Properties of novel aldose reductase inhibitors, M16209 and M16287, in comparison with known inhibitors, ONO-2235 and sorbinil

Properties and efficacies of novel aldose reductase (AR) inhibitors, M16209 (1-(3-bromobenzo[b]furan-2-ylsulfonyl)hydantoin) and M16287 (1-(3-chlorobenzo[b]furan-2-ylsulfonyl)hydantoin), were examined in vitro and in vivo, compared with known AR inhibitors, ONO-2235 and sorbinil. These four compounds inhibited partially purified aldose reductases from various origins, and the potencies of M16209 and M16287 were on the whole similar to ONO-2235, and were greater than that of sorbinil. The IC50 values of the four AR inhibitors did not substantially depend on the substrate used. Kinetic studies of inhibition of partially purified bovine lens (BLAR) revealed that M16209, M16287 and sorbinil were uncompetitive with glyceraldehyde and noncompetitive with nicotineamide adenine dinucleotide phosphate (NADPH), whereas ONO-2235 was noncompetitive with both glyceraldehyde and NADPH. Aldose reductase became less sensitive to the four inhibitors as enzyme purification progressed, although the susceptibility to inhibition was partially reversed by incubation with dithiothreitol. In addition, the four compounds slightly affected those enzymes of carbohydrate and glutathione metabolism which were tested. M16209 and M16287 prevented sorbitol accumulation in isolated rat tissues as potently as ONO-2235 and sorbinil. M16209 and M16287 were effective in the prevention of galactosemic cataracts and amelioration of diabetic neuropathy with almost the same potency, while ONO-2235 was effective only in neuropathy, and sorbinil was effective in galactosemic cataracts and diabetic neuropathy with a different potency. These results indicate that M16209 and M16287 are potent aldose reductase inhibitors, which could be applicable to treatment for diabetic complications.     

Synthesis and activity of a new series of (Z)-3-phenyl-2-benzoylpropenoic acid derivatives as aldose reductase inhibitors

During the course of studies directed towards the discovery of novel aldose reductase inhibitors for the treatment of diabetic complications, we synthesized a series of new (Z)-3-phenyl-2-benzoylpropenoic acid derivatives and tested their in vitro inhibitory activities on rat lens aldose reductase. Of these compounds, (Z)-3-(3,4-dihydroxyphenyl)-2-(4-methylbenzoyl)propenoicacid (3k) was identified as the most potent inhibitor, with an IC50 of 0.49 microM. The theoretical binding mode of 3k was obtained by simulation of its docking into the active site of the human aldose reductase crystal structure.     

Synthesis and aldose reductase inhibitory activity of triazine derivatives possessing acetic acid group.

N-Acetic acid derivatives of 6-aryl-pyrazolo-triazin-4-ones were synthesized for evaluation as new aldose reductase inhibitors. The intrinsic activity of each compound was assessed by measuring the inhibition of enzymatic activity in an isolated pig lens enzyme preparation. All the prepared compounds exhibited a significant in vitro aldose reductase inhibitory effect (10(-6) M less than or equal to IC50 less than or equal to 10(-4) M). Furthermore, biological activity (log 1/IC50) for most of the data sets could be correlated directly to electronic and steric parameters. Finally, spatial configuration of the most active derivative 6c (IC50 = 2 x 10(-6) M) was compared with that of tolrestat and with pharmacophor requirements of the aldose reductase inhibitor site using a molecular modeling system.     

Characterization of aldose reductase and aldehyde reductase from the medulla of rat kidney

Aldose reductase and aldehyde reductase from the medulla of the rat kidney have been purified to homogeneity by using affinity chromatography, gel filtration and chromatofocusing. The molecular weights of aldose reductase and aldehyde reductase by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were found to be 37000 and 39000, respectively. The isoelectric points of aldose reductase and aldehyde reductase were found to be 5.4 and 6.2 by chromatofocusing, respectively. The major differences of amino acid compositions between both enzymes were found in serine, alanine and aspartic acid. Substrate specificity studies showed that aldose reductase utilized aldo-sugars such as D-glucose and D-galactose, but aldehyde reductase did not use them. The Km values of aldose reductase for various substrates were lower than those of aldehyde reductase. Aldose reductase utilized both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, whereas aldehyde reductase utilized only NADPH. The presence of the sulfate ion resulted in a dramatic activation of aldose reductase whereas it did not affect aldehyde reductase activity. These enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldose reductase was more susceptible than aldehyde reductase to inhibition by the aldose reductase inhibitors.     

