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.     

Synthesis and biological activity of cyclotetrapeptide analogues of the natural HDAC inhibitor FR235222

In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the development of promising anticancer candidates, we have recently undertaken a molecular modelling study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure–activity relationship investigation, made in order to identify the key structural elements essential for the activity. Progress made in structure elucidation of HDAC active site, together with accurate docking calculations, provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should assure an optimal interaction between the synthetic ligands and the biological target. Following the computer aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3–8), bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their synthesis and their inhibition activity on different HDAC isoforms.     

FR290581, a novel sordarin derivative: Synthesis and antifungal activity

Sordarin is a unique natural product antifungal agent that is an inhibitor of elongation factor 2. To improve biological activity, we synthesized various compounds by novel modification of the aglycone, sordaricin. As a result, we have discovered the novel sordarin derivative FR290581. This compound exhibited superior activity and a good pharmacokinetic profile, and also displayed good in vivo activity against Candida albicans.     

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.     

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.     

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.     

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.     

Drug design based on biosynthetic studies: synthesis, biological activity, and kinetics of new inhibitors of 2,3-oxidosqualene cyclase and squalene epoxidase

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.     

Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the mitochondrial fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the microsomal fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase.     

Synthesis and biological activity of novel oxazolidinones

A number of 5-substituted derivatives of Ranbezolid, a novel oxazolidinone were synthesized. Antibacterial activity of the compounds against a number of sensitive and resistant bacteria showed promising results.     

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.     

Synthesis and biological evaluation of novel propylamine derivatives as orally active squalene synthase inhibitors

Squalene synthase inhibitors are potentially superior hypolipidemic agents. We synthesized novel propylamine derivatives, as well as evaluated their ability to inhibit squalene synthase and their lipid-lowering effects in rats. 1-Allyl-2-[3-(benzylamino)propoxy]-9H-carbazole (YM-75440) demonstrated potent inhibition of the enzyme derived from HepG2 cells with an IC(50) value of 63 nM. It significantly reduced both plasma total cholesterol and plasma triglyceride levels following oral dosing to rats with a reduced tendency to elevate plasma transaminase levels.     

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.     

Synthesis and biological activity of analogs of a nonapeptide inhibitor of peptidyldipeptidase

The synthesis of 5 analogues of the effective inhibitor of peptidyl dipeptidase, teprotide, has been carried out. The inhibitory and bradykinin-potentiating activity of these compounds has been assayed. N-Terminal pyroglutamic acid and positive charge of arginine in position 4 were found to be essential for biological activity of the inhibitor.     

Synthesis and biological activity of novel MIF antagonists

Two series of novel furan and indole compounds were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds from both series inhibited the enzymatic activity of MIF at levels equal to or significantly better than ISO-1 (an early MIF inhibitor). The majority of the compounds that robustly inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells were from the furan series (compounds 5, 9, 13, 15, and 16). In contrast, compounds that markedly inhibited the MIF-induced production of pro-inflammatory cytokines were predominantly from the indole series (compounds 26, 29, and 32).     

Synthesis and biological activity of novel carbocyclic nucleosides

The syntheses of several novel carbocyclic nucleosides which incorporate the cyclopentene moiety of neplanocin A will be presented. These include modified pyrimidine derivatives of the very potent antitumor agent cyclopentenyl cytosine and carbocyclic analogues of the ketohexose nucleosides psicofuranine and psicofuranosyl cytosine.