Showing papers on "Lanosterol published in 1999"
TL;DR: From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.
Abstract: Sterol 14α-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14α-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H37Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14α-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14α-demethylases. It catalyzes 14α-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and 1H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14α-methyl group and Δ8(9)-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14α-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.
216 citations
98 citations
TL;DR: Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates, and there was little or no accumulation of lanosterol or 3-ketosterols.
Abstract: 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 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.
96 citations
TL;DR: Multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.
Abstract: Lanosterol 14α-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs ≤32 μg ml−1) or dose-dependent resistance (MICs 8–16 μg ml−1) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.
91 citations
TL;DR: It is concluded that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34cdc2 activity, and cholesterol starvation decreases P34CDc2 kinase activity and arrests the cell cycle at G2.
Abstract: 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-α demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [14C]-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 (IC50 = 0.1 μM), as assessed by both [3H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or lo...
78 citations
TL;DR: The mRNA levels of squalene synthase (an enzyme preceding CYP51 in cholesterol biosynthesis in testis of CREM-/- mice are unchanged as compared with wild-type animals, showing that regulation by CREMtau is not characteristic for all cholesterogenic genes expressed during spermatogenesis.
Abstract: Lanosterol 14α-demethylase (CYP51) produces MAS sterols, intermediates in cholesterol biosynthesis that can reinitiate meiosis in mouse oocytes. As a cholesterogenic gene, CYP51 is regulated by a sterol/sterol-regulatory element binding protein (SREBP)-dependent pathway in liver and other somatic tissue. In testis, however, cAMP/cAMP-responsive element modulator CREMτ-dependent regulation of CYP51 predominates, leading to increased levels of shortened CYP51 mRNA transcripts. CREM−/− mice lack the abundant germ cell-specific CYP51 mRNAs in testis while expression of somatic CYP51 transcripts is unaffected. The mRNA levels of squalene synthase (an enzyme preceding CYP51 in cholesterol biosynthesis in testis of CREM−/− mice are unchanged as compared with wild-type animals, showing that regulation by CREMτ is not characteristic for all cholesterogenic genes expressed during spermatogenesis. The− 334/+314 bp CYP51 region can mediate both the sterol/SREBP-dependent as well as the cAMP/CREMτ-dependent transcript...
76 citations
TL;DR: Although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, the difference was less than 10‐fold, and increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP 51 and the potential consequences are discussed.
Abstract: Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.
76 citations
TL;DR: The wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences and the expression level was fivefold reduced, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions.
Abstract: CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14α-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C.E. & Halkier, B.A. (1997) Plant J.11, 191–201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun.230, 381–385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32–39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum–wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450·(L culture)−1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14α-demethylation of obtusifoliol very actively (Km,app = 197 µm, kcat = 1.2 min−1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 µm) and eburicol (Ks = 2.5 µm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 µm), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and γ-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 µm) and γ-ketotriazole (0.40 µm) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.
48 citations
TL;DR: The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 from Saccharomyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium, is described and it is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model.
Abstract: The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 (CYP51) from Succhuromyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial P450 of known crystal structure) is described. It is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model such that the known mono-oxygenation reaction, leading to C14−demethylation of lanosterol, is the preferred route of metabolism for this particular substrate. Key amino acid contacts within the CYP51 active site appear to orientate lanosterol for oxidative attack at the C14−methyl group, and the position of the substrate relative to the haem moiety is consistent with the phenyl-iron complexation studies reported by Tuck et al. [J. Biol. Chem., 267, 13175-13179 (1992)l. Typical azole inhibitors, such as ketoconazole, are able to fit the putative active site of CYP51 by a combination of haem ligation, hydro...
35 citations
TL;DR: A series of phospholipids, including previously undescribed compounds 4-7, were isolated by a bioactivity-guided fractionation from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis in human liver cells.
