Inhibition and activation of porcine squalene epoxidase.

Pig liver squalene epoxidase (SE) has been partially purified from solubilized microsomes by DEAE-Sephacel and Blue Sepharose 4B chromatography. This stable and reproducible preparation was used to investigate the mechanism of several substrate-like inhibitors of SE and to study the effects of pH, metals, detergents, and cofactors on enzyme activity. Most divalent (1 mM) and trivalent (0.1 mM) metal cations had little effect on SE at pH 7.4; only ferrous and cupric ions showed ca. 50% reduction in SE activity. Interestingly, at pH 8.8, EDTA (10 mM) shows 1.8-fold enhancement of enzyme activity. Among the detergents, Triton X-100 was clearly superior for solubilization and purification of porcine SE; Tween 80, Lubrol-PX, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, octyl beta-glucoside, and three different Zwittergents were much less effective for SE solubilization. Partially purified pig liver SE showed maximal activity at pH 8.8-9.0. Trisnorsqualene alcohol and trisnorsqualene cyclopropylamine were noncompetitive inhibitors at pH 8.8, with Ki values of 4 microM and 180 nM, respectively; these two inhibitors were not substrates for SE. In contrast, 26-hydroxysqualene was both a competitive inhibitor with a Ki value of 4 microM at pH 8.8 and a substrate for SE. An unexpected enhancement (up to 350%) of SE activity was observed at pH 7.4 following preincubation with selected nonpolar derivatives of farnesol and farnesoic acid. At pH 8.8, this effect was less dramatic but still evident.     

Inhibition of vertebrate squalene epoxidase by isoprenyl gallates and phenylalkyl gallates

Galloy esters with 'substrate-like' isoprenoid or phenylalkyl side chains were newly synthesized and tested for the enzyme inhibition activities toward recombinant rat squalene epoxidase. Isoprenyl gallates (4-6) showed good inhibition (IC50 = 1.5 5.1 microM), as potent as previously reported substrate analogues, while phenylalkyl gallates (7-10) were moderate inhibitors of the enzyme (IC50 = 12-61 microM).     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.     

Potent and selective inhibition of SARS coronavirus replication by aurintricarboxylic acid

The severe acute respiratory syndrome virus (SARS) is a coronavirus that instigated regional epidemics in Canada and several Asian countries in 2003. The newly identified SARS coronavirus (SARS-CoV) can be transmitted among humans and cause severe or even fatal illnesses. As preventive vaccine development takes years to complete and adverse reactions have been reported to some veterinary coronaviral vaccines, anti-viral compounds must be relentlessly pursued. In this study, we analyzed the effect of aurintricarboxylic acid (ATA) on SARS-CoV replication in cell culture, and found that ATA could drastically inhibit SARS-CoV replication, with viral production being 1000-fold less than that in the untreated control. Importantly, when compared with IFNs alpha and beta, viral production was inhibited by more than 1000-fold as compared with the untreated control. In addition, when compared with IFNs alpha and beta, ATA was approximately 10 times more potent than IFN alpha and 100 times more than interferon beta at their highest concentrations reported in the literature previously. Our data indicated that ATA should be considered as a candidate anti-SARS compound for future clinical evaluation.     

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.     

Purification of squalene epoxidase from rat liver microsomes

Squalene epoxidase was purified from rat liver microsomes by DEAE-cellulose, alumina C_ν gel, hydroxylapatite, CM-Sephadex C-50 and Cibacron Blue Sepharose 4B in the presence of Triton X-100. The specific activity was increased 50 fold with a yield of about 10%. On SDS-polyacrylamide gel electrophoresis, the preparation gave one major band and one minor band with apparent molecular weights of 47,000 and 27,000 daltons, respectively. The protein of 47,000 was the most probable candidate for squalene epoxidase. Squalene epoxidase activity could be reconstituted in the squalene epoxidase preparation with the addition of NADPH-cytochrome P-450 reductase, FAD, and Triton X-100.     

In vitro assay of squalene epoxidase of Saccharomyces cerevisiae

We describe a simple assay for measuring squalene epoxidase specific activity in Saccharomyces cerevisiae cell-free extracts, by using [14C] farnesyl pyrophosphate as substrate. Cofactor requirements for activity are FAD and NADPH or NADH, NADPH being the preferred reduced pyridine nucleotide. Squalene epoxidase activity is localized in microsomal fraction and no supernatant soluble factor is required for maximum activity. Microsomal fraction converted farnesyl pyrophosphate into squalene, squalene 2,3-epoxide and lanosterol, showing that squalene 2,3-epoxide-lanosterol cyclase is also a microsome-bound enzyme. We show also that squalene epoxidase activity is not inhibited by ergosterol or lanosterol, but that enzyme synthesis is induced by oxygen.     

