The effect of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor ML-236b on phytosterol synthesis in Acer pseudoplatanus tissue culture.

Sycamore cell cultures were incubated with various labelled sterol precursors. ML-236B, a fungal metabolite, caused virtually total inhibition of acetate or leucine incorporation into sterols, while mevalonate incorporation was unaffected. Sterol synthesis from endogenous precursors, measured by incorporation of [Me-14C] methionine into the side chain, continued at a reduced rate for at least 6 h after addition of the inhibitor.

Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes.

Promastigotes of 36 World Health Organization reference (and other) strains of 6 species and 10 subspecies of Leishmania were cultured in the presence of 3 antimycotic azole drugs (ketoconazole, itraconazole, fluconazole) and their population growth determined. A representative of each subspecies was also analyzed for its sterol composition. For all strains the order of azole drug activity with respect to both growth and sterol biosynthesis inhibition was itraconazole greater than or equal to ketoconazole greater than fluconazole. The inhibitory actions of the three azole drugs were greater on L. donovani and L. braziliensis subspecies and on L. mexicana amazonensis than on L. aethiopica, L. major, L. tropica and L. mexicana mexicana. The nature of the changes in sterol composition caused by the drugs was the same for all strains. The normal, major endogenous sterols of the promastigotes (5-dehydroepisterol and ergosterol) were reduced in amount to 1-2% of the total free sterols and were replaced by endogenous 14 alpha-methyl sterols and exogenous cholesterol. The changes occurred rapidly, were drug concentration dependent and coincided with growth inhibition. Six strains of those Leishmania species less sensitive to the azole drugs could be subcultured indefinitely at reduced growth rates in the presence of a ketoconazole concentration causing the same extraordinary alterations in sterol composition. This suggested that the bulk membrane functions of sterols in leishmanias can be served by 14 alpha-methyl sterols and cholesterol, albeit imperfectly, while traces of 14 alpha-desmethyl sterols are needed for uncharacterized metabolic functions.

Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes.

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2-14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14 alpha-methyl sterols accumulated in free and esterified forms (cholesterol much greater than 4 alpha,14 alpha-dimethylcholesta-8,24-dien-3 beta-ol much greater than 14 alpha-methylcholesta-8,24-dien-3 beta-ol congruent to 14 alpha-methylergosta-8,24(28)-dien-3 beta-ol much greater than 4 alpha,14 alpha-dimethylergosta-8,24(28)-dien-3 beta-ol; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14 alpha-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7, 24(28)-trien-3 beta-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6-2H]cholesterol or [4-14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14 alpha-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.

Sterols of Leishmania species. Implications for biosynthesis.

The major sterol of promastigotes of stocks of Leishmania tropica, L. donovani and 3 subspecies of L. mexicana has been identified as ergosta-5,7,24(28)-trien-3 beta-ol; and of an L. major stock as ergosta-7,24(28)-dien-3 beta-ol. 24-Methylcholesta-5,7,22-trien-3 beta-ol and 24-ethylcholesta-5,7,22-trien-3 beta-ol were minor constituents, and traces of ergosta-5,7,22,24(28)-tetraen-3 beta-ol and a C27-diene were also recognized in some species. Lanosterol and 4,4-dimethylcholesta-8,24-dien-3 beta-ol were detected in all species studied, and squalene was identified in a stock of L. tropica. The sterol composition of members of the genus Leishmania and the sterol biosynthetic pathways it implies are characteristic of yeast and other fungi.

Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells.

Murine macrophage tumor cells infected with Leishmania mexicana mexicana were exposed to the antimycotic drug ketoconazole and to [2-14C]mevalonate, then the amastigotes were isolated, collected, purified, and their free sterols were analyzed by chromatographic and mass spectrometric methods. Control amastigotes contained as products of de novo biosynthesis C28 4-desmethyl sterols (episterol, 5-dehydroepisterol), C29 4-desmethyl sterols (stigmasta-7,24 (28)-dien-3 beta-ol, stigmasta-5,7,24(28)-trien-3 beta-ol), 4-methyl sterols (4 alpha, 14 alpha-dimethylzymosterol, obtusifoliol) and a 4,4-dimethyl sterol (lanosterol). Present also were macrophage sterols (cholesterol, desmosterol) and a putative product of the C-24 alkylation of desmosterol by amastigotes (24-methylenecholesterol). Amastigotes from macrophages exposed to ketoconazole showed notable changes in the proportions, concentrations and specific activities of their free sterols; increased for 4 alpha, 14 alpha-dimethylzymosterol and decreased for the endogenous C28 and C29 4-desmethyl sterols. Such changes were observed at a ketoconazole concentration as low as 0.01 microgram ml-1. By contrast, uninfected macrophages accumulated only small amounts of lanosterol of high specific activity at a ketoconazole concentration of 10 micrograms ml-1. the ketoconazole-induced alterations in amastigote sterols parallel those previously reported in fungi and L. m. mexicana promastigotes, and suggest a biochemical mechanism for the anti-leishmanial activity of the drug in which changes in sterol composition are linked to disturbances of cell membrane structure and function, and hence to cytotoxicity.

