Sterols in blood of normal and Smith-Lemli-Opitz subjects.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols. The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.

Sterol synthesis. Synthesis of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one and its effects on HMG-CoA reductase activity in Chinese hamster ovary cells, on ACAT activity in rat jejunal microsomes, and serum cholesterol levels in rats.

3 beta-Hydroxycholest-5-en-7-one (I; 7-ketocholesterol) is an oxysterol of continuing interest in biology and medicine. In the present study, we have prepared a side-chain fluorinated analog, 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI), with the anticipation that the F7 substitution would block major metabolism of the 7-ketosterol, and thereby enhance its potential in vivo effects on serum cholesterol levels and other parameters. Chromium trioxide/dimethyl pyrazole oxidation of the acetate derivative of the previously described 25,26,26,26,27,27,27-heptafluorocholest-5-en-3 beta-ol (Swaminathan et al., 1993. J. Lipid Res. 34, 1805-1823) followed by mild alkaline hydrolysis gave VI. The effects of VI on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in Chinese hamster ovary (CHO-K1) cells, on acyl coenzyme A-cholesterol acyltransferase (ACAT) activity in rat jejunal microsomes, and on serum cholesterol levels and other parameters in male Sprague-Dawley rats were determined and compared with those obtained with I and with another alpha, beta-unsaturated ketosterol, i.e. 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (II). I and VI showed essentially the same potency, considerably less than that of II, in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells. Whereas addition of II to rat jejunal microsomes inhibited ACAT activity (IC50 approximately 3 microM), I and VI had no effect under the conditions studied (from 1 to 16 microM). Dietary administration of I, at levels of 0.1 and 0.15%, had no effect on food consumption, gain in body weight, or serum cholesterol levels. At 0.2%, I caused a modest decrease in body weight gain and a slight decrease in serum cholesterol levels (relative to ad libitum but not pair-fed control animals). The F7-7-ketosterol VI, at 0.26% in diet (the molar equivalent of 0.2% I), had no effect on food consumption, body weight, or serum cholesterol levels. Administration of I (0.1, 0.15 or 0.2% in diet) caused increases in the weight of small intestine. In contrast, no effect of VI (0.26% in diet) on small intestinal weight was observed.

An updated look at the analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) and GC are commonly used methods for the identification and quantitation of sterols from samples of biological origin. To investigate the utility and limitations of these methods, we have determined gas chromatographic mobilities and mass spectral properties of 5alpha-cholestan-3beta-ol and 26 unsaturated C27 sterols as their acetate and trimethylsilyl (TMS) ether derivatives by GC and GC-MS. The GC retention data showed that numerous sterols were essentially coeluted on capillary GC columns coated with either 5% phenyl-95% methyl polysiloxane or polyethylene glycol, although the peaks were more widely dispersed on the latter column. Mass spectra of many groups of sterol isomers were also quite similar. Sterol mixtures of any complexity are likely to contain coeluting components, and attempts to establish structures based on mass spectra that may represent a mixture of sterol isomers could easily lead to errors. Our results demonstrate that GC and GC-MS alone cannot generally be used for rigorous structure determinations of individual components in mixtures of unsaturated sterols. However, all but a few of the 26 sterols could be distinguished by their combined chromatographic mobilities on the two GC columns coupled with critical examination of their mass spectra. GC-MS analysis of appropriate sterol subclasses or preferably individual sterol components obtained by prior purification by other methods may provide valuable supporting evidence for the identification of sterol structures. Reliability of identification is dependent upon careful attention to GC and MS conditions, calibration of GC and MS data with authentic sterol standards, and consideration of possible decomposition under GC conditions and of the effect of overloading on GC retention times.

Sterol synthesis. A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis.

Sterol intermediates in the biosynthesis of cholesterol have recently assumed a very prominent position in a number of important problems in medicine and biology. In studies of these matters, the separation and identification of the sterol intermediates present formidable challenges, a situation which does not appear to be generally appreciated. High performance liquid chromatography (HPLC) is a simple and rapid approach for the separation of the concerned compounds. Reversed phase HPLC is very commonly used for this purpose. In the present studies, we have evaluated the capabilities of reversed phase, normal phase, and silver ion HPLC for the separation of sterols. Using an extensive collection of authentic sterols, our studies indicate very limited capabilities of reversed phase and normal phase HPLC for the separation of C27 sterols differing in the number and location of olefinic double bonds. In contrast, silver ion HPLC provided remarkable separations of the same compounds, either as the free sterols or their acetate derivatives. These findings, coupled with the results of recent studies of the properties of the same compounds by gas chromatography and by nuclear magnetic resonance and mass spectroscopy, have important implications regarding current application of methodologies for the separation, identification, and quantitation of sterol intermediates in cholesterol biosynthesis as critical portions of investigations on a number of current and emerging problems in biology and medicine.

Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

We report that silver ion HPLC provides remarkable separations of C27 sterols differing only in the number or location of olefinic double bonds. This technique has been extended to LC-MS, analysis of purified components by GC, GC-MS, and 1H NMR, and to its use on a semipreparative scale. The application of this methodology for the demonstration of the catalysis, by rat liver microsomes, of the conversion of 7-dehydrocholesterol to cholesta-5,8-dien-3 beta-ol is also presented.

Inhibitors of sterol synthesis. Metabolism-based design and construction of a new analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and its effects in cultured mammalian cells and in rats.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent regulator of cholesterol metabolism. In the present study, the 7 alpha-methyl-25,26,26,26,27,27,27-heptafluoro analog (X) of I has been synthesized with the goal of blocking not only the side chain oxidation of I but also its conversion to cholesterol. X was prepared in seven steps from the known 7 alpha-methyl analog (IX) of I. Treatment of the acetate of IX with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid gave 3 beta-acetoxy-7 alpha-methyl-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII) in remarkably high (68%) yield. Dehydration of XII via the orthonitrophenylselenide to the 23-ene, followed by addition of (CF3)2CFI gave (23R)-3 beta-acetoxy-7 alpha-methyl-23-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (XV). Reductive deiodination of XV with tributyltin hydride, followed by hydrolysis of the acetate gave 3 beta-hydroxy-7 alpha-methyl-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (X). The F7-7 alpha-methyl-15-ketosterol X lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells with a potency equivalent to that of I. X showed significant hypocholesterolemic action upon oral administration to rats, with a potency far in excess of the 7 alpha-methyl-15-ketosterol IX lacking the F7 substitution. In marked contrast to I, X showed little or no suppression of food consumption in rats. Upon oral administration of X to rats, low levels of X (relative to cholesterol), characterized by chromatographic and gas chromatography-mass spectrometric methodologies, were observed in serum, liver, and small intestine. No material was observed with the expected properties of F7-7-methylcholesterol (or potential intermediates in its possible formation from X). In contrast to I, X lowered serum cholesterol levels at dosages at which no effect on food consumption was observed.

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholestan-15-one and its 17 beta-epimer and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

3 beta-Hydroxy-5 alpha-cholestan-15-one (2a) and its 14 beta-epimer 2b were prepared from 3 beta-acetoxy-5 alpha-cholest-8(14)-ene (3). Hydroboration of 3 at 45-50 degrees C gave a mixture of 5 alpha,14 alpha-cholestane-3 beta,15 alpha-diol and 5 alpha,14 beta-cholestane-3 beta,15 beta-diol, which were separated on silica gel as their 3 beta-tert-butyldimethylsilyl ethers 5a and 5b. Oxidation of 5a with pyridinium chlorochromate, followed by desilylation with tetrabutylammonium fluoride gave 2a. Analogous transformations of 5b gave 2b contaminated with 2a. Desilylation of 5b followed by oxidation with pyridinium chlorochromate resulted in a mixture composed mainly of 5 alpha,14 beta-cholestane-3,15-dione and 2b. Successive chromatographic separations on silica gel and reversed phase media gave 2b of high purity. Compound 2a was also prepared by lithium-ammonia reduction of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (96% yield) and by selective reduction of 5 alpha-cholestane-3,15-dione with lithium tri-tert-butoxyaluminum hydride (90% yield). Isomers 2a and 2b were readily epimerized under acidic or basic conditions or under conditions used for gas chromatographic analysis. The purities of 2a and 2b were measured from nuclear magnetic resonance (NMR) spectra; chromatographic methods gave less reliable estimates of purity. NMR data also showed that ring C of the 14 beta sterols is predominantly in a chair conformation. The effects of 2a and 2b on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase have been studied in Chinese hamster ovary cells.

Inhibitors of sterol synthesis: synthesis and spectral properties of derivatives of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one fluorinated at carbon 7 or carbon 9 and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells.

