Conversion Of Lanosterol Research Articles

The role of cytochrome P450 in the regulation of cholesterol biosynthesis.

A ubiquitously expressed member of the cytochrome P450 superfamily, CYP51, encodes lanosterol 14alpha-demethylase, the first step in the conversion of lanosterol into cholesterol in mammals. The biosynthetic intermediates of lanosterol 14alpha-demethylation are oxysterols, which inhibit HMG-CoA reductase and sterol synthesis in mammalian cells in vitro. These oxysterols (5alpha-lanost-8-en-3beta,32-diol and 3beta-hydroxy-5alpha-lanost-8-en-32-al) are efficiently converted into cholesterol in vitro and are generally considered to be natural cholesterol precursors. When added to hepatocytes in high concentrations, besides their conversion into cholesterol, they are also rapidly metabolized into more polar sterols and into steryl esters. The 15alpha- and 15beta-hydroxy epimers of 5alpha-lanost-8-en-3beta-ol are also rapidly metabolized into more polar sterols and steryl esters but are not converted efficiently into cholesterol. Polar sterol formation from all these oxysterols is dependent on an active form of cytochrome P450. Oxysterols are potent regulators of the activities of transcription factors of the sterol regulatory element-binding protein family and of liver X-receptor alpha. It is proposed that the rapid, cytochrome P450-dependent metabolism of naturally occurring regulatory oxysterols provides a route for their deactivation so that they become incapable of affecting gene transcription. Inhibition of cytochrome P450 by the drug ketoconazole prevents the inactivation of such oxysterols, leading to a prolonged suppression of hepatic HMG-CoA reductase in vivo and in vitro.

Biosynthesis of cholesterol and lanosterol in isolated dog hepatocytes: incorporation of [1,2-13C2]- and [2-13C2H3]-acetate, and [1-13C]acetate and [1-2H2]ethanol

Since isolated dog hepatocytes have been shown to exhibit potent activity for the synthesis of cholesterol from acetic acid, an analysis of cholesterol and lanosterol biosynthesized from [1,2-13C2]- or [2-13C2H3] acetate in fresh isolated hepatocytes has been attempted in order to clarify the incorporation and distribution of the acetate carbon and hydrogen into these sterols. Lanosterol was obtained by adding SSF-109, a sterol biosynthesis inhibitor, to the hepatocytes suspension. 13C NMR spectroscopic analysis of the [13C]- and [13C2H]-labelling patterns of these compounds confirmed the following: (i) the involvement of two 1,2-hydride shifts, 20-H from C-17 and 17-H from C-13; (ii) two 1,2-methyl migrations, 13-methyl group (C-18) from C-14 and 14-methyl group (C-32) from C-8 in the cyclization of 2,3-oxidosqualene to form lanosterol; and (iii) stereospecific hydrogenation from the Si-face at C-25 in the conversion of lanosterol into cholesterol. The proton-decoupled 13C–1H COSY spectrum confirmed the stereospecific hydrogen attack at C-7, C-15 and C-24. The deuterium atoms of [1-2H2]ethanol were incorporated into lanosterol at C-2, C-6, C-11, C-12, C-16 and C-23, which arise from C-5 of mevalonic acid.

Endocrine effects of the new anti-mycotic drug amorolfine Ro 14-4767/002 after oral application

Antifungal drugs interfering with ergosterol biosynthesis in the fungal cell wall may also affect mammalian steroidogenesis by unselective inhibition of P-450 dependent monooxygenase reactions, as is known for a variety of azole compounds [1]. Other antimycotic compounds like morpholines inhibit Δ114-reduction and Δ18-Δ7-isomerization in the conversion of lanosterol, which is different from the mode of action of most azole antimycotics [2]. Therefore, the newly developed morpholine derivative amorolfine Ro 14-4767/002 [4-(3-(p-(1,1-dimethylpropyl)phenyl)2-methyl-propyl-2,6-cis-dimethylmorpholine-hydrochloride] was expected to cause lesser side-effects in the steroidogenic pathways of humans than other azole compounds.

Aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits conversion of lanosterol to C-27 sterols in mouse L cells.