Potent and selective gene inhibition using antisense oligodeoxynucleotides

The development of antisense technology as a generally useful tool relies on the use of potent agents and the utilization of many controls in experiments. Here we describe our experience using oligodeoxynucleotides (ODNs) containing C-5 propynyl pyrimidine and phosphorothioate modifications as broadly applicable gene inhibition agents in cell culture. Methods include selection of antisense sequences, synthesis and purification of ODNs, choice of controls, delivery methods (microinjection, cationic lipid transfection, and electroporation), and analysis of gene inhibition.     

Properties of a particulate squalene epoxidase from Candida albicans

The properties and requirements of squalene epoxidase and effects of some inhibitors were investigated in the pathogenic yeast Candida albicans. A washed 'microsomal' fraction converted radiolabelled squalene to 2,3-oxidosqualene and lanosterol. Minimum requirements for activity were molecular oxygen, NADH or NADPH, and FAD. Epoxidase activity was stimulated by up to 100% by addition of the soluble cytoplasmic fraction, which itself contained negligible epoxidase activity. This stimulation was most powerful at low concentrations of enzyme, or high concentrations of squalene. Divalent cations did not stimulate activity and EDTA was not inhibitory. An apparent Km for squalene of 50 microM was determined in the presence of soluble cytoplasm. Epoxidase activity was destroyed by Triton X-100, deoxycholate or Cu2+, and partially inhibited by thiol reagents, rotenone and antimycin A. The enzyme was not inhibited by cyanide or by several inhibitors of cytochrome P-450.     

Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.     

Green tea polyphenols: novel and potent inhibitors of squalene epoxidase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction.     

Site-directed mutagenesis of conserved aromatic residues in rat squalene epoxidase

Squalene epoxidase catalyzes the conversion of squalene to (3S)2,3-oxidosqualene, which is a rate-limiting step of the cholesterol biogenesis. To evaluate the importance of conserved aromatic residues, 15 alanine-substituted mutants were constructed and tested for the enzyme activity. Except F203A, all the mutants significantly lost the enzyme activity, confirming the importance of the residues, either for correct folding of the protein, or for the catalytic machinery of the enzyme. Further, interestingly, F223A mutant no longer accepted (3S)2,3-oxidosqualene as a substrate, while Y473A mutant converted (3S)2,3-oxidosqualene to (3S,22S)2,3:22,23-dioxidosqualene twice more efficiently than wild-type enzyme. It is remarkable that the single amino acid replacement yielded mutants with altered substrate and product specificities. These aromatic residues are likely to be located at the substrate-binding domain of the active-site, and control the stereochemical course of the enzyme reaction.     

Potent and selective inhibition of membrane-type serine protease 1 by human single-chain antibodies

Specific human antibodies targeting proteases expressed on cancer cells can be valuable reagents for diagnosis, prognosis, and therapy of cancer. To this end, a phage-displayed antibody library was screened against a cancer-associated serine protease, MT-SP1. A protein inhibitor of serine proteases that binds to a defined surface of MT-SP1 was used in an affinity-based washing procedure. Six antibodies were selected on the basis of their ELISA profiles and ability to serve as useful immunological reagents. The apparent K(i), indicative of the potency of the antibodies at inhibiting human MT-SP1 activity, ranged from 50 pM to 129 nM. Two of the antibodies had approximately 800-fold and 1500-fold selectivity when tested against the most homologous serine protease family member, mouse MT-SP1, that exhibits 86.6% sequence identity. Surface plasmon resonance was used as an independent means of determining the binding constants of the six antibodies. Association rates were as high as 1.15 x 10(7) s(-)(1) M(-)(1), and dissociation rates were as low as 3.8 x 10(-)(4) s(-)(1). One antibody was shown to detect denatured MT-SP1 with no cross reactivity to other family members in HeLa or PC3 cells. Another antibody recognized the enzyme in human prostate tissue samples for immunohistochemistry analysis. The mode of binding among the six antibodies and the protease was analyzed by competition ELISA using three distinctly different inhibitors that mapped the enzyme surface. These antibodies constitute a new class of highly selective protease inhibitors that can be used to dissect the biological roles of proteolytic enzymes as well as to develop diagnostic and therapeutic reagents.     

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.     

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.     

NB-598: a potent competitive inhibitor of squalene epoxidase

NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bith iophen-5-yl)methoxy]benzene-methanamine, was found to inhibit human microsomal squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate dose dependently in Hep G2 cells and increased the intracellular radioactivity of squalene. A single oral administration of NB-598 inhibited cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administration of NB-598 to dogs decreased serum total and low density lipoprotein cholesterol levels and increased serum squalene levels. After termination of treatment, the reduced serum cholesterol and increased squalene levels returned to their control values.