Discovery of novel tricyclic compounds as squalene synthase inhibitors

In the present article, we have reported the design, synthesis, and identification of highly potent benzhydrol derivatives as squalene synthase inhibitors (compound 1). Unfortunately, the in vivo efficacies of the compounds were not enough for acquiring the clinical candidate. We continued our investigation to obtain a more in vivo efficacious template than the benzhydrol template. In our effort, we focused on a benzoxazepine ring and designed a new tricyclic scaffold by the incorporation of heterocycle into it. Prepared pyrrolobenzoxazepine derivatives showed further efficient in vitro and in vivo activities.     

Inhibition of squalene epoxidase by allylamine antimycotic compounds. A comparative study of the fungal and mammalian enzymes.

The inhibition of squalene epoxidase by the allylamine antimycotic agents naftifine and compound SF 86-327 was investigated, with particulate enzyme preparations from the pathogenic yeasts Candida albicans and Candida parapsilosis and from rat liver. Both naftifine and compound SF 86-327 were potent inhibitors of the Candida epoxidases and showed apparently non-competitive kinetics with respect to the substrate squalene. The Ki values for naftifine and compound SF 86-327 in the C. albicans system were 1.1 microM and 0.03 microM respectively. The C. parapsilosis enzyme was slightly more sensitive to inhibition. Varying the concentrations of cofactors or the soluble cytoplasmic fraction (S200) had no effect on the inhibition. The epoxidase from rat liver was much less sensitive (Ki for compound SF 86-327 was 77 microM). The inhibition was also qualitatively different from that in Candida, being competitive with respect to squalene and also with respect to the S200 fraction. S200 fraction derived from C. albicans also antagonized the inhibition of the epoxidase from liver, but the liver S200 fraction did not affect inhibition of the Candida enzyme by compound SF 86-327. There was no evidence for an irreversible or mechanism-based inhibition of either the fungal or the mammalian epoxidase. The selective inhibition of squalene epoxidase was sufficient to account for the known antimycotic action of the compounds.     

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.     

Acetylenic amines as inhibitors of microsomal monooxygenases

A new class of compounds: acetylenic amines, possessing structural similarity with the known inhibitor SKF-525A, and their saturated analogues, has been studied for its effect on the microsomal cytochrome P-450-dependent monooxygenases (MM). Significant differences in sensitivity of different substrate oxidation reactions in experiments with the mouse liver MM were observed. It was shown that the acetylenic amines investigated 13-30 times exceeded their saturated analogues as to the ability to inhibit aminopyrine and benzo[a]pyrene oxidation, and differed but slightly from their analogues with respect to p-nitroanisole and paraoxon oxidation. Benzo[a]pyrene hydroxylase of the house-fly abdomens was less sensitive to the compound investigated than that of the mouse liver, however, in contrast to it exhibited selectively to the diphenyl derivatives and not to the monophenyl ones.     

beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.     

Thiol-modifying inhibitors for understanding squalene cyclase function.

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by site-directed mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3-carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene) showed that the cysteine residue at the channel constriction was covalently modified by the squalene-like inhibitor. Time-dependent inactivation of the C25S/C50S/C455S/C537S mutant by a number of squalene analogues and other agents with thiol-modifying activity suggested that modifying C435 caused the obstruction of the channel constriction thus blocking access of the substrate to the active site. The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC-ESI-MS measurements. The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate-protonating residue, was substituted by cysteine. The inhibitory effect was evaluated in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene. Among the inhibitors tested, the substrate analogue squalene-maleimide proved to be a very effective time-dependent inhibitor.     

Syntheses and biological evaluation of novel quinuclidine derivatives as squalene synthase inhibitors

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerization of two molecules of farnesyl diphosphate to form squalene and is involved in the first committed step in cholesterol biosynthesis. Inhibition of this enzyme is therefore an attractive target for hypocholesterolemic strategies. A series of quinuclidine derivatives incorporating a tricyclic system was synthesized and evaluated for their ability to inhibit squalene synthase in vitro. A 9H-fluorene moiety was found to be optimal as the tricyclic system for potent inhibitory activity. Improved activity can be achieved with a conformationally constrained three-atom linkage connecting the tricyclic system with the quinuclidine nucleus. Among these compounds, (Z)-3-[2-(9H-fluoren-2-yloxy)ethylidene]-quinuclidine hydrochloride 31 was found to be a potent inhibitor of squalene synthase derived from hamster liver and human hepatoma cells with IC(50) values of 76 and 48 nM, respectively. Oral dosing of compound 31 demonstrated effective reduction of plasma non-HDL cholesterol levels in hamsters.     

Enzyme inhibitors and other bioactive compounds from marine actinomycetes

Several enzyme-inhibitor-producing actinomycetes were isolated from various samples collected from the marine environment and characterized. Most of them produced novel compounds that are useful in medicine and agriculture. Actinomycete strain no. 18, which produces antibiotics against Gram-positive bacteria only in the presence of seawater, was isolated from sediment sampled from neritic sea water and characterized. The production of antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v). Thus, the production was seawater-dependent. The production of tetrodotoxin (TTX), known otherwise as puffer fish toxin, was investigated in various actinomycetes collected from the marine environment. Of 10 isolates from various sea areas, 9 produced TTX as judged by their retention times on high-performance liquid chromatography (HPLC). To our knowledge, this is the first report of actinomycetes from the marine environment that produce TTX.     

Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.     

Detergents as selective inhibitors and inactivators of enzymes

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.     

Assay of the possible organization of particle-bound enzymes with squalene synthetase and squalene oxidocyclase systems

Lanosterol was biosynthesized in pig liver homogenate from [4,8,12-(14)C(3)]farnesyl pyrophosphate and [4S-4-(3)H]NADPH through the intermediary formation of squalene labelled asymmetrically with (3)H. The biosynthetic lanosterol, freed from labelled 24,25-dihydrolanosterol, which was also synthesized, was converted into 24,25-dihydrolanosteryl acetate and subjected to chemical degradations to locate the position(s) of the (3)H label in the molecule. The ratio of (3)H at C-11 to that at C-12 was found to be 1.28. Although a certain inequality of labelling was thus indicated, experimental uncertainties did not permit the conclusion that the asymmetrically labelled squalene might have been cyclized preferentially from one end.     

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.