Aphidicolin, a fungal metabolite which is a specific inhibitor of DNA polymerase alpha, inhibited the incorporation of [14C]acetate into desmosterol in mouse L cells by 50% at a concentration of 8.8 microM. It had no effect on acetate metabolism into fatty acids or CO2. The site of inhibition was determined to be distal to the formation of mevalonic acid since aphidicolin also inhibited the incorporation of [14C]mevalonolactone into desmosterol but had no effect on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into total nonsaponifiable lipids. High pressure liquid chromotographic analysis of the distribution of radioactivity among the nonsaponifiable lipids formed from [14C]acetate in the presence of aphidicolin indicated an accumulation of lanosterol accompanied by a proportional decrease in radiolabeled desmosterol and two of its precursors, delta 5,7,24-cholestatrienol, and 4 alpha-methyl-delta 8,24-cholestadienol. In cells exposed to aphidicolin, lanosterol accumulation was rapid (15 min) and reversible after a 3-h exposure when cells were rinsed and fresh medium added. It was concluded that aphidicolin inhibits the conversion of lanosterol to C-27 sterols. Although the exact mechanism of this inhibition has not yet been determined, addition of aphidicolin to 20,000 X g supernatant fractions of mouse liver homogenates inhibited the incorporation of [14C]mevalonolactone into cholesterol in a concentration-dependent manner, suggesting that aphidicolin may act directly on one or more of the enzymatic steps involved in lanosterol demethylation. The ubiquitous occurrence of an aphidicolin binding site on eukaryotic DNA alpha polymerases and the inhibitory action of aphidicolin at a proposed secondary regulatory site in sterol biosynthesis (lanosterol metabolism) suggest that a naturally occurring compound may exist which can regulate both DNA replication and cholesterogenesis.

Mechanism of action of Denmert, an inhibitor of sterol biosynthesis

Effects of the fungicide Denmert on sterol biosynthesis were examined. The rates of inhibition of ergosterol formation were roughly correlated to its fungitoxic activity among four fungal species tested. In cell-free homogenates of yeast (S. cerevisiae), the fungicide blocked the conversion of lanosterol to 14-desmethyl-lanosterol. It was evident that Denmert directly affects the enzymatic formation of ergosterol, selectively inhibiting the demethylation at the C-14 position of the sterol nucleus. The fungicide triarimol was found to exhibit the same effects on sterol biosynthesis as Denmert in cell-free homogenates of yeast.

Uptake of sterols by Phytophthora infestans, their intracellular distribution and metabolism

Sterols were not detected in the mycelium of Phytophthora infestans when grown on a chemically denned sterol-free medium. The incorporation of various exogenously supplied sterols into the mycelium was demonstrated. The amounts of each sterol incorporated did not correlate with the ability of that sterol to stimulate vegetative growth of the fungus on agar medium. Inter-conversions within the mycelium of one sterol to another could not be detected with cholesterol, cholestanol, stigmasterol or β -sitosterol. There was some evidence for the conversion of lanosterol to cholesterol. The fate of exogenously supplied ergosterol was unclear. Radiolabel, supplied as [4- 14 C] cholesterol to the growing mycelium, became associated predominantly with a particulate subcellular fraction. The ability of Triton X-100 to solubilize this radiolabel suggested an association with membranes. A significant proportion of the radio-label was associated with the supernatant/oily fraction of the mycelial contents. Preliminary studies of the metabolism of radiolabelled cholesterol by P. infestans showed that some conversion to metabolites of lower polarity, probably by esterification, had occurred.

Removal of the C 4 methyl groups from triterpenoids. Conversion of lanosterol into 14α-Methylcholesta-4,8-dien-3-one and the modification of ring A of methyl glycyrrhetinate

14α-Methylcholesta-4,8-dien-3-one (9a) has been synthesized from lanosterol by a route involving the ?abnormal? Beckmann rearrangement of the oxime of 5α-lanost-8-en-3-one to the 3,4-seco nitrile (1a), from which the steroidal ring A was formed via the 3,5-dioxo 4,5-seco intermediate (8a). The formation of (8a) from the seco nitrile (1a) was readily achieved in a high-yielding sequence, which entailed stepwise degradation of the isopropenyl group, protection of the introduced 5- oxo function, and reaction of the cyano group with a Grignard reagent. The application of the above route to the modification of ring A of methyl glycyrrhetinate has also been investigated. Although the required triketone (8b) could be obtained, the method was rather low- yielding due to attack by the Grignard reagent on (11b) occurring at both C3 and C 11.

Hydrogen exchange and double bond formation in cholesterol biosynthesis.