Abstract: A series of phospholipids, including previously undescribed compounds 4-7, were isolated by a bioactivity-guided fractionation from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis in human liver cells. These compounds were identified as lyso-PAF analogues (1-5) and lysophosphatidylcholines (6, 7) based on NMR and MS analyses. Compounds 1-7 specifically blocked the conversion of lanosterol into cholesterol in the Chang liver cell.
35 citations
TL;DR: The results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols, and that Are2p constitutes a new member of the karmellae-inducing protein family.
Abstract: The sterol-acyl transferase encoded by the gene ARE2 was transcriptionally deregulated in the yeast Saccharomyces cerevisiae to understand its role in sterol storage and sterol enrichment. Our results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols. ARE2 overexpression had no impact on the accumulation of the early sterols such as lanosterol, but influenced the later intermediates and the end product ergosterol. Thus an enhanced conversion of free sterols to their esterified counterparts may provide a tool to increase the overall sterol content of the yeast cell. We have previously shown that the overexpression of a truncated version of the key enzyme of the early sterol pathway, HMG-CoA reductase (HMG1), leads to an increase in the early sterols such as lanosterol and zymosterol. The simultaneous deregulation of both genes in one strain produces a cumulative effect in that both early and late sterols are enhanced. Karmellae-like structures can be detected when Are2p is overexpressed. Are2p therefore constitutes a new member of the karmellae-inducing protein family.
TL;DR: Lanosterol analogs which function as dual-action inhibitors of cholesterol biosynthesis promise to be useful not only as tools for dissecting the cellular regulation of cholesterol metabolism, but also as models for the development of safe, effective hypocholesterolemic agents.
Abstract: Drugs which suppress hepatic cholesterol biosynthesis are important therapeutic tools for lowering serum cholesterol, a major risk factor in coronary heart disease. With the goal of developing molecules that will effectively shut down cholesterol biosynthesis in hepatic tissue but allow for the buildup of the isoprenes needed for the biosynthesis of polyisoprenes other than sterols, we have designed and evaluated a series of lanosterol analogs to act as dual-action inhibitors of cholesterol biosynthesis. These sterols were predicted to act as competitive inhibitors of lanosterol 14alpha-methyl demethylase (P-450DM) and as partial suppressors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in the pathway. Compounds which have been identified as dual-action inhibitors of cholesterol biosynthesis include analogs of the intermediates generated during the removal of the 14alpha-methyl group of lanosterol by P-450DM, aminolanosterols with the amine nitrogen placed in the vicinity of C-32, and lanosterol analogs with a ketone or oxime functionality at C-15. While some dual-action inhibitors require an active P-450DM for suppression of HMGR activity, others do not. The inability of some compounds to suppress HMGR activity in cells which lack P-450DM activity suggests either that these compounds require P-450DM for conversion to an active metabolite which then suppresses HMGR activity, or that they cause the accumulation of the natural demethylation intermediates resulting in the suppression of HMGR activity. Lanosterol analogs, in contrast to 25-hydroxycholesterol, do not inhibit transcription of the HMGR gene. Rather, they inhibit translation of the HMGR mRNA, and in most cases also accelerate the degradation of enzyme protein. The potential pharmacological utility of cholesterol biosynthesis inhibitors may be determined at least in part by their effects on LDL receptor (LDLR) activity. The transcriptional regulator 25-hydroxycholesterol suppresses both HMGR and LDLR activities, while the post-transcriptional regulatory lanosterol analogs exhibit a more desirable profile, lowering HMGR levels without suppressing LDLR expression, and in some cases actually enhancing cellular LDL metabolism. Lanosterol analogs which function as dual-action inhibitors of cholesterol biosynthesis promise to be useful not only as tools for dissecting the cellular regulation of cholesterol metabolism, but also as models for the development of safe, effective hypocholesterolemic agents.
TL;DR: This method can be used for evaluating the accumulation of MAS in huFF and its correlation to oocyte quality and fertilization parameters in in vitro fertilization programmes.