The activity of ketoconazole and other azoles against Trypanosoma cruzi: biochemistry and chemotherapeutic action in vitro.

Trypanosoma cruzi epimastigotes in culture medium, and amastigotes and trypomastigotes in cultured human diploid lung cells were exposed to the antimycotic agent ketoconazole and their growth and/or sterol biosynthesis observed. Propagation of epimastigotes and amastigotes was impaired by concentrations of ketoconazole achievable in human serum, and amastigotes were more sensitive than were epimastigotes. Epimastigotes and trypomastigotes (non-dividing stage) displayed changes in their membrane sterol content such that the amounts of normal, end-product sterols (ergosterol, ergosta-5,7-dien-3 beta-ol, 24-ethylcholesta-5,7,22-trien-3 beta-ol, 24-ethylcholesta-5,7-dien-3 beta-ol) were notably decreased and the amounts of 14 alpha-methyl sterol precursors of these sterols (24-methylenedihydrolanosterol, obtusifoliol, lanosterol) were increased. Other azole drugs, itraconazole and fluconazole, when tested on epimastigotes, evoked the same qualitative pattern of changes in free sterols. Itraconazole was nearly as potent as ketoconazole, but fluconazole was significantly less potent. The nature of the sterols found in T. cruzi and the actions of azole drugs on their biosynthesis were similar in many respects to those observed in fungi and in Leishmania species. By analogy, it would seem that the primary mechanism of action of azole drugs on T. cruzi life-cycle stages is the impairment of the cytochrome P-450 sterol 14 alpha-demethylase. The consequent loss of normal sterols and accumulation of 14 alpha-methyl sterols may be responsible for the coincident retardation or cessation of growth.

Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa.

Current treatments for diseases caused by apicomplexan and trypanosomatid parasites are inadequate due to toxicity, the development of drug resistance and an inability to eliminate all life cycle stages of these parasites from the host. New therapeutics agents are urgently required. It has recently been demonstrated that type II fatty acid biosynthesis occurs in the plastid of Plasmodium falciparum and Toxoplasma gondii and inhibitors of this pathway such as triclosan and thiolactomycin restrict their growth. Furthermore, Trypanosoma brucei has recently been demonstrated to use type II fatty acid biosynthesis for myristate synthesis and to be susceptible to thiolactomycin. As this pathway is absent from mammals, it may provide an excellent target for novel antimicrobial agents to combat these diverse parasites. Leishmania and Trypanosoma parasites produce ergosterol-related sterols by a biosynthetic pathway similar to that operating in pathogenic fungi and their growth is susceptible to sterol biosynthesis inhibitors. Thus, inhibition of squalene 2,3-epoxidase by terbinafine, 14alpha-methylsterol 14-demethylase by azole and triazole compounds and delta(24)-sterol methyl transferase by azasterols all cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytoxic consequences. However, Leishmania parasites can survive with greatly altered sterol profiles induced by continuous treatment with low concentrations of some inhibitors and they also have some ability to utilise and metabolise host sterol. These properties may permit the parasites to evade treatment with sterol biosynthesis inhibitors in some clinical situations and need to be taken into account in the design of future drugs.

Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents.

The antifungal drug miconazole and the cholesterol-lowering agent lovastatin (mevinolin) were used in combination to assess their potency as anti-leishmanial agents. The drug combination was synergistic, being more potent in terms of inhibition of promastigote proliferation, macrophage infection and amastigote numbers. In promastigote cultures the effect was more marked in Leishmania amazonensis than L. donovani. Analysis of the sterol compositions of both promastigote and amastigote cultures revealed the inhibition of sterol 14 alpha-demethylation by miconazole and showed some apparent evidence of inhibition of sterol biosynthesis by lovastatin.

A 37-kDa peroxidase secreted from liverworts in response to chemical stress.

