Squalene Epoxidase Inhibitor Research Articles

Effects of Seed Treatment with SAN 789 F, a Homopropargylamine Fungicide, on Germination and Contents of Squalene and Sterols of Wheat Seedlings

Wheat seedlings ( Triticum aestivum L. cv. Arina) contained mainly sitosterol and campesterol as the principal sterol compounds (71 and 21% of the total amount of free sterols, respectively), whereas stigmasterol (3%) and other unidentified sterols (together 5%) were much less abundant. Squalene, an important intermediate of sterol biosynthesis, was only found in traces. Seed treatment with SAN 789 F ( N-(5,5-dimethyl-3-hexynyl)- N-ethyl-1-naphthalenemethanamine), a homopropargylamine fungicide, induced two primary effects on sterol biosynthesis: First, squalene accumulated temporarily in large amounts within 48 hr after treatment, indicating an inhibition of squalene epoxidase; second, the content of free sterols slightly decreased within 14 days after treatment, probably as a consequence of squalene epoxidase inhibition. An additional effect of the seed treatment was an inhibition of growth occurring immediately after germination, whereas later, seedlings started to regrow. In contrast to a seed treatment with a wettable powder formulation (10 WP), seeds treated with an encapsulated "slow-release" formulation (CS 250) accumulated much less squalene. In addition, the growth of seedlings was only slightly inhibited by treatment with the CS 250 formulation. A reduced growth inhibition was also achieved when seedlings were treated between 3 and 7 days after germination. These results suggest that accumulation of squalene rather than a deficiency of free sterols is responsible for the inhibition of germination induced by the treatment with SAN 789 F. The inhibitory effects of the fungicide on growth and on squalene epoxidase were drastically reduced, when the homopropargylamine delivery to the seed was delayed by either a late application or the use of an encapsulated formulation.

Synthesis and structure-activity relationships of side-chain-substituted analogs of the allylamine antimycotic terbinafine lacking the central amino function.

Terbinafine is a therapeutically used inhibitor of fungal squalene epoxidase that has prompted extensive derivatization programs for structure-activity relationship studies. In the present study, derivatives of terbinafine were synthesized that lack the central tertiary amino group but have polar substitutents at the tert-butyl residue of the side chain. Evaluation of the antifungal potential revealed that representatives of this novel structural type can also exhibit broad antifungal activity, indicating that the central amino function of allylamine antimycotics is not essential for inhibition of fungal growth. Potency appears to correlate with the polarity of the introduced functional groups, while broad antifungal activity seems to be restricted to compounds with basic substituents. The dimethylamino-substituted "carba-analog" of terbinafine (8k) showed the best antimycotic profile within the whole series.

Metabolism of [3H] Farnesol to Cholesterol and Cholesterogenic Intermediates in the Living Rat Eye

Adult rats were injected intravitreally with all-trans [1-3H]farnesol, with or without co-injection of the squalene epoxidase inhibitor NB-598. Retinas were isolated 16 h later and their lipids were extracted, saponified, and analyzed by radio-HPLC. Most (≥90%) of the nonsaponifiable radioactivity was recovered as unmetabolized [3H]farnesol; however, about 6-8% of the radioactivity in control retinas exhibited the chromatographic behavior of sterols, including cholesterol. Unlike the controls, the NB-598-treated retinas exhibited substantial accumulation of both [3H]squalene and squalene mass. Calculations indicate that most of the squalene mass was derived from metabolism of endogenous precursors, with an in vivo biosynthetic rate of 46±17.5 pmol/retina/h. Retinas from eyes injected with all-trans [1-3H]geranylgeraniol yielded only the unmetabolized precursor in the nonsaponifiable extracts. These results suggest that farnesol can be "activated" in vivo (presumably to the corresponding allylic pyrophosphate) in the retina and subsequently metabolized to sterols and sterol precursors.