The conversion of lanosterol║to cholesterol requires a considerable number of intermediary steps involving loss or uptake of hydrogen atoms and formation and migration of nuclear double bonds. Detailed discussions on the intermediary steps in cholesterol biosynthesis are reported in several reviews (Olson 1965; Frantz & Schroepfer 1967; Goad 1970). In the present report some mechanisms in the formation of cholesterol and its sterol precursors from lanosterol are discussed. The relation between in vitro and in vivo pathways of cholesterol biosynthesis and the composition and metabolism of sterols in biological tissues is underlined.

Recent investigations on the nature of sterol intermediates in the biosynthesis of cholesterol

Lanosterol(4,4,14α-trimethyl-cholesta-8,24-dien-3β-ol) has been proposed as the primary product of the cyclization of 2,3-epoxysqualene in animal tissues. Enzymic conversion of lanosterol to cholesterol requires reduction of the ∆ 24 double bond, removal of the three extra methyl groups, and shift of the nuclear double bond from ∆ 8 position to the ∆ 5 position. Until very recently, all of the proposed sterol intermediates in the biosynthesis of cholesterol possessed nuclear double bonds in the ∆ 8 , ∆ 7 , ∆ 5,7 or ∆ 5 positions. Consideration of possible mechanisms for the removal of the methyl group at carbon atom 14 of sterol precursors led to our demonstration of the presence of cholest-8(14)-en-3β-ol in animal tissues and establishment of the convertibility of this sterol to cholesterol in rat liver homogenate preparations. Reports (from other laboratories) of the stereospecific loss of the 15α-hydrogen of lanosterol upon enzymic conversion to cholesterol led to the demonstration of the convertibility of cholesta-8,14-dien-3β-ol, cholesta-7,14-dien-3β-ol, 14α-methyl-cholest-7-en-3β,15-diol, cholest-8(14)-en-3β,15α-diol, and cholest-8(14)-en-3β,15β-diol to cholesterol in rat liver preparations. We have recently developed chromatographic methods permitting the resolution of all of the C 27 sterols in question. The results of recent experiments directed towards an understanding of the detailed metabolism of these compounds are presented herein.

Conversion of lanosterol into a compound with the carbon skeleton of fusidic acid.

A recently established method for the monodemethylation of triterpenes at C-4 has been used to convert lanosterol into 3β-acetoxy-9,11α-epoxy-4α, 14α-dimethyl-5α-cholestane. A boron trifluoride-catalysed ‘backbone’ rearrangement of this epoxide produced 3β-acetoxy-29-nor-8α, 14β-dammara-9(11), 13(17)-diene and 3β-acetoxy-4α, 14α-dimethyl-5α, 9β-cholestan-11-one.

Steroids. XXXV. Removal of the 4-methyl groups in 4,4,14alpha-trimethyl-steroids: conversion of lanosterol into 14alpha-methylcholest-4-en-3-one.

3,4-Seco-5α-lanost-4(30)-en-3-onitrile, obtained by ‘second-order’ Beckmann cleavage of 3-hydroxyimino-5α-lanostane, has been used as an intermediate in the conversion of lanosterol into 14α-methylcholest-4-en-3-one.

Biosynthesis of skin sterols. V. Effect of arsenite inhibition and the accumulation of labeled lanosta-7,24-dien-3β-ol in rat skin

Slices of epidermis from rat skin were incubated with arsenite and various radioactively labeled substrates. The following sites of inhibition of sterol biosynthesis were observed: the formation of mevalonate from acetate; the formation of squalene from mevalonate; the conversion of squalene to lanosterol; and the conversion of lanosterol and companions to cholesterol and companions. The last inhibition resulted in an accumulation of labeled Δ 7-sterols; in high concentrations of arsenite, more than 50% of the activity was in lanosta-7,24-dien-3β-ol. The arsenite did not affect the accumulation of labeled lanosterol. The formation of cholesterol was markedly inhibited by 1.7 × 10 −4 M arsenite, whereas the formation of cholest-7-en-3β-ol was inhibited only in arsenite solutions of higher concentrations. The accumulation of labeled lanosta-7,24-dien-3β-ol and the depressed formation of product sterols suggested an intermediary role for this sterol in the biosynthesis of skin sterols. 2,3-Dimercaptopropanol in the presence of equimolar amounts of arsensite enhanced the observed inhibition. Partial reversal of arsenite inhibition by 2,3-dimercaptopropanol was not observed even in the presence of a tenfold excess of the dithiol. 2,3-Dimercaptopropanol inhibited sterol formation from mevalonate in the absence of arsenite.