A peroxidase was purified from the culture medium of a suspension culture of Marchantia polymorpha (liverwort) after treatment with bornyl acetate, which acts as a chemical stress agent to the cells. The peroxidase was characterised as a glycoprotein of molecular mass 37-kDa having a pl of about 10 and an optimal pH of 6.5. The peroxidase was thermally stable at 50 degrees C for up to 60 min. The partial amino acid sequence of the peroxidase was determined and found to be dissimilar to the amino acid sequences of other higher plant peroxidases. The oxidative polymerization of lunularin by this peroxidase was examined and the formation of a dimer, a trimer and a tetramer was demonstrated by negative ion Fast Atom Bombardment (FAB)-mass spectroscopy of the reaction products.

Negative ion ammonia chemical ionization and electron impact ionization mass spectrometric analysis of steryl fatty acyl esters.

Synthesis of steryl palmitates, varied in the nature of the steryl moiety, provided model compounds for investigation of the mass spectrometric behavior of steryl long-chain fatty acyl esters. The structure of the steryl moiety was varied according to: (i) position and degree of unsaturation in the steroid nucleus and C-17 side-chain, (ii) position and degree of methylation, (iii) presence or absence of a 9 beta, 19-cyclopropane ring. Compounds were chosen so as to be representative of biochemically important steryl esters. Electron impact (EI) behavior of steryl palmitate esters closely resembles that of their short-chain (e.g. acetate) counterparts. M+.ions were generally weak or absent and the major high mass ions arose from characteristic fragmentations of the steroid nucleus following loss of the acyl moiety ([M-RCO2H]+.). Fragment ions characteristic of the acyl moiety were lacking. Negative ion chemical ionization (NICI) using ammonia as reagent gas, on the other hand, afforded spectra containing characteristic fragment ions [RCO2]-, [RCO2-18]-, and [RCO2-19]- from which the nature of the fatty acyl moiety can be readily deduced. Hence, NICI and EI provide complementary means of ionization for the mass spectrometric determination of structures of steryl esters.

Identification of 27-nor-(24R)-24-methylcholesta-5,22-dien-3ß-ol and brassicasterol as the major sterols of the marine dinoflagellateGymnodinium simplex.

The major 4α-monomethyl sterol of the dinoflagellateGymnodinium simplex was identified as (24S)-4α,24- dimethylcholestan-3ß-ol. The major 4-demethyl sterols were characterized as (24R)-24-methylcholesta-5,22-dien-3ß-ol (brassicasterol) and 27-nor-(24R)-24-methylcholesta-5,22-dien-3ß-ol. The latter sterol has the opposite configuration at C-24 to that assigned to occelasterol, which has the same basic structure and has previously been reported as a constituent of the sterols of a marine worm. 24-Nor-cholesta-5,22-dien-3ß-ol was also identified along with several other trace sterols. The co-occurrence of 27-nor-(24R)-cholesta-5,22-dien-3ß-ol together with 24-nor-cholesta-5,22-dien-3ß-ol and brassicasterol provides new evidence for the biosynthetic origins of the two former nor-sterols. It is suggested that they may be produced de novo by a route involving nor-isoprenoid pyrophosphates and nor-squalene as intermediates, rather than as bacterial degradation products of brassicasterol (or related sterols) as previously suggested in the literature.

Biosynthesis of 24-methylcholest-5-en-3ß-ol and 24-ethylcholest-5-en-3ß-ol inZea mays.

The relative rates of synthesis of 24-methylcholest-5-en-3ß-ol and 24-ethylcholest-5-en-3ß-ol inZea mays shoots were determined using [2-(14)C]mevalonic acid and [methyl-(14)C]methionine as substrates. The 24-ethylsterol had a higher specific activity and it apparently was synthesized at about 3-4 times the rate of the 24-methylsterol.(1)H NMR spectroscopy showed that the 24-ethylsterol was predominantly the 24α-epimer but the 24-methylsterol was a mixture of the 24α-epimer (30-40%) and the 24ß-epimer (60-70%). The results are discussed in relation to the involvement of Δ(24(28))-, Δ(23), Δ(25)- and Δ(24(25))-sterol intermediates in 24-methyl- and 24-ethylsterol production.

Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies.

A simple method is described for the preparation of [6,7,7(-2)H3] sterols and steroids. The synthesis starts with a Δ(5)-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7(-2)H3]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7(-2)H3]-Δ(5) sterol or steroid. [6,7,7(-2)H3]Cholesterol, [6,7,7(-2)H3]pregnenolone and [6,7,7(-2)H3]3ß-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7(-2)H3]progesterone was prepared from the [6,7,7(-2)H3]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7(-2)H3]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7(-2)H3]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3ß-hydroxypregnan-20-one. However, the 5ß-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7(-2)H3]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one.