Export of steryl esters from lipid particles and release of free sterols in the yeast, Saccharomyces cerevisiae

Fatty acyl esters of the yeast specific sterol, ergosterol, are exclusively stored in lipid particles. Under conditions of sterol deficiency, e.g., in the presence of terbinafine, an inhibitor of fungal squalene epoxidase, steryl esters are hydrolyzed, and sterols are set free for membrane formation. Lipid particles do not contain steryl-ester hydrolase activity themselves; the highest specific activity of this enzyme is found in the plasma membrane. Therefore, steryl esters have to be exported from lipid particles to their site of hydrolytic cleavage. This process of translocation and metabolic conversion was studied in vivo. Addition of nocodazole to terbinafine-treated cells did not disturb the mobilization of steryl esters, indicating that this process is not mediated by microtubuli-dependent vesicle flux. Under the influence of inhibitors of cellular energy production (azide and fluoride) and protein biosynthesis (cycloheximide) mobilization of steryl esters came to an halt. These results support the view that ongoing membrane proliferation may be a driving force for the release of sterols from steryl esters of lipid particles.

Stereocontrolled synthesis of fluorosqualenes and fluoroepoxy-squalenes as inhibitors of squalene epoxidase and 2,3-oxidosqualene cyclase

Z-Fluorosqualene derivatives having one or more fluorine atoms at the terminal methyls of the squalene skeleton have been synthesized. A highly stereoselective synthesis, based on a Wittig reaction, was developed together with a new method for obtaining bifunctional derivatives of squalene. The compounds tested showed poor inhibitory activity on squalene epoxidase and 2,3-oxidosqualene cyclase from microsomes of Saccharomyces cerevisiae, Candida albicans and rat liver, and on S. cerevisiae and 3T3 fibroblast cell cultures.

Effects of NB-598, a squalene epoxidase inhibitor, on the secretion of apolipoprotein B

Effects of NB-598, a squalene epoxidase inhibitor, on the secretion of apolipoprotein B

Mevalonic acid-dependent degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo and in vitro.

The microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to rapid degradation when cells are incubated with sterols or mevalonic acid (MVA). It has been shown that this rapid degradation is dependent upon both a sterol and another MVA-derived metabolite (Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 258, 8929-8937). In the current study, inhibitors of the isoprene biosynthetic pathway were used to define further this mevalonic acid derivative involved in the accelerated degradation of HMG-CoA reductase. The accelerated degradation of HMG-CoA reductase in met-18b-2 cells, which is induced by the addition of MVA, was inhibited by the presence of the squalene synthase inhibitor, zaragozic acid/squalestatin, or the squalene epoxidase inhibitor, NB-598. Accelerated degradation of HMG-CoA reductase was observed when NB-598-treated cells were incubated with both MVA and sterols. In contrast, the addition of MVA and sterols to zaragozic acid/squalestatin-treated cells did not result in rapid enzyme degradation. This MVA- and sterol-dependent degradation of HMG-CoA reductase persisted in cells permeabilized with reduced streptolysin O. Finally, the selective degradation of HMG-CoA reductase was also observed in rat hepatic microsomes incubated in vitro in the absence of ATP and cytosol. We conclude that the MVA-derived component that is required for the accelerated degradation of HMG-CoA reductase is derived from farnesyl disphosphate and/or squalene in the isoprenoid biosynthetic pathway. We propose that this component has a permissive effect and does not, by itself, induce the degradation of HMG-CoA reductase. We also conclude that the degradation of HMG-CoA occurs in the endoplasmic reticulum, and, once the degradation of HMG-CoA reductase has been initiated by MVA and sterols, all necessary components for the continued degradation of HMG-CoA reductase reside in the endoplasmic reticulum.

In vivo biosynthesis of cholesterol in the rat retina

Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of [ 3H]acetate. HPLC analysis of retinal non-saponifiable lipid extracts revealed co-elution of radioactivity with endogenous cholesterol mass within 4.5 h post-injection. Incorporation of [ 3H]acetate into cholesterol was markedly reduced by co-injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB-598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo.

