

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non-ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.

Potent inhibition of cholesterol biosynthesis in 3T3 fibroblasts by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new 2,3-oxidosqualene cyclase inhibitor.

N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new compound rationally designed to inhibit the 2,3-oxidosqualene cyclase (M. Taton et al., Biochem. biophys. Res. Commun. 138, 764, 1986) was studied as an inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment of cells, which were grown for 2 days in a delipidated medium, resulted in a dramatic decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 of 20 nM was calculated, which classes this drug amongst the most powerful cholesterol biosynthesis inhibitors acting at the 2,3-oxidosqualene-lanosterol cyclase tested so far on mammalian cells. The inhibition of the C27-sterols synthesis was correlated with the accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene indicating that the cyclase was indeed an intracellular target of the drug. A minor secondary target was identified as the sterol-8-ene isomerase. Cells treated with the inhibitor also accumulated sterols more polar than cholesterol which could originate, for example, from the metabolization of 2,3:22,23-dioxidosqualene. Treatment of the cells with increasing concentrations of the drug resulted in a progressive reduction of the HMG-CoA reductase activity (up to 50% of control). The drug affected normal growth of the fibroblasts and growth arrest was correlated with a decrease in cellular cholesterol content to less than 50% of control. This work indicates that N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol is a potent and promising new tool in the inhibition of cholesterol biosynthesis in mammalian cells.

Partial purification of 2,3-oxidosqualene-lanosterol cyclase from hog-liver. Evidence for a functional thiol residue.

2,3-Oxidosqualene-lanosterol cyclase is an intrinsic microsomal protein which can be solubilized by ionic (deoxycholate) and nonionic (emulphogene) detergents with good yields. The hog-liver microsomal cyclase was purified approximately 140-fold by chromatography on DEAE-cellulose and hydroxylapatite. The partially purified enzyme was inactivated by N-ethylmaleimide, following pseudo-first order kinetics, indicating that a cysteine residue is essential for activity.

The squalene-2,3-epoxide cyclase as a model for the development of new drugs.

The 2,3-oxido squalene (SO) cyclases represent a group of enzymes which convert SO into polycyclic triterpenoids such as lanosterol, cycloartenol, cucurbitadienol and beta-amyrin. Taking into account the postulated model of the enzymatic cyclization of SO, we have investigated the possibility of designing compounds that would be selective and potent inhibitors of SO cyclases. Due to the fundamental role of sterols in animal, higher plant and fungal tissues, these inhibitors might behave as very selective (ipocholesterolemic, antifungal or phytotoxic) drugs. Our first approach was the synthesis and biological evaluation of 2-aza-2,3-dihydrosqualene and its derivatives which, being protonated at physiological pH, would present some similarities to the C-2 carbon ion generated by the opening of the oxirane ring of SO. Microsomes from different sources (germinated pea cotyledons, maize seedlings, rat liver and yeasts) were utilized to determine the inhibition values (I50: concentration of inhibitor producing 50% inhibition at a given substrate concentration). From the results obtained so far we conclude that 2-aza-2-dihydrosqualene and its derivatives strongly inhibited the cyclases, the site of the enzyme responsible for binding to the inhibitor is quite sensitive to the steric hindrance, and the degree of the inhibitory activity is greater in higher plants than in rat liver or fungi.

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3-dihydrosqualene and derivatives, and by other ammonium-containing molecules.

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.