Abstract: A chromatographic assay for 4,4-dimethyl-5alpha-cholesta-8,14, 24-triene-3beta-ol (FF-MAS), and its reduced species, 4, 4-dimethyl-5alpha-cholesta-8,24-triene-3beta-ol (T-MAS), has been established for analysis of human follicular fluid (huFF). The assay also quantifies lanosterol, free cholesterol and progesterone. It was established using a pool of more than 100 individual follicular fluids from women undergoing in vitro fertilization treatment. Both FF-MAS and T-MAS were found in huFF, and can be quantified with HPLC equipped with photodiode array (PDA) detection. The examination wavelength for each analyte was chosen at the absorption maximum between 200 and 300 nm. Spike-recovery experiments revealed mean recoveries of 91 +/- 7.3% for lanosterol, 103 +/- 5.1% for FF-MAS, 104 +/- 5.5% for T-MAS, 103 +/- 4.5% for free cholesterol and 85 +/- 5.1% for progesterone. The lower recovery value for progesterone was due to a sub-optimal extraction procedure for this particular analyte, as indicated by re-extraction. The minimum amounts of FF-MAS required for quantification were 4 ng/mL and 23 ng/mL for T-MAS and lanosterol. FF-MAS was assayed to approximately 1.6 microM. T-MAS and lanosterol was assayed to about half of this value. No esterification of either MAS or lanosterol could be detected in huFF. Less than 10% of cholesterol was underivatized cholesterol, as more than 10 times the amount of free cholesterol could be assayed after extended saponification. This method can be used for evaluating the accumulation of MAS in huFF and its correlation to oocyte quality and fertilization parameters in in vitro fertilization programmes.
TL;DR: The identification of euphorbol and pneumocysterol in P. carinii hominis suggests that 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and the inefficacy of azole drugs against P.carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus.
Abstract: Pneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct Δ7, 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C32 sterol were found in human-derived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C31 and C32 sterols were identified as euphorbol (24-methylenelanost-8-en-3β-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3β-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen’s membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.
TL;DR: 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), which is much less effective in supporting vital cell functions (including barrier properties of natural membranes) compared to cholesterol.
Journal Article•
TL;DR: Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance.
Abstract: We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.
TL;DR: Cloned rat and human CYP51 cDNAs are cloned and catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes.
Abstract: Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.
TL;DR: Administration of SKP-450, a novel antihypertensive agent to hypercholesterolemic Syrian hamsters led to a significant reduction in plasma lipids in a dose-dependent manner, i.e., a 10.8% to 29% reduction in low-density lipoprotein cholesterol at doses of 0.3 to 10 mg/kg.
Journal Article•
TL;DR: A series of lanosterol and cholesterol derivatives with modified side chain structures, which might interfere with sterol C24-methyltransferase in the ergosterol biosynthesis as substrate analogs, have been synthesized as discussed by the authors.
Abstract: A series of lanosterol and cholesterol derivatives with modified side chain structures, which might interfere with sterol C24-methyltransferase in the ergosterol biosynthesis as substrate analogs, have been synthesized. The in vitro bioassay studies have shown that some of these compounds, in particular with C24-amino- and thio-functionalities, possess potent antifungal activities, in vivo . Bioassays have also been carried out for the leading compounds.
TL;DR: A convergent asymmetric synthesis led to 14-fluoro-2,3-oxidosqualene (14-FOS, 16 ), which was cyclized by bacterial squalene:hopane cyclase to a monocarbocyclic product with a bridged ether and a 2:3 mixture of bicyclic alcohols.
Abstract: A convergent asymmetric synthesis led to ( 3S )-14-fluoro-2,3-oxidosqualene (14-FOS, 16 ), which was cyclized by bacterial squalene:hopane cyclase to a monocarbocyclic product with a bridged ether and a 2:3 mixture of bicyclic alcohols. 14-FOS was neither a substrate nor an inhibitor for vertebrate oxidosqualene:lanosterol cyclase.