Effects of NB-598, a potent squalene epoxidase inhibitor, on the apical membrane uptake of cholesterol and basolateral membrane secretion of lipids in Caco-2 cells

Caco-2 cells grown on membrane filters were used as a model to study the effects of NB-598, an inhibitor of squalene epoxidase, on cholesterol absorption from the intestinal epithelia. NB-598 (10 μM) inhibited the synthesis of sterol and sterol ester from [ 14C]acetate without affecting the synthesis of other lipids such as phospholipids (PL), free fatty acids (FFA) and triacylglycerol (TG). When labeled lipid was apically loaded as a micellar lipid solution into Caco-2 cell cultures, NB-598 reduced basolaterally secreted radioactivity in cholesterol, cholesterol ester, PL and TG. Furthermore, NB-598 suppressed the basolateral secretion of apolipoprotein (apo) B. When microsomes prepared from control Caco-2 cells were incubated with 10 μM NB-598, acyl CoA:cholesterol acyltransferase (ACAT) activity was inhibited slightly. After incubating Caco-2 cells with 10 μM NB-598, a slight reduction in cellular ACAT activity was also observed. These results suggest that suppression of the secretion of particles containing apo B and reduction of cellular ACAT activity in the intestinal epithelia are part of the mechanism of the cholesterol-lowering effect of NB-598.

An inhibitor of squalene epoxidase, NB-598, suppresses the secretion of cholesterol and triacylglycerol and simultaneously reduces apolipoprotein B in HepG2 cells

NB-598, a specific inhibitor of squalene epoxidase, suppressed the secretion of cholesterol and triacylglycerol from HepG2 cells into the medium. L-654,969, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibited the secretion of cholesterol as potently as NB-598, but did not suppress the secretion of triacylglycerol. Both compounds decreased the intracellular cholesterol content almost equally, and neither of the compounds reduced the intracellular triacylglycerol content. The suppression of lipid secretion by NB-598 was associated with a significant reduction in apolipoprotein (apo) B secretion into the medium. Therefore, the suppression of lipid secretion by NB-598 may be caused by a reduction in the number of triacylglycerol-rich lipoprotein particles. In contrast, the suppression of cholesterol secretion by L-654,969 may be due to a modulation of lipoprotein lipid composition, since this agent did not reduce the secretion of apo B or triacylglycerol. The secretion of apo A-I was unaffected by either NB-598 or L-654,969. Pulse chase studies using [ 35S]methionine showed that the suppression of apo B secretion by NB-598 depended on an enhancement of intracellular degradation of apo B. These results indicate that the secretion of apo B from HepG2 cells is not regulated by the lipid synthesis alone, and suggest that the mechanism of the hypolipidemic effect of NB-598 involves the suppression of triacylglycerol-rich lipoprotein secretion from the liver as well as an inhibition of cholesterol synthesis in the liver.

Enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae.

Squalene epoxidase is a microsomal membrane-associated enzyme that acts as an important regulator in the sterol biosynthetic pathway. In this study, the enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae were examined. Unlike Candida squalene epoxidase, S. cerevisiae squalene epoxidase required NADPH for enzyme reaction. However, S. cerevisiae enzyme reaction did not require FAD or autologous S105 fraction. Unlike rat squalene epoxidase, the activity of S. cerevisiae was reduced by Triton X-100, a nonionic detergent. Terbinafine, an inhibitor of fungal squalene epoxidase, inhibited the enzyme in a non-competitive manner, while NB-598, an inhibitor of mammalian squalene epoxidase, barely inhibited it in a partially non-competitive manner. Thus, the properties of squalene epoxidase from S. cerevisiae were different from those of squalene epoxidase from rats and Candida, which were previously known. We propose that a species difference of squalene epoxidase exists not only between animals and fungi but between Candida and Saccharomyces.

