Inhibition of some hepatic glycosidases by the diseco nucleoside, 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analog

The in vivo and in vitro effects of 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analogue on alpha- and beta-glucosidases, beta-glucuronidase as well as alpha-amylase have been investigated. alpha-Glucosidase is the enzyme that is markedly affected in vivo and in vitro in a dose-dependent manner. The compounds showed a reversible inhibition of a competitive type for alpha-glucosidase. Moreover, they exert a relatively potent inhibition on alpha-glucosidase with a Ki magnitude of 3.6 x 10(-4), 9.5 x 10(-5) M.     

Purin-6-yl 6-deoxy-1-thio-beta-D-glucopyranoside. Enzymology, chemistry, and cytotoxicity

Purin-6-yl 6-deoxy-1-thio-beta-D-glucopyranoside (4) is a substrate for almond beta-glucosidase and a weak competitive inhibitor of bovine liver beta-D-glucuronidase (Ki approximately 20mM). Both 4 and purine-protonated 4 undergo hydrolysis catalyzed by dilute acid in the pH range 0.17-2.59. These results are compared with those previously obtained with ammonium (purin-6-yl 1-thio-beta-D-glucopyranosid)uronate, (purin-6-yl 1-thio-beta-D-glucopyranosid)uronamide, purin-6-yl 1-thio-beta-D-glucopyranoside, and purin-6-yl 2-deoxy-1-thio-beta-D-glucopyranoside, and it is concluded that the data support an involvement of substituents at C-5 in producing productive Michaelis-complex conformers. The 6-deoxyglucoside is more active than the D-glucosiduronic acid in an L1210 mouse screen.     

Aglycon specificity profiling of alpha-glucosidases using synthetic probes

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

The effect of debrisoquin on MAO A and MAO B activities

To examine the mode of action of debrisoquin (DEB), we studied the effect of this drug in vitro on MAO A and MAO B enzyme activities. DEB was shown to be a competitive inhibitor of highly purified human MAO A and MAO B enzyme activities. DEB inhibited placental MAO A with a Ki value of 0.5 microM and liver MAO B with a Ki value of 8.8 microM, 18-fold greater effect on the A form. Kynuramine was used as substrate for both enzymes. Additional studies using a dilution technique showed that DEB was a reversible inhibitor of both forms of the enzyme. The results of this study show that DEB is a potent competitive and reversible inhibitor of both MAO A and MAO B enzymes.     

Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6'

Sucrose derivatives modified at position 6 (6-deoxysucrose, 6-thiosucrose, 6,6'-dithiodisucrose, and 6,6'-dideoxy-6,6'-difluorosucrose) were tested as inhibitors of the two Streptococcus mutans 6715 glucosyltransferases. 6-Deoxysucrose was the best inhibitor studied, competitively inhibiting the soluble-D-glucan forming enzyme (GTF-S) and the insoluble-D-glucan forming enzyme (GTF-I) with Ki values one order of magnitude lower than the sucrose Km values. 6-Thiosucrose was also a competitive inhibitor for both enzymes. 6,6'-Dithiodisucrose and 6,6'-dideoxy-6,6'-difluorosucrose only inhibited GTF-I; 6,6'-dithiodisucrose gave mixed inhibition and 6,6'-dideoxy-6,6'-difluorosucrose gave uncompetitive inhibition. 6-Thiosucrose was a substrate for both enzymes to produce acceptor products when acceptors were present. GTF-I synthesized de novo a water-insoluble, (1----3)-6-thio-alpha-D-glucan from 6-thiosucrose.     

Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.     

Neuroprotective effects of ONO-1924H, an inhibitor of poly ADP-ribose polymerase (PARP), on cytotoxicity of PC12 cells and ischemic cerebral damage

N-[3-(4-Oxo-3,4-dihydro-phthalazin-1-yl)phenyl]-4-(morpholin-4-yl) butanamide methanesulfonate monohydrate (ONO-1924H) is a novel inhibitor of poly ADP-ribose polymerase (PARP). In this study, we examined the effects of ONO-1924H on cytotoxicity induced by hydrogen peroxide in PC12 cells in vitro and cerebral damage and neurological deficits induced by middle cerebral artery (MCA) thrombus occlusion in vivo in rat. In the in vitro cytotoxicity assay, exposure to 0.5 mmol/L hydrogen peroxide induced cell death in differentiated PC12 cells. ONO-1924H, a PARP inhibitor (Ki=0.21 micromol/L), reduced cell death in a concentration-dependent manner that was correlated with inhibition of PARP activation. A 50% reduction in cell death (EC50) was achieved with 2.4 micromol/L ONO-1924H. In the MCA occlusion model, ONO-1924H was injected intravenously at doses of 3, 10 and 30 mg/kg/h for 3 h, and cerebral damage and neurological deficits were estimated 24 h after MCA occlusion. ONO-1924H treatment led to a significant decrease in cerebral damage in the 10 mg/kg/h-treated group (P < 0.05) and the 30 mg/kg/h-treated group (P < 0.01). Further, ONO-1924H at doses of 30 mg/kg/h significantly (P < 0.05) improved neurological deficits. These findings suggest that the novel PARP inhibitor, ONO-1924H, affords effective neuroprotection and is a useful therapeutic candidate for the treatment of ischemic stroke.     

Isolation and characterization of alpha-glucosidase inhibitor from the fungus Ganoderma lucidum

An alpha-glucosidase inhibitor, SKG-3, was isolated from the fruiting bodies of Ganoderma lucidum and its physico-chemical properties were characterized. It was a highly specific and effective reversible inhibitor of alpha-glucosidase. It showed very potent inhibitory activity against alpha-glucosidase with an IC50 value of 4.6 micro g/ml, but no activity for any other glycosidases tested. Enzyme activity could be recovered upon dialysis, thus providing evidence for the reversibility of the inhibition. A Lineweaver-Burk plot indicated that the SKG-3 inhibition of alpha-glucosidase was competitive.     

Selective inhibition of human cathepsin S by 2,4,6-trisubstituted 1,3,5-triazine analogs

We report herein the synthesis and biological evaluation of a new series of 2,4,6-trisubstituted 1,3,5-triazines as reversible inhibitors of human cysteine cathepsins. The desired products bearing morpholine and N-Boc piperidine, respectively, were obtained in three to four steps from commercially available trichlorotriazine. Seventeen hitherto unknown compounds were evaluated in vitro against various cathepsins for their inhibitory properties. Among them, compound 7c (4-(morpholin-4-yl)-6-[4-(trifluoromethoxy)anilino]-1,3,5-triazine-2-carbonitrile) was identified as the most potent and selective inhibitor of cathepsin S (Ki??=??2??±??0.3?nM). Also 7c impaired the autocatalytic maturation of procathepsin S. Molecular docking studies support that 7c bound within the active site of cathepsin S, by interacting with Gly23, Cys25 and Trp26 (S1 subsite), with Asn67, Gly69 and Phe70 (S2 subsite) and with Gln19 (S1′ pocket).     

Hypoglycemic activity of the antioxidant saponarin, characterized as alpha-glucosidase inhibitor present in Tinospora cordifolia

Tinospora cordifolia, used in anti-diabetic herbal drug preparations, was reported [12] to contain an alpha-glucosidase inhibitor, characterized as saponarin (apigenin-6-C-glucosyl-7-O-glucoside). The leaf extract had appreciable antioxidant and hydroxyl radical scavenging activities and contained the flavonoid in the range of 32.1 +/- 1.5-45.5 +/- 3.5 mg/g of dry solid. Saponarin showed mixed competitive inhibition on activities of alpha-glucosidase and sucrase of different origins. IC(50), Ki and ki' values determined were 48 muM, 8 muM and 19.5 microM respectively for intestinal maltase and 35 microM, 6 microM and 13 microM respectively for intestinal sucrase. When given orally to maltose-fed rat, saponarin showed hypoglycemic activity in the range of 20-80 mg/kg compared to 100-200 mg/kg for acarbose as reported.     

Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, baker's yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either baker's yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.     

6-hydroxyflavonoids as alpha-glucosidase inhibitors from marjoram (Origanum majorana) leaves

A methanol extract of marjoram leaves strongly inhibited rat intestinal alpha-glucosidase. Five 6-hydroxyflavonoids, 6-hydroxyapigenin (scutellarein; IC50 for sucrose hydrolysis by rat intestinal alpha-glucosidase, 12 microM), 6-hydroxyapigenin-7-O-beta-D-glucopyranoside (> 500 microM), 6-hydroxyluteolin-7-O-beta-D-glucopyranoside (300 microM), 6-hydroxyapigenin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (>500 microM), and 6-hydroxyluteolin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (> 500 microM), were isolated as active principles and related compounds. The two feruloylglucosides are novel compounds.     

A new 6-C-alkylation from an alkyl mannofuranoside 5,6-cyclic sulfate

Methyl 6-C-alkyl-6-deoxy-alpha-D-mannofuranoside derivatives have been synthesized from methyl 2,3-O-isopropylidene-5,6-O-sulfuryl-alpha-D-mannofuranoside (1). In a Path A, reaction of the 5,6-cyclic sulfate 1 with 2-lithio-1,3-dithiane afforded 2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane (2). Treatment of 2 with n-butyllithium then alkyl iodide gave the corresponding 2-(methyl 5-O-alkyl-6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl )-1,3- dithiane. Reaction of 2 with n-butyllithium and 5,6-cyclic sulfate 1 furnished 2-[methyl 6-deoxy-2,3-O-isopropylidene-5-O-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-manno-furanosid-6-yl)-alpha-D - mannofuranosid-6-yl]-1,3-dithiane. 2-(Methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid- 6-yl)-1,3-dithiane was converted into the lithiated anion, which after treatment with alkyl halide afforded the corresponding 2-alkyl-C-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-1,3- dithiane. In a Path B, 5,6-cyclic sulfate 1 reacted with 2-alkyl-2-lithio-1,3-dithiane derivatives, which led after acidic hydrolysis to 2-alkyl-2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane accompanied by methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranos-5-u loside as the by-product. This methodology was applied to synthesize 2-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-2- (methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane.     

Stereoselective inhibition of human placental aromatase

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.     

Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling.     

In vitro inhibition of simvastatin metabolism in rat and human liver by naringenin.

We demonstrated that naringenin (NRG), the aglycon form of naringin present in grapefruit juice inhibits in vitro the metabolism of simvastatin (SV), a HMG-CoA reductase inhibitor. SV undergoes an important first pass metabolism and this is thought to be partly responsible for its low bioavailability after oral administration. SV is a prodrug that requires metabolic activation through hydrolysis by esterases. In addition, SV is a substrate for cytochrome P450 enzymes. NRG, a potent inhibitor of cytochrome P450 enzymes, interferes with the isoenzymes of cytochrome P450 involved in the hepatic metabolism of SV. NRG inhibits the metabolism of SV in rat hepatocytes (the intrinsic clearance of SV decreases from 26.2 microl/min/10(6) cells in absence of NRG to 4.15 microl/min/10(6) cells in presence of 50 microM NRG). This inhibition is more pronounced in hepatocytes (Ki value approximately 5 microM) than in liver microsomes (Ki approximately 23 microM and approximately 30 microM in human and rat liver microsomes respectively). Therefore, the hepatocytes seem to be the best approach for in vitro interaction study between SV and NRG ; and this should be taken into account in the in vitro/in vivo extrapolation. If this interaction were confirmed in man, the doses of SV should be reduced when co-administered with grapefruit juice because of increased bioavailability of SV.     

Kinetics of beta-lactamase inhibition by clavulanic acid]

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.     

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin I converting enzyme inhibitors containing unnatural alpha-amino acid analogues of phenylalanine

The activity of three angiotensin I converting enzyme (ACE) inhibitors with unique related structures was assessed in vitro and in vivo. The three compounds were (S)(-)-1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-3-isoquinoline carboxylic acid (EU-4865), 1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-1- isoquinolinecarboxylic acid (EU-4881), and (S)(-)-1,2,3,4-tetrahydro-1-(3-mercapto-1-oxopropyl)-2- quinolinecarboxylic acid (EU-5031). In vitro EU-4881 was a competitive inhibitor that lacked potency (IC50 = 1980 nM) against purified ACE. The other two compounds were equipotent (IC50 = 41 nM) against purified ACE but differed in their inhibition kinetics. EU-4865 (Ki = 38 nM) was a noncompetitive inhibitor, and EU-5031 (Ki = 6.9 nM) was a competitive inhibitor. Against caveolae membrane-bound ACE EU-4881 also lacked potency (IC50 = 2852 nM). In vivo in the conscious acute aortic coarctate (AAC) rat it also lacked potency, having an ED30 (oral dose decreasing blood pressure 30 mmHg) greater than 100 mg/kg. The activity of EU-4865 and EU-5031 in the caveolae membrane-bound ACE and AAC rat reflected their different Ki values rather than their similar IC50 values. In vitro, EU-4865 and EU-5031 had IC50 values of 19 and 6.7 nM, respectively, and in vivo, they had ED30 values of 52 and 1.1 mg/kg, respectively. These results suggest that ACE has a binding requirement for a carboxy-terminus, hydrophobic amino acid that is important for in vivo activity.