Showing papers on "Lanosterol published in 2016"

Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase.

Abstract: Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance.

Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

Abstract: Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

The Investigational Drug VT-1129 Is a Highly Potent Inhibitor of Cryptococcus Species CYP51 but Only Weakly Inhibits the Human Enzyme

Abstract: Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 μM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 μM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 μM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes.

Roles of Sterol Derivatives in Regulating the Properties of Phospholipid Bilayer Systems.

Abstract: Liposomes are considered an ideal biomimetic environment and are potential functional carriers for important molecules such as steroids and sterols. With respect to the regulation of self-assembly via sterol insertion, several pathways such as the sterol biosynthesis pathway are affected by the physicochemical properties of the membranes. However, the behavior of steroid or sterol molecules (except cholesterol (Chl)) in the self-assembled membranes has not been thoroughly investigated. In this study, to analyze the fundamental behavior of steroid molecules in fluid membranes, Chl, lanosterol, and ergosterol were used as representative sterols in order to clarify how they regulate the physicochemical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Membrane properties such as surface membrane fluidity, hydrophobicity, surface membrane polarity, inner membrane polarity, and inner membrane fluidity were investigated using fluorescent probes, including 1-(4-trimethylammoniumphenyl)-6-p...

Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals.

Abstract: Azole antifungals, known as demethylase inhibitors (DMIs), target sterol 14α-demethylase (CYP51) in the ergosterol biosynthetic pathway of fungal pathogens of both plants and humans. DMIs remain the treatment of choice in crop protection against a wide range of fungal phytopathogens that have the potential to reduce crop yields and threaten food security. We used a yeast membrane protein expression system to overexpress recombinant hexahistidine-tagged S. cerevisiae lanosterol 14α-demethylase and the Y140F or Y140H mutants of this enzyme as surrogates in order characterize interactions with DMIs. The whole-cell antifungal activity (MIC50 values) of both the R- and S-enantiomers of tebuconazole, prothioconazole (PTZ), prothioconazole-desthio, and oxo-prothioconazole (oxo-PTZ) as well as for fluquinconazole, prochloraz and a racemic mixture of difenoconazole were determined. In vitro binding studies with the affinity purified enzyme were used to show tight type II binding to the yeast enzyme for all compounds tested except PTZ and oxo-PTZ. High resolution X-ray crystal structures of ScErg11p6×His in complex with seven DMIs, including four enantiomers, reveal triazole-mediated coordination of all compounds and the specific orientation of compounds within the relatively hydrophobic binding site. Comparison with CYP51 structures from fungal pathogens including Candida albicans, Candida glabrata and Aspergillus fumigatus provides strong evidence for a highly conserved CYP51 structure including the drug binding site. The structures obtained using S. cerevisiae lanosterol 14α-demethylase in complex with these agrochemicals provide the basis for understanding the impact of mutations on azole susceptibility and a platform for the structure-directed design of the next-generation of DMIs.

Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis

Abstract: Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.

Amino acid substitution in Cryptococcus neoformans lanosterol 14-α-demethylase involved in fluconazole resistance in clinical isolates.

Abstract: The molecular basis of fluconazole resistance in Cryptococcus neoformans has been poorly studied. A common azole resistance mechanism in Candida species is the acquisition of point mutations in the ERG11 gene encoding the enzyme lanosterol 14-α-demethylase, target of the azole class of drugs. In C. neoformans only two mutations were described in this gene. In order to evaluate other mutations that could be implicated in fluconazole resistance in C. neoformans we studied the genomic sequence of the ERG11 gene in 11 clinical isolates with minimal inhibitory concentration (MIC) values to fluconazole of ≥16μg/ml. The sequencing revealed the G1855A mutation in 3 isolates, resulting in the enzyme amino acid substitution G484S. These strains were isolated from two fluconazole-treated patients. This mutation would not intervene in the susceptibility to itraconazole and voriconazole.

Coumarin Antifungal Lead Compounds from Millettia thonningii and Their Predicted Mechanism of Action.

Abstract: Fungal pathogens continue to pose challenges to humans and plants despite efforts to control them. Two coumarins, robustic acid and thonningine-C isolated from Millettia thonningii, show promising activity against the fungus Candida albicans with minimum fungicidal concentration of 1.0 and 0.5 mg/mL, respectively. Molecular modelling against the putative bio-molecular target, lanosterol 14α-demethylase (CYP51), revealed a plausible binding mode for the active compounds, in which the hydroxyl group binds with a methionine backbone carboxylic group blocking access to the iron catalytic site. This binding disrupts the synthesis of several important sterols for the survival of fungi.

Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea.

Abstract: The red seaweed Laurencia dendroidea belongs to the Rhodophyta, a phylum of eukaryotic algae that is widely distributed across the oceans and that constitute an important source of bioactive specialized metabolites. Laurencia species have been studied since 1950 and were found to contain a plethora of specialized metabolites, mainly halogenated sesquiterpenes, diterpenes and triterpenes that possess a broad spectrum of pharmacological and ecological activities. The first committed step in the biosynthesis of triterpenes is the cyclization of 2,3-oxidosqualene, an enzymatic reaction carried out by oxidosqualene cyclases (OSCs), giving rise to a broad range of different compounds, such as the sterol precursors cycloartenol and lanosterol, or triterpene precursors such as cucurbitadienol and β-amyrin. Here, we cloned and characterized the first OSC from a red seaweed. The OSC gene was identified through mining of a L. dendroidea transcriptome dataset and subsequently cloned and heterologously expressed in yeast for functional characterization, which indicated that the corresponding enzyme cyclizes 2,3-oxidosqualene to the sterol precursor cycloartenol. Accordingly, the gene was named L. dendroidea cycloartenol synthase (LdCAS). A phylogenetic analysis using OSCs genes from plants, fungi and algae revealed that LdCAS grouped together with OSCs from other red algae, suggesting that cycloartenol could be the common product of the OSC in red seaweeds. Furthermore, profiling of L. dendroidea revealed cholesterol as the major sterol accumulating in this species, implicating red seaweeds contain a ‘hybrid’ sterol synthesis pathway in which the phytosterol precursor cycloartenol is converted into the major animal sterol cholesterol.

Further improvement in ganoderic acid production in static liquid culture of Ganoderma lucidum by integrating nitrogen limitation and calcium ion addition

Abstract: To further improve the ganoderic acid (GA) production, a novel integrated strategy by combining nitrogen limitation and calcium ion addition was developed. The effects of the integrated combination on the content of GA-T (one powerful anticancer compound), their intermediates (squalene and lanosterol) and on the transcription levels of GA biosynthetic genes in G. lucidum fermentation were investigated. The maximum GA-T content with the integrated strategy were 1.87 mg/ 100 mg dry cell weight, which was 2.1–4.2 fold higher than that obtained with either calcium ion addition or nitrogen limitation alone, and it is also the highest record as ever reported in submerged fermentation of G. lucidum. The squalene content was increased by 3.9- and 2.2-fold in this case compared with either individual strategy alone. Moreover, the transcription levels of the GA biosynthetic genes encoding 3-hydroxy-3-methyglutaryl coenzyme A reductase and lanosterol synthase were also up-regulated by 3.3–7.5 and 1.3–2.3 fold, respectively.

Increased lanosterol turnover: a metabolic burden for daunorubicin-resistant leukemia cells.

Abstract: The cholesterol metabolism is essential for cancer cell proliferation. We found the expression of genes involved in the cholesterol biosynthesis pathway up-regulated in the daunorubicin-resistant leukemia cell line CEM/R2, which is a daughter cell line to the leukemia cell line CCRF-CEM (CEM). Cellular (2)H2O labelling, mass spectrometry, and isotopomer analysis revealed an increase in lanosterol synthesis which was not accompanied by an increase in cholesterol flux or pool size in CEM/R2 cells. Exogenous addition of lanosterol had a negative effect on CEM/R2 and a positive effect on sensitive CEM cell viability. Treatment of CEM and CEM/R2 cells with cholesterol biosynthesis inhibitors acting on the enzymes squalene epoxidase and lanosterol synthase, both also involved in the 24,25-epoxycholesterol shunt pathway, revealed a connection of this pathway to lanosterol turnover. Our data highlight that an increased lanosterol flux poses a metabolic weakness of resistant cells that potentially could be therapeutically exploited.

Commands and method of treating cancer via RHO pathway

Abstract: Lanosterol derivatives useful as anti-cancer agent, which can inhibit the growth of lung cancer cells, liver cancer cells, mammary cancer cells, brain cancer cells and pancreatic cancer cells, possibly by acting on the RHO pathway. These lanosterol derivatives are represented by compound LD030:

The oxidosqualene cyclase from the oomycete Saprolegnia parasitica synthesizes lanosterol as a single product.

Abstract: The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol or cycloartenol. This is catalyzed by an oxidosqualene cyclase. Lanosterol and cycloartenol are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the oxidosqualene cyclase therefore determines the final composition of the end sterols of an organism. Despite the functional importance of oxidosqualene cyclases, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics and metabolite analysis suggest that lanosterol is produced. However, this catalytic activity has never been experimentally demonstrated. Here we show that the oxidosqualene cyclase of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the oxidosqualene cyclase from S. parasitica produces lanosterol exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete oxidosqualene cyclases for disease control in aquaculture.

4-Methylzymosterone and Other Intermediates of Sterol Biosynthesis from Yeast Mutants Engineered in the ERG27 Gene Encoding 3-Ketosteroid Reductase

Abstract: Studies in the post-squalene section of sterol biosynthesis may be hampered by the poor availability of authentic standards. The present study used different yeast strains engineered in 3-ketosteroid reductase (Erg27p) to obtain radioactive and non-radioactive intermediates of sterol biosynthesis hardly or not available commercially. Non-radioactive 3-keto 4-monomethyl sterones were purified from non-saponifiable lipids extracted from cells bearing point-mutated 3-ketosteroid reductase. Two strategies were adopted to prepare the radioactive compounds: (1) incubation of cell homogenates of an ERG27-deletant strain with radioactive lanosterol, (2) incubation of growing cells of a strain expressing point-mutated 3-ketosteroid reductase with radioactive acetate. Chemical reduction of both radioactive and non-radioactive 3-keto sterones gave the physiological 3-β OH sterols, as well as the non-physiological 3-α OH isomers. This combined biological and chemical preparation procedure provided otherwise unavailable or hardly available 4-mono-methyl intermediates of sterol biosynthesis, paving the way for research into their roles in physiological and pathological conditions.

Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

Abstract: Aims The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase, as a promising target for treatment of hypercholesterolemia. Methods and Results The human-yeast hybrid squalene synthase, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in S. cerevisiae strain deleted of its own squalene synthase gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end-products, ubiquinone and dolichol, was down-regulated. Conclusions The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. Significance and Impact of the Study The yeast strain expressing hybrid squalene synthase with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties. This article is protected by copyright. All rights reserved.

Constituents of the Leaves of Pandanus utilis.

Abstract: Nineteen compounds, including seven triterpenoids (1-7), five steroids (8-12), four cyclohexenone derivatives (13-16), two benzenoid glycosides (17 and 18) and one lignan (19), were isolated and separated from the leaves of Pandanus utilis through bioactivity-guided fractionation. Among them, one new lanosterol- type triterpenoid was found and named as (24R)-24-methyl-5a-4-demethyllanosta-9(11),25-dien-3β-ol (1). The structures of the isolates were determined by mass and spectroscopic analyses, and the compounds were subjected to anti-inflammatory, anti-oxidative and cytotoxic assays.

Compositions for the treatment of cataracts

Abstract: In one embodiment, the present application discloses an aqueous ophthalmic composition for the treatment of eye diseases, lesions and injuries, comprising: a) one steroid, or a combination of at least two steroids selected from the group consisting of lanosterol, dihydrolanosterol, 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol, 4,4-dimethylcholesta-8,24- dien-3β-ol, 4,4-dimethylcholesta-8-en-3β-ol, 4,4-dimethylcholesta-8(9),14-dien-3β-ol, 14-desmethyl lanosterol, lathosterol, Δ 7,24 -cholestadienol, cholesterol, cholesta-7-enol, cholesteryl ester, 7-dehydrocholesterol, desmosterol, 7-dehydrodesmosterol, zymosterol, 27-hydroxycholesterol, cholesta-7,24-dien-3-P-ol, cholesta-8(9)-en-3-β-ol, 5α-cholestan-3β-ol-6- one, 5-cholesten-3β,25-diol, 5-cholesten-3p,25-OS03H (5-cholesten-3β,25-sulfate), 5- cholesten-3β-OSO 3 H,25-ol (5-cholesten-3β-sulfate,25-ol), 5-cholesten-3β,25-diol, disulfate, and their esters thereof, or a pharmaceutically acceptable salt thereof, in a concentration effective for the treatment and/or prophylaxis of the eye diseases, lesions and injuries; and b) a pharmaceutical excipient; and methods of treatment using such compositions.

Lucid ganoderma lanosterol derivatives and preparation method and application thereof

Abstract: Four lanosterol derivatives are obtained through separation from polyporaceae lucid ganoderma. On the basis of authentication, the derivatives are triterpene compounds of the novel structure; activity rating is performed by applying an in-vitro anti-tumor cell proliferation screening system, it is found that the lucid ganoderma lanosterol derivatives can effectively restrain proliferation of MDA-MB-231 and HepG2, and the Lucid ganoderma lanosterol derivatives have the effects of preventing and treating tumor, and can be applied to preparing anti-tumor drugs.

Coordinate regulation of unsaturat protein synthesis in Mycoplasma cap

Abstract: The effect of cholesterol, epicoprostanol, and phosphatidylcholine on phospholipid, RNA, and protein synthesis was investigated in the sterol auxotroph Mycoplasma capricolum. Cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with either 2 ug of choles- terol or 2.2 ,g of egg phosphatidylcholine per ml. In such cells cholesterol caused a sequential stimulation of phospholipid, RNA, and protein synthesis. Enhanced oleate incorporation into phos- pholipid occurred early; the rates of RNA and protein synthesis increased later. In cells supplemented with phosphatidylcholine only RNA and protein syntheses were enhanced. The addition of 2 ,ug of epicoprostanol per ml to cells, growing on lanosterol promptly inhibited the rate of unsaturated phospholipid synthesis and subsequently the rate of growth. Inhibition of both processes was relieved by supplying 2 ,ug of cholesterol or 2.2 /g of phos- phatidylcholine per ml along with the inhibitory sterol. The results suggest that cholesterol in small amounts exerts a positive regu- latory effect and epicoprostanol exerts a negative one on unsat- urated phospholipid synthesis and, in turn, that RNA and protein synthesis are coordinately controlled with phospholipid synthesis. The previously reported phenomenon of sterol synergism and the postulated novel role of sterols in membranes (Dahl, J. S., Dahl, C. E. & Bloch, K. (1981)J. Biol. Chem.. 256, 87-91) are here de- fined in terms of specific metabolic events.

Comment to: Effect of lanosterol on human nuclei.

Abstract: Cite this article as: Sayin N, Kara N, Pekel G, Altinkaynak H. Response to: Choroidal thickness changes after dynamic exercise as measured by spectraldomain optical coherence tomography. Indian J Ophthalmol 2016;64:474-5. This is an open access article distributed under the terms of the Creative Commons Attribution‐NonCommercial‐ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‐commercially, as long as the author is credited and the new creations are licensed under the identical terms. of choroidal and retinal thickness measurements using optical coherence tomography in non‐diabetic haemodialysis patients. Int Ophthalmol 2013;33:533‐9.

Design, synthesis and antifungal activity of some benzyl benzimidazole derivatives

Abstract: Fungal infections are increasing rapidly due to an increase in number of immuno-compromised hosts. Azoles are first line drugs for treatment of fungal infections due to their high therapeutic index but development of resistance due to extensive use of azoles and toxicity has significantly reduced the efficacy of azole antifungal agents and led to the search for new azoles. Azoles act by competitive inhibition of cytochrome P 450 (CYP450) enzyme, Lanosterol 14α-demethylase a key enzyme in biosynthesis of sterol in fungi.

Synthesis, Molecular docking and evaluation of antifungal activity of some benzyl benzimidazole derivatives

Abstract: A novel series of benzyl benzimidazoles substituted with piperazines were designed by study of active site of the fungal enzyme of cytochrome P 450 family lanosterol 14 α demethylase and synthesized. The molecular docking study of these compounds was performed at the active site of this enzyme. The synthesised compounds were charecterised by spectral analysis (IR,1H NMR).The screening of the synthesised compounds for in vitro antifungal activity revealed activity against c Candida albicans of some of the compounds comparable to that of ketoconazole.