Mammalian squalene epoxidase inhibitors and structure-activity relationships

Mammalian squalene epoxidase inhibitors and structure-activity relationships

Four Δ5, 7-sterols from terbinafine treated celery cell suspension cultures

Incubation of celery cell suspension cultures with the squalene epoxidase inhibitor, terbinafine, caused an accumulation of squalene, a depletion of steryl esters and the appearance of appreciable amounts of di- and tri-unsaturated sterols in the free sterol fraction. The four major unsaturated sterols were isolated by preparative reverse phase HPLC and identified by UV, 1H NMR and mass spectrometry as campesta-5,7-dien-3β-ol, stigmasta-5,7-dien-3β-ol, [24(241)Z]-stigmasta-5,7,24(241)-trien-3β-ol and (22E)-stigmasta-5,7,22-trien-3β-ol. Small amounts of Δ8- and Δ8, 14-sterols were also observed to accumulate in the terbinafine treated cultures. These results apparently reveal other sites of terbinafine inhibition of the sterol biosynthetic pathway in plants at the Δ7-reductase and Δ14-reductase steps, in addition to its primary inhibitory effect on squalene epoxidase.

Effects of squalene epoxidase inhibitors on Candida albicans.

The relationship between sterol biosynthesis inhibition, membrane integrity, and cell growth inhibition in Candida albicans was examined for five squalene epoxidase inhibitors. The compounds were the thiocarbamates tolnaftate and tolciclate and the allylamines naftifine, terbinafine, and SDZ 87-469. All compounds inhibited sterol biosynthesis, with the concentrations that caused a 50% decrease in the total sterol-to-squalene ratio ranging from less than or equal to 0.01 microM for terbinafine and SDZ 87-469 to 500 microM for tolnaftate. At 100 microM, the compounds also caused up to a 30% release of intracellular [14C]aminoisobutyric acid. With terbinafine and SD2 87-469, aminoisobutyric acid release further increased in cells grown at concentrations that inhibited ergosterol biosynthesis. It is suggested that inhibition of ergosterol synthesis may render the C. albicans membrane susceptible to further damage, including direct damage from squalene epoxidase inhibitors.

Patent Evaluation: Novel Inhibitors of Squalene Epoxidase and/or Oxidosqualene Cyclase

SummaryNovelty: Novel piperidyl ethers and thioethers are disclosed as squalene epoxidase inhibitors. The compounds are claimed for the treatment of ischaemia and fungal infections.Biology: In rat liver homogenates, the best compound has an IC50 of 5 μM against [3H]-squalene.Chemistry: Twenty-one examples are given for the preparation. Characterization is by mp, MS and elemental analysis. The preferred compound is 2-(1-piperidyl)pentylisopentyl sulphone.

Terbinafine: Mode of action and properties of the squalene epoxidase inhibition

Terbinafine (Lamisil) has primarily fungicidal action against many fungi as a result of its specific mechanism of squalene epoxidase inhibition. Treated fungi accumulate squalene while becoming deficient in ergosterol, an essential component of fungal cell membranes. The cidal action is closely associated with the development of high intracellular squalene concentrations, which are believed to interfere with fungal membrane function and cell wall synthesis. In the case of Candida albicans, growth inhibition with terbinafine appears to result from the ergosterol deficiency. The filamentous form of this fungus is more susceptible than the yeast form. Measurement of ergosterol biosynthesis by incorporation of radiolabelled precursors indicates a correlation between inhibition of growth and ergosterol biosynthesis in a range of pathogenic fungi. Terbinafine is a potent non-competitive inhibitor of squalene epoxidase from Candida (Ki = 30 nM). In contrast, inhibition of rat liver squalene epoxidase only occurs at higher drug concentrations (Ki = 77 microM), and is competitive with squalene. Thus, terbinafine has no effect on cholesterol biosynthesis in vivo. Squalene epoxidase is not an enzyme of the cytochrome P-450 type, thereby avoiding potential inhibition of this class of enzymes.

