Accumulation Of Lanosterol Research Articles

Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene.

This work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.

Lipid saturation induces degradation of squalene epoxidase for sterol homeostasis and cell survival.

A fluid membrane containing a mix of unsaturated and saturated lipids is essential for life. However, it is unclear how lipid saturation might affect lipid homeostasis, membrane-associated proteins, and membrane organelles. Here, we generate temperature-sensitive mutants of the sole fatty acid desaturase gene OLE1 in the budding yeast Saccharomyces cerevisiae Using these mutants, we show that lipid saturation triggers the endoplasmic reticulum-associated degradation (ERAD) of squalene epoxidase Erg1, a rate-limiting enzyme in sterol biosynthesis, via the E3 ligase Doa10-Ubc7 complex. We identify the P469L mutation that abolishes the lipid saturation-induced ERAD of Erg1. Overexpressed WT or stable Erg1 mutants all mislocalize into foci in the ole1 mutant, whereas the stable Erg1 causes aberrant ER and severely compromises the growth of ole1, which are recapitulated by doa10 deletion. The toxicity of the stable Erg1 and doa10 deletion is due to the accumulation of lanosterol and misfolded proteins in ole1 Our study identifies Erg1 as a novel lipid saturation-regulated ERAD target, manifesting a close link between lipid homeostasis and proteostasis that maintains sterol homeostasis under the lipid saturation condition for cell survival.

Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

Effect of Hypoxia on the Function of the Human Serotonin1A Receptor.

Cellular hypoxia causes numerous pathophysiological conditions associated with the disruption of oxygen homeostasis. Under oxygen-deficient conditions, cells adapt by controlling the cellular functions to facilitate the judicious use of available oxygen, such as cessation of cell growth and proliferation. In higher eukaryotes, the process of cholesterol biosynthesis is intimately coupled to the availability of oxygen, where the synthesis of one molecule of cholesterol requires 11 molecules of O2. Cholesterol is an essential component of higher eukaryotic membranes and is crucial for the physiological functions of several membrane proteins and receptors. The serotonin1A receptor, an important neurotransmitter G protein-coupled receptor associated with cognition and memory, has previously been shown to depend on cholesterol for its signaling and function. In this work, in order to explore the interdependence of oxygen levels, cholesterol biosynthesis, and the function of the serotonin1A receptor, we developed a cellular hypoxia model to explore the function of the human serotonin1A receptor heterologously expressed in Chinese hamster ovary cells. We observed cell cycle arrest at G1/S phase and the accumulation of lanosterol in cell membranes under hypoxic conditions, thereby validating our cellular model. Interestingly, we observed a significant reduction in ligand binding and disruption of downstream cAMP signaling of the serotonin1A receptor under hypoxic conditions. To the best of our knowledge, our results represent the first report linking the function of the serotonin1A receptor with hypoxia. From a broader perspective, these results contribute to our overall understanding of the molecular basis underlying neurological conditions often associated with hypoxia-induced brain dysfunction.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans.

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, resulting in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hypha-inducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hypha-inducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

Eudesmane sesquiterpenes from Chinese liverwort are substrates of Cdrs and display antifungal activity by targeting Erg6 and Erg11 of Candida albicans

We isolated twelve sesquiterpenes from Chinese liverwort Tritomaria quinquedentata (Huds.) Buch., including four new compounds. Among them, five eudesmane sesquiterpenes were ineffective against Candida albicans wild strain SC5314 but active towards efflux pumps-deficient strain DSY654 using Alamar blue assay. Further test of the most active agent of ent-isoalantolactone (8, ent-iLL) showed that it also inhibited the yeast-to-hyphal switch of DSY654 cells. The intracellular content measurement using high performance liquid chromatography (HPLC) revealed that ent-iLL was intracellularly accumulated in C. albicans when efflux pumps were deficient or inhibited by Cdrs inhibitor riccardin D, suggesting that the activity of ent-iLL was compromised by efflux pumps Cdrs. Moreover, ent-iLL potentially inhibited the activity of Erg11 and Erg6 of DSY654 and thereby resulted in the alteration of sterol composition by decreasing ergosterol contents and increasing zymosterol and lanosterol accumulation. Our study demonstrated that eudesmane sesquiterpenes, as the substrates of Cdrs, could interfere with sterol synthesis of C. albicans to exert antifungal activity when co-applied with Cdrs inhibitors.

Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14α-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages invitro, decreases IFNβ-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNβ-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi

Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis.

Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14α-demethylase (CYP51).

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.

Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway

We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, and prevented formation of multivesicular bodies and virus infection. Loading of lipid starved cells with cholesterol increased infection to some extent but could not completely restore EV1 infection to control levels. Cold Triton X-100 treatment did not solubilize the α2-MVBs suggesting, together with cholesterol labeling, that the cytoplasmic endosomes were enriched in detergent-resistant lipids in contrast to αV integrin labeled control endosomes in the clathrin pathway. Cholesterol aggregation leading to increased ion permeability caused a significant reduction in EV1 uncoating in endosomes as judged by sucrose gradient centrifugation and by neutral red-based uncoating assay. In contrast, the replication step was not dependent on cholesterol in contrast to the reports on several other viruses. In conclusion, our results showed that the integrin internalization pathway is dependent on cholesterol for uptake of collagen, EV1 and integrin, for maturation of endosomal structures and for promoting EV1 uncoating. The results thus provide novel information for developing anti-viral strategies and more insight into collagen and integrin trafficking.

Mouse Knockout of the Cholesterogenic Cytochrome P450 Lanosterol 14α-Demethylase (Cyp51) Resembles Antley-Bixler Syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.

Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a K mapp of 38 μM and k catapp of 0.14 min −1. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27- 13C]- or [24- 2H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of 1H and 13CNMR and mass spectroscopy established the regiospecific conversion of the pro- Z methyl group of the Δ 24(25)-substrate to the pro- R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol ( K i 14 nM) and 26,27-dehydrolanosterol ( K i 54 μM and k inact of 0.24 min −1) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC 50, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C 1-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

The Effect of Plagiochin E Alone and in Combination with Fluconazole on the Ergosterol Biosynthesis of Candida albicans

Plagiochin E (PLE), a macrocyclic bis(bibenzyl) isolated from the liverwort Marchantia polymorpha, has been reported to have antifungal activity and resistance reversal effects on Candida albicans. In order to understand the underlying mechanisms, we studied the effects of PLE alone and in combination with fluconazole (FLC) on the ergosterol biosynthetic pathway against both FLC-sensitive and FLC-resistant strains by analyzing the sterol content and the ergosterol pathway gene (ERG) expression. Relative quantitative analysis of different ergosterol precursors was carried out by employing the hyphenated technique of gas chromatography-high resolution mass spectrometry (GC-HR-MS). We observed that for FLC-resistant strain PLE itself can cause the accumulation of lanosterol and the decrease of 14alpha-methylfecosterol. When it combined with FLC, a significant decrease was observed in ergosterol formation and corresponding accumulation of 14alpha-methylated sterols was also found. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the transcription level of ERG11 was decreased in FLC-resistant strain when exposed to PLE alone or PLE plus FLC. These results suggest that PLE potentiates FLC antifungal activity by interfering with the FLC-targeted ergosterol biosynthesis pathway.

Hypoxia Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase through Accumulation of Lanosterol and Hypoxia-Inducible Factor-mediated Induction of Insigs

Endoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the cholesterol precursor lanosterol. Conversion of lanosterol to cholesterol requires removal of three methyl groups, which consumes nine molecules of dioxygen. Here, we report that oxygen deprivation (hypoxia) slows demethylation of lanosterol and its metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. In addition, hypoxia increases the amount of Insig-1 and Insig-2 in a response mediated by hypoxia-inducible factor (HIF)-1alpha. Accumulation of lanosterol together with increased Insigs accelerates degradation of reductase, which ultimately slows a rate-determining step in cholesterol synthesis. These results define a novel oxygen-sensing mechanism mediated by the combined actions of methylated intermediates in cholesterol synthesis and the hypoxia-activated transcription factor HIF-1alpha.

Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase

Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14alpha-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.

Pyridines and pyrimidines mediating activity against an efflux-negative strain of Candida albicans through putative inhibition of lanosterol demethylase.

The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific beta-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 micro g. ml(-1)). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 micro g. ml(-1)), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation.

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2.

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT-4 and HL-60 cell lines were cultured in a cholesterol-deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14-alpha demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [(14)C]-acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC(50) = 0.1 microM), as assessed by both [(3)H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34(cdc2) and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34(cdc2) immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34(cdc2) phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34(cdc2) and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34(cdc2) activity.

Selective modification of sterol composition of hepatomas: new opportunities for chemotherapy

The absence of feedback regulation of cholesterol biosynthesis in hepatomas suggests the possibility of substitution of cholesterol with its biosynthetic precursors (7-dehydrocholesterol or lanosterol) selectively in hepatomas without the accumulation of these precursors in liver and other normal tissues, by a combination of a high cholesterol diet and specific inhibitors of cholesterol biosynthesis (AY-9944 for accumulation of 7-dehydrocholesterol and ketoconazole for accumulation of lanosterol). We suggest: (1) using a selective accumulation of 7-dehydrocholesterol in hepatoma plasma membranes to increase the sensitivity of hepatoma cells to polyene antibiotics (amphotericin B, nystatin), because polyene antibiotics have higher affinity to 7-dehydrocholesterol compared to cholesterol; (2) using a selective accumulation of lanosterol in hepatoma cells to increase the sensitivity of hepatoma cells to different antitumor agents, because lanosterol is much less effective in supporting vital cell functions (including barrier properties of natural membranes) compared to cholesterol.