Isolation and structural determination of squalene synthase inhibitor from Prunus mume fruit

Squalene synthase plays an important role in the cholesterol biosynthetic pathway. Inhibiting this enzyme in hypercholesterolemia can lower not only plasma cholesterol but also plasma triglyceride levels. A squalene synthase inhibitor was screened from Prunus mume fruit, and then purified via sequential processes of ethanol extraction, HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, and crystallization. The squalene synthase inhibitor was identified as chlorogenic acid with a molecular mass of 354 Da and a molecular formula of C16H18O9 based on UV spectrophotometry, 1H and 13C NMRs, and mass spectrometry. Chlorogenic acid inhibited the squalene synthase of pig liver with an IC50 level of 100 nM. Since chlorogenic acid was an effective inhibitor against the squalene synthase of an animal source, it may be a potential therapeutic agent for hypercholesterolemia.     

Cloning, expression and characterization of squalene synthase from Inonotus obliquus

The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.     

Development of a radiometric spot-wash assay for squalene synthase.

The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Identification of novel mammalian squalene synthase inhibitors using a three-dimensional pharmacophore

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1-4] head to head fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway, namely the ubiquinones (co-enzyme Q(10)), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transferase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter system are pro-drug type inhibitors and exhibit promising biological activity.     

Measurement of squalene in human tissues and plasma: validation and application.

A method is described for accurate and reproducible measurement of squalene in plasma, feces, urine, bile, and tissue that depends on isolation by alumina column chromatography after mild saponification and on measurement by gas-liquid chromatography. Recoveries from all tissues exceeded 80% and from plasma 96%; losses were accurately corrected by appropriate additions of squalene as an overall recovery standard.     

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.     

Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats.

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.     

Solubilization and partial purification of squalene synthase from daffodil microsomal membranes

A squalene synthase was solubilized from daffodil (Narcissus pseudonarcissus L.) microsomes with CHAPS, a zwitterionic non-denaturating detergent. By successive chromatography on DEAE Sephacel and APP Sepharose a fraction enriched in this enzyme (21-fold) was prepared.     

Discovery of a new 2-aminobenzhydrol template for highly potent squalene synthase inhibitors

To obtain small and efficient squalene synthase inhibitors, a flexible 2-aminobenzhydrol open form structure was designed and showed potent inhibitory activity comparable to 4,1-benzoxazepin compounds. Further chemical modification led to the discovery of a novel template with a strong squalene synthase inhibitory activity, and its basic structure-activity relationship was revealed. The X-ray crystallographic data of compound 12 bound to the active site of squalene synthase provided an important insight into the binding mode of this alternative template that formed 11-membered ring conformations with an intramolecular hydrogen bond.     

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.     

Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.     

Agmatine deiminase from cucumber seedlings is a mono-specific enzyme: purification and characteristics

Agmatine deiminase was purified to homogeneity from cucumber seedlings. The purification procedures included treatment with DE52, ammonium sulfate precipitation, DE52 column chromatography, Superdex 200 column chromatography, and agmatine-(CNBr)-diaminohexane-CNBr-activated-Sepharose 4B column chromatography. The purified agmatine deiminase exhibited a specific activity of 242nkat/mg protein at 30 degrees C, pH 7.0, with a yield of 33%. The molecular mass of the native enzyme was 67kDa, as estimated by Superdex 200 column chromatography. On the other hand, SDS-PAGE showed that the molecular masses of the subunits with 1% SDS and 5% of 2-mercaptoethanol treatment and with additional N-glycosidase F treatment were 47 and 36kDa, respectively. These results suggest that agmatine deiminase from cucumber is a glycoprotein. The Km of the enzyme for agmatine was 16microM and arcaine was a potent competitive inhibitor of the enzyme, with a Ki of 7.1microM. The enzyme was stable for 2 months at 4 degrees C. The enzyme does not have putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. The characteristics of the enzyme purified from cucumber were like those of the enzyme from maize. These results indicate that agmatine deiminase is distinctly different from putrescine synthase in higher plants.     

Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal

Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.     

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Towards the characterization of squalene synthase activity in extracts of Zymomonas mobilis

Abstract Extracts of Zymomonas mobilis in the presence of NADPH converted tritium-labelled farnesyl diphosphate (FPP) into squalene, resulting from the activity of squalene synthase, as well as diploptene and diplopterol, derived from further squalene cyclisation. An unidentified isoprenoid representing up to 70% of the conversion products of FPP and different from presqualene alcohol was also formed, even in the absence of NADPH. Addition of squalestatin 1, an inhibitor of squalene synthase, blocked biosynthesis from FPP of the three former triterpenes, in accordance with the role of squalene synthase in their formation, as well as that of the unknown compound.     

Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.     

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase

Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.