Effect of an inhibitor of squalene epoxidase, NB-598, on lipid metabolism in Hep G2 cells.

NB-598, a potent inhibitor of squalene epoxidase, inhibited cholesterol synthesis from [14C]acetate and induced intracellular squalene accumulation in Hep G2 cells. NB-598 inhibited cholesterol synthesis from [14C]acetate, [3H]mevalonate, and [3H]squalene, but not from [3H]2,3-oxidosqualene in Hep G2 cells. It reduced cholesterol ester synthesis remarkably in the absence of exogenous cholesterol. This compound did not have any effect on the synthesis of ubiquinone and dolichol. When Hep G2 cells were prelabeled with micellar [3H]cholesterol, NB-598 did not affect the excretion of bile acid incorporated from [3H]cholesterol. However, NB-598 decreased the secretion of free and esterified cholesterol, triacylglycerol, and phospholipids, and increased the secretion of squalene. NB-598 is thought not only to inhibit cholesterol synthesis, but also to inhibit the secretion of lipids.

Effect of squalene on the structure and function of fungal membranes

Terbinafine (Lamisil®) is a potent and specific inhibitor of fungal squalene epoxidase. Fungi treated with the drug accumulate large quantities of squalene in the form of lipid droplets but, due to its hydrophobicity, squalene partitions also into cellular membranes. Organelle membranes from terbinafine-treated Trichophyton cells can contain up to 100 times more squalene than found in controls. In artificial phospholipid membranes, squalene destabilizes the bilaminar structure by inducing transition to the inverted micellar HII phase. Thus, it can be assumed that high squalene concentrations produce disturbances in the structure of cellular membranes and thereby interfere with essential membrane functions. Biochemical and ultrastructural analyses of Trichophyton mentagrophytes treated with terbinafine have provided evidence of perturbations of vital membrane-associated functions subsequent to squalene accumulation. Synthesis of membrane phospholipids was markedly reduced whereas the rate of degradation of...

Selective action of allylamines and its therapeutic implications

Terbinafine (Lamisil®) is an orally active antimycotic of the allylamine class with activity against a wide range of pathogenic fungi, especially dermatophytes. The primary mechanism of action of these compounds is the specific inhibition of fungal squalene epoxidase. Measurement of ergosterol biosynthesis by incorporation of radiola-belled precursors indicates a correlation between inhibition of growth and of ergosterol biosynthesis in a range of pathogenic fungi. Fungi treated with these drugs accumulate squalene while becoming deficient in ergosterol, an essential component of fungal cell membranes. The characteristic primary fungicidal action of terbinafine and related allylamines is closely associated with the development of high intracellular squalene concentrations, which are thought to interfere with fungal membrane function and cell wall synthesis. Terbinafhe is a potent non-competitive inhibitor of the microsomal squalene epoxidase from Candida (Ki = 30 nM). In contrast, inhibition of rat liver squalene epoxidase only occurs at very high drug concentrations (Ki = 77 μM) and is also qualitatively different, being competitive with squalene. Terbinafine thus has no effect on cholesterol biosynthesis in vivo. Squalene epoxidase is not an enzyme of the cytochrome P-450 type and, therefore, the allylamines have no intrinsic potential for inhibition of this class of enzymes.

Terminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidase

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTTerminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidaseWilliam R. Moore, Gerald L. Schatzman, Esa T. Jarvi, Raymond S. Gross, and James R. McCarthyCite this: J. Am. Chem. Soc. 1992, 114, 1, 360–361Publication Date (Print):January 1, 1992Publication History Published online1 May 2002Published inissue 1 January 1992https://doi.org/10.1021/ja00027a056RIGHTS & PERMISSIONSArticle Views536Altmetric-Citations110LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (281 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts