Showing papers on "Lanosterol published in 2015"
TL;DR: In this paper, the authors identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts.
Abstract: The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people1, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.
331 citations
TL;DR: This structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.
Abstract: Infections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 A. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.
120 citations
TL;DR: The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.
111 citations
TL;DR: The identification of CbQ and glycosyltransferase from S. grosvenorii for the first time is reported and the results suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.
Abstract: Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.
62 citations
TL;DR: Lanosterol 25 mM solution did not reverse opacification of human age-related cataractous nuclei and showed progression or no change in the lens opacity at the end of 6 days.
Abstract: Aim: To study the effect of lanosterol on age-related cataractous human lens nuclei. Materials and Methods: Forty age-related cataractous nuclei removed during manual small incision cataract surgery were obtained and randomly immersed in 25 mM lanosterol solution or in control solution and stored at room temperature for 6 days. Pre- and post-immersion photographs were graded by two masked observers and collated for the regression or progression of lens opacity. Results: Both lanosterol and control groups showed progression or no change in the lens opacity at the end of 6 days. Conclusion: Lanosterol 25 mM solution did not reverse opacification of human age-related cataractous nuclei.
40 citations
TL;DR: The elaborate, state-of-the-art two-dimensional QM/MM MD simulations have clearly shown that the cyclization of the A-C rings involves a nearly concerted, but highly asynchronous cyclization, to yield a stable intermediate with "6-6-5" rings.
Abstract: The remarkable cyclization mechanism of the formation of the 6-6-6-5 tetracyclic lanosterol (a key triterpenoid intermediate in the biosynthesis of cholesterol) from the acyclic 2,3-oxidosqualene catalyzed by oxidosqualene cyclase (OSC) has stimulated the interest of chemists and biologists for over a half century. Herein, the elaborate, state-of-the-art two-dimensional (2D) QM/MM MD simulations have clearly shown that the cyclization of the A–C rings involves a nearly concerted, but highly asynchronous cyclization, to yield a stable intermediate with “6-6-5” rings followed by the ring expansion of the C-ring concomitant with the formation of the D-ring to yield the “6-6-6-5” protosterol cation. The calculated reaction barrier of the rate-limiting step (≈22 kcal mol−1) is comparable to the experimental kinetic results. Furthermore all previous experimental mutagenic evidence is highly consistent with the identified reaction mechanism.
37 citations
TL;DR: Findings indicate that pronounced physiological feedback regulation of cholesterol biosynthesis in cholesterol resistant animals occurs at the level of translational efficiency without substantial reduction in hepatic HMG-CoA reductase transcription.
31 citations
TL;DR: This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 and SMT2 isoforms in red and green algae.
29 citations
TL;DR: Determination of the sterol content found that PYR inhibited the ergosterol synthesis dose dependently and caused the accumulation of squalene and lanosterol, and the heterocyclic hydroxamic acid in PYR was the key group for the antifungal action.
28 citations
TL;DR: Although the compound has a high MIC80 value in liquid medium, it shows significant antifungal activity against Candida albicans in solid plate assays and is found to target chitin in the cell wall and lanosterol 14α-demethylase of the ergosterol biosynthesis pathway.
Abstract: Compounds constructed by the grafting of amino acid and triazole with an indole moiety were synthesized and investigated for antifungal activities wherein one of the compounds gave highly promising results. Although the compound has a high MIC80 value in liquid medium, it shows significant antifungal activity against Candida albicans in solid plate assays. The compound is found to target chitin in the cell wall and lanosterol 14α-demethylase of the ergosterol biosynthesis pathway. The inhibitory effect of the compound becomes more pronounced in combination with azoles and CFW. The compound is also found to significantly affect chitin levels, cell morphology and cell viability in combination with ketoconazole.
11 citations
TL;DR: The phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae) and suggests that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.
Abstract: Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.
TL;DR: The antifungal activity for most of the synthesized compounds was more potent than that of miconazole and fluconazole, and in silico study results showed the high binding affinity to lanosterol 14α-demethylase for compounds V and VII.
Abstract: Fungal infections are a main reason for the high mortality rate worldwide. It is a challenge to design selective antifungal agents with broad-spectrum activity. Lanosterol 14α-demethylase is an attractive target in the design of antifungal agents. Seven compounds were selected from a number of designed compounds using a rational docking study. These compounds were synthesized and evaluated for their antifungal activity. In silico study results showed the high binding affinity to lanosterol 14α-demethylase (-24.49 and -25.83 kcal/mol) for compounds V and VII, respectively; these values were greater than those for miconazole (-18.19 kcal/mol) and fluconazole (-16.08 kcal/mol). Compound V emerged as the most potent antifungal agent among all compounds with a half maximal inhibitory concentration of 7.01, 7.59, 7.25, 31.6, and 41.6 µg/mL against Candida albicans, Candida parapsilosis, Aspergillus niger, Trichophyton rubrum, and Trichophyton mentagrophytes, respectively. The antifungal activity for most of the synthesized compounds was more potent than that of miconazole and fluconazole.
TL;DR: In this work, homology models of the CYP51 enzymes from Candida albicans were constructed based on the X-ray crystal structure of CYP 51 from Saccharomyces cerevisiae.
Abstract: The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby inhibit fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole.In this work, homology models of the CYP51 enzymes from Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Saccharomyces cerevisiae .
Patent•
04 Nov 2015
TL;DR: In this paper, a method for separating lanosterol and lanostenol is described, which is characterized by comprising of the following steps: dissolving a lanosterols and linostenols mixed crude product in a solvent to prepare a feed solution, carrying out fractional extraction by the use of an extraction agent, washing with a washing agent, collecting a heavy-phase extract and concentrating the extract to obtain a lanostol concentrate, and carrying out solvent crystallization to get a pure lanostols product; collecting a light-phase raffinate to get lan
Abstract: The invention discloses a method for separating lanosterol and lanostenol. The method is characterized by comprising the following steps: dissolving a lanosterol and lanostenol mixed crude product in a solvent to prepare a feed solution, carrying out fractional extraction by the use of an extraction agent, washing with a washing agent, collecting a heavy-phase extract and concentrating the extract to obtain a lanosterol concentrate, and carrying out solvent crystallization to obtain a pure lanosterol product; collecting a light-phase raffinate to obtain a lanostenol concentrate, and carrying out solvent crystallization to obtain a pure lanostenol product, wherein the solvent for dissolving the mixed crude product is one of petroleum ether, hexane or octane; the extraction agent is one or a mixture of methanol, octanol, sulfolane or N,N-dicarboamide; and the washing agent is one of petroleum ether, hexane or octane. The solvent used in the invention is an industrial commonly-used solvent. The method provided by the invention has advantages of simple process, stable operation, high productivity, high product quality and low production cost, will not cause environmental pollution, and is suitable for industrial production.