As part of a program to prepare delta 8(14)-15-ketosterols that cannot readily be metabolized to cholesterol or side-chain oxygenated species, we have prepared 3 beta-hydroxy-7 alpha-fluoro-5 alpha-cholest-8(14)-en-15-one (VII) and the 9 alpha-hydroxy (IV), 9 alpha-fluoro (VI) and 7 alpha-fluoro (VIII) derivatives of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (II). Sterol IV was prepared by oxidation of the delta 8,14 dienol ethyl ether of the 3 beta-acetate of II with m-chloroperbenzoic acid, followed by mild alkaline hydrolysis of the 3 beta-acetate derivative of IV. Treatment of IV with hydrogen fluoride-pyridine gave VI. The 7 alpha-fluoro-15-ketosterols VII and VIII were synthesized by treating the 3 beta,15-bis-trimethylsilyl delta 7,14-dienol ether derivative of the appropriate delta 8(14)-15-ketosterol with N-fluoropyridinium triflate, followed by hydrolysis of residual trimethylsilyl ethers and purification by high-performance liquid chromatography. The combined results of 1H and 13C nuclear magnetic resonance (NMR) chemical shifts, 1H-1H coupling constants, 1H-19F long-range coupling constants and molecular modeling indicated that a 7 alpha-fluoro, 9 alpha-fluoro or 9 alpha-hydroxy substituent has negligible effect on the conformation of the 15-ketosterols. 1H and 13C-NMR data are also given for delta 6,8(14)- and delta 8(14),9(11)-15-ketosterols, synthetic byproducts that could not be detected readily in samples of the fluoro-15-ketosterols by chromatographic methods. Mass spectra of VI and of previously reported 9 alpha-fluoro and 9 alpha-hydroxy-delta 8(14)-15-ketosterols showed abundant M-62 or M-60 ions that appear to correspond to loss of ketene and HF or H2O. The 9 alpha-hydroxy-F7-15-ketosterol IV, the 7 alpha-fluoro-15-ketosterol VII and the 7 alpha-fluoro-F7-15-ketosterol VIII were of equivalent potency to the parent 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. The 9 alpha-fluoro-F7-15-ketosterol VI showed high potency but appeared to be slightly less active than I.

Inhibitors of sterol synthesis. Effects of a new fluorinated analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one in rats.

3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (VII), an analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in which conversion to 26- and 25-oxygenated metabolites is blocked by the F7-substitution, was administered to male Sprague-Dawley rats at levels of from 0.025 to 0.15% by weight in a ground chow diet. Administration of VII resulted in lowering of the levels of serum cholesterol at dosages as low as 0.025% by weight in diet. In marked contrast to I, VII had little or no effect on food consumption. Whereas administration of I at a level of 0.1% by weight in diet resulted in a cessation of growth, VII, at approximately the same molar concentration in diet, had only slight or no effect on changes in total body weight. Significant levels of 25,26,26,26,27,27,27-heptafluorocholesterol (VIII) were observed in serum and liver, indicating the conversion of VII to VIII. Characterization of VIII in liver was based upon the results of gas chromatography, low and high resolution mass spectral studies, infrared spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The levels of VIII in serum appeared to be related to dosage and duration of administration of VII.

Inhibitors of sterol synthesis: 3 beta-hydroxy-25,26,26,26,27,27,27- heptafluoro-5 alpha-cholestan-15-one, an analog of a potent hypocholesterolemic agent in which its major metabolism is blocked.

The chemical synthesis of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro- 5 alpha-cholestan-15-one (IV) has been pursued to provide an analog of the potent hypocholesterolemic agent 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in which its major metabolism is blocked. Reduction of 3 beta-acetoxy-5 alpha-chola-8(14),23-dien-15-one with lithium in liquid ammonia gave 3 beta-hydroxy-5 alpha-chol-23-en-15-one (VI). Addition of (CF3)2CFI to VI in the presence of triethylborane gave 3 beta-hydroxy-23R-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholestan- 15-one, which was reduced to IV with tributyltin hydride. IV was found to be highly active in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells, in lowering acyl coenzyme A:cholesterol acyltransferase activity in jejunal microsomes, and in lowering serum cholesterol levels in rats.

Inhibitors of sterol synthesis. Effects of fluorine substitution at carbon atom 25 of cholesterol on its spectral and chromatographic properties and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

25-Fluorocholesterol (III) was prepared by treatment of 25-hydroxycholesterol (IV) with hydrogen fluoride-pyridine. Compounds III, IV, and cholesterol (I) were fully characterized by 1H and 13C NMR, and stereochemical assignments were established for the C-22 and C-23 protons. The side-chain proton assignments, which apply to most other sterols with a saturated eight-carbon side chain, were based on conformational analysis and comparisons with NMR data for 25,26,26,26,27,27,27-heptafluorocholesterol (II). The chromatographic behavior of I, II, and III were compared on thin-layer chromatography, high performance liquid chromatography, and gas chromatography. Major fragment ions in electron-impact mass spectra of III were analogous to ions of either cholesterol or desmosterol, and a similar analogy was observed for the trimethylsilyl ethers. The 25-hydroxysterol IV and the 25-fluorosterol III differed markedly in their effects on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. Whereas 25-hydroxycholesterol caused a approximately 66% lowering of reductase activity in cells at 0.1 microM, the 25-fluorosterol III had no effect at this concentration.

Inhibitors of sterol synthesis: effects of a 7 alpha-alkyl analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells and on serum cholesterol levels and other parameters in rats.

The 7 alpha-methyl analog (II) of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15- one (I) was prepared by chemical synthesis and evaluated with respect to its effects on HMG-CoA reductase activity in CHO-K1 cells and on serum cholesterol levels in rats. The 7 alpha-methyl substitution had no detectable effect on the potency of I in lowering HMG-CoA reductase activity in the cultured cells. In contrast, the 7 alpha-methyl substitution had a marked effect on the action of I in the suppression of food consumption in rats. Whereas II was less potent than I in lowering serum cholesterol levels in rats, it did so at dosage levels at which only slight or moderate effects on food consumption were observed. Full 1H and 13C-NMR assignments for II and intermediates in its synthesis have been presented. Conformational analysis, based on 1H-1H coupling constants, NMR shieldings and force-field calculations, indicated that the 7 alpha-methyl substitution had virtually no effect on the conformation of the 15-ketosterol apart from minor distortions of ring B.

Inhibitors of sterol synthesis. Chemical synthesis and properties of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one and 25,26,26,26,27,27,27-heptafluorocholesterol and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells.

A side-chain fluorinated delta 8(14)-15-ketosterol has been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VII) as part of a program to prepare new analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. 3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (VIII) was prepared in five steps from VII in 38% overall yield. Dehydration of VII via the ortho-nitrophenylselenide to the 23-ene, followed by addition of (CF3)2CFI gave 3 beta-acetoxy-23R-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one. Reductive deiodination with tributyltin hydride, followed by hydrolysis of the acetate gave VIII. 25,26,26,26,27,27,27-Heptafluorocholest-5-en-3 beta-ol (XXI) was prepared in eight steps in 31% overall yield from 3 alpha,6 alpha-diacetoxy-5 beta-cholanic acid (XIII). Compound XIII was reduced with borane-methyl sulfide to the corresponding 24-hydroxysteroid, which was converted to 3 alpha,6 alpha-diacetoxy-25,26,26,26,27,27,27-heptafluoro-5 beta-cholestane (XVIII) by reactions analogous to those developed for the preparation of VIII from VII. Conversion of XVIII via the 3 alpha,6 alpha-diol to the 3 alpha,6 alpha-ditosylate, followed by heating with potassium acetate in dimethylformamide and subsequent hydrolysis gave XXI. Full 1H and 13C NMR assignments are presented for VIII, XXI, and intermediates involved in their synthesis. 13C NMR assignments for 3 alpha,6 alpha-dihydroxy-5 beta-steroids have been corrected, and stereochemical assignments were established for the side-chain methylene protons of VIII, XXI, and most synthetic intermediates. Compound VIII lowered the levels of HMG-CoA reductase activity in CHO-K1 cells and in HepG2 cells with a potency comparable to that of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I). In contrast, 25,26,26,26,27,27,27-heptafluorocholest-5-en-3 beta-ol had little or no effect on reductase activity in CHO-K1 cells. These combined results indicate that metabolism of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) to 26- and 25-oxygenated species is not required for the suppressive action of I on the levels of HMG-CoA reductase activity in CHO-K1 cells and HepG2 cells.

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.

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.

Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2-aza-2,3-dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor.

The effect of 2-aza-2,3-dihydrosqualene, a new compound designed to inhibit the 2,3-oxidosqualene-lanosterol cyclase [A. Duriatti et al., Biochem. Pharmac. 34, 2765 (1985)] was studied as inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment with the drug of cells which were grown for 2 days in a delipidated medium resulted in a marked decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 = 0.3 microM was calculated when the cells were preincubated for a period of 4 hr with 2-aza-2,3-dihydrosqualene. This inhibition was correlated with an intracellular 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 precursor-product relationship of the accumulated [14C]squalene oxide(s) and the [14C]sterols was demonstrated in chase experiments in the absence of drug. Sterols more polar than cholesterol were also detected in treated fibroblasts and in the cells which underwent chase experiments; they were mainly composed of 24,25-epoxycholesterol. The C27-[14C]sterols of [14C]acetate pulse labeled cells consisted in a mixture of desmosterol and cholesterol; treatment of the cells with 2-aza-2,3-dihydrosqualene resulted in a decreased conversion of desmosterol into cholesterol indicating that the delta 24-sterol reductase might be another target of the drug. 2-Aza-2,3-dihydrosqualene at 1 microM affected normal growth of 3T3 fibroblasts, this effect could be prevented by addition of exogeneous cholesterol (50 microM). Growth arrest of the treated cells was correlated with a decrease in cellular sterol content to less than 40% of controls. About 30% of the C27-sterol fraction, of the treated cells, was desmosterol. Our work demonstrates that 2-aza-2,3-dihydrosqualene is a valuable new inhibitor of cholesterol biosynthesis in mammalian cells.