Improvement of nerve conduction velocity in mutant diabetic mice by aldose reductase inhibitor without affecting nerve myo-inositol content

The sciatic motor nerve conduction velocity of mutant diabetic C57BL/Ks mice was significantly improved from 30.0 +/- 1.4 to 38.0 +/- 4.6 m/s by treatment with the aldose reductase inhibitor 1-[(beta-naphthyl)sulfonyl]hydantoin (30 mg/kg/d) for 2 weeks. The treatment, however, did not cause any significant change in myo-inositol concentration in the sciatic nerve. The results indicate that the ameliorating effect of the aldose reductase inhibitor on nerve conduction velocity in mutant diabetic mice is not due to alteration of myo-inositol content in the nerve.     

Electrophoretic studies of blood globin preparations

Isoelectric focusing (IEF) across an urea gradient, and titration curves obtained by IEF-electrophoresis with and without urea, were used to characterize porcine and bovine haemoglobin and globins prepared either by the cold-acetone method (native globin) or by a new method based on haem precipitation with a dilute carboxymethylcellulose (CMC) solution at acidic pH. CMC-treated bovine globins dissociated at moderately low urea concentration into alpha and beta subunits. In native bovine globin and in CMC-treated porcine globin, one intense band consisting of both alpha and beta subunits was stable to urea at the isoelectric point (pI), but was dissociated into subunits below the pI. Common to all titration curves of the globins was a marked reduction in mobility at pH below 5.0 in the case of bovine globin and below 6.0 in the case of porcine globin because of the formation of an aggregate. In all globin samples except spray-dried bovine globin the main band remained stable between the pI and the pH of aggregation.     

Bioactive constituents from Chinese natural medicines. XV. Inhibitory effect on aldose reductase and structures of Saussureosides A and B from Saussurea medusa

The 80% aqueous acetone extract from the whole plant of Saussurea medusa MAXIM. was found to inhibit rat lens aldose reductase (IC50=1.4 microg/ml). From this extract, flavonoids, lignans, and quinic acid derivatives were isolated together with two new ionone glycosides, saussureosides A and B. Their absolute stereostructures were elucidated on the basis of chemical and physicochemical evidence including the application of modified Mosher's method. In addition, some isolates were found to show an inhibitory effect on aldose reductase.     

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.     

Microheterogeneity of human plasma lecithin: cholesterol acyltransferase examined by isoelectric focusing in immobilized pH gradients

The microheterogeneity of highly purified human plasma lecithin:cholesterol acyltransferase (LCAT) has been examined by electrophoresis in immobilized pH gradients in Immobiline-polyacrylamide gels of the pH ranges 4-7 and 4.2-4.9. Seven isoforms were obtained with LCAT isolated from pools of normal plasma. Using this technique the apparent pI values at 15 degrees C for the isoforms in the pH 4.2-4.9 gradient were 4.37, 4.42, 4.48, 4.53, 4.60, 4.67 and 4.74. (SD = +/- 0.03 for all). The most intensely stained band in the isoform pattern corresponded to the isoform with a pI value of 4.48.     

Synthesis and aldose reductase inhibitory activity of N-1,N-4-disubstituted 3,4-dihydro-2(1H)-quinoxalinone derivatives

Synthetic routes have been developed for the preparation of 4-acylated, 4-benzenesulfonylated, and 4-methylated 3,4-dihydro-2(1H)-quinoxalinone-1-acetic acids. One example of the corresponding propionic acid has also been made. These compounds have been evaluated for their ability to inhibit bovine lens aldose reductase in vitro. Some members from this series also show weak activity in vivo, inhibiting sorbitol formation in sciatic nerves of streptozotocin-diabetic rats.     

Aldose reductase inhibitory, anti-cataract and antioxidant potential of selected medicinal plants from the Marathwada region, India

The water, ethanol and chloroform extracts of selected plants such as Adhatoda vasica (L.) (Acanthaceae), Caesalpinia bonduc (L.), Cassia fistula (L.) (Caesalpiniaceae) and Biophytum sensitivum (L.) (Oxalidaceae) were evaluated for rat lens aldose reductase inhibitory (RLAR) potential, anti-cataract and antioxidant activities. All the samples inhibited the aldose reductase considerably and exhibited anti-cataract activity, while C. fistula (IC(50), 0.154 mg mL(-1)) showed significant RLAR inhibitory activity as compared to the other tested samples, and was further found to be more effective in maintaining sugar-induced lens opacity in the rat lens model. The antioxidant potential of plant extracts was determined using DPPH (2,2-diphenyl-1-picryl hydrazine), hydroxyl (OH), nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) scavenging activities, along with determination of reducing power, ferrous ion chelating ability and inhibition of polyphenol oxidase (PPO). The extracts of the tested plant showed significant free radical scavenging activities and inhibited the activity of enzyme PPO, a model oxidising enzyme. The plant samples were found to possess considerable amounts of vitamin C, total polyphenols and flavonoids.     

Capillary gas chromatographic-electron-capture assay for the aldose reductase inhibitor imirestat in lens and plasma

A sensitive and selective gas chromatographic-electron-capture assay was developed for the determination of the aldose reductase inhibitor imirestat in lens and plasma. The method involves solid-phase extraction of drug and internal standard from the plasma specimen or lens sample homogenate using "Baker"-10 SPE extraction columns followed by derivatization with pentafluorobenzyl bromide and further purification. Derivatives of drug and internal standard were separated on a fused-silica capillary column and analyzed using a 63Ni electron-capture detector. The limit of detection was 2.5 ng per lens or ml of plasma. The method was used to evaluate the pharmacokinetics of imirestat in human subjects and to quantitate imirestat in animal lens tissue following topical ocular administration.     

Structural requirements of flavonoids and related compounds for aldose reductase inhibitory activity

The methanolic extracts of several natural medicines and medicinal foodstuffs were found to show an inhibitory effect on rat lens aldose reductase. In most cases, flavonoids were isolated as the active constituents by bioassay-guided separation, and among them, quercitrin (IC(50)=0.15 microM), guaijaverin (0.18 microM), and desmanthin-1 (0.082 microM) exhibited potent inhibitory activity. Desmanthin-1 showed the most potent activity, which was equivalent to that of a commercial synthetic aldose reductase inhibitor, epalrestat (0.072 microM). In order to clarify the structural requirements of flavonoids for aldose reductase inhibitory activity, various flavonoids and related compounds were examined. The results suggested the following structural requirements of flavonoid: 1) the flavones and flavonols having the 7-hydroxyl and/or catechol moiety at the B ring (the 3',4'-dihydroxyl moiety) exhibit the strong activity; 2) the 5-hydroxyl moiety does not affect the activity; 3) the 3-hydroxyl and 7-O-glucosyl moieties reduce the activity; 4) the 2-3 double bond enhances the activity; 5) the flavones and flavonols having the catechol moiety at the B ring exhibit stronger activity than those having the pyrogallol moiety (the 3',4',5'-trihydroxyl moiety).     

Polyacrylamide gel electrophoretic studies on the self-association of crotamine: characterization and molecular dimension of n-mer species

Polyacrylamide gel electrophoresis (PAGE) at pH 4.4 was used to study the concentration dependence of absolute mobility of crotamine. Within amounts ranging from 5-65 micrograms the toxin appeared in at least three n-mer species which were characterized by their geometrical mean radius R and molecular weight Mr estimation. The R and Mr values of crotamine bands were obtained from equations described in the literature and by using standard polypeptides and proteins submitted to the same experimental conditions. When amounts of up to 20 micrograms were assayed by PAGE the bands had a monomer molecular weight value of 4650 and R was 1.08 nm. From 20-35 micrograms the toxin migrated as dimer (Mr 10,000) with an R value of 1.42 nm. However, amounts higher than 35 micrograms crotamine were mostly resolved in a "two-band" pattern with R and Mr values corresponding to higher associated species.     

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.     

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.     

Water leaves extracts of <Emphasis Type="Italic">Cornus mas</Emphasis> and <Emphasis Type="Italic">Cornus kousa</Emphasis> as aldose reductase inhibitors: the potential therapeutic agents

The Cornaceae family is mainly known for the research connected with its fruits and flowers, but the biological activities of leaves of Cornus species are not very intensively studied. Present work is focused on inhibition of rat lens aldose reductase, cytotoxicity, and study of oxidant efficacy on NIH-3T3 cells of the water extracts of the leaves of Cornus mas and Cornus kousa. The obtained results showed effective inhibition of isolated rat lens aldose reductase in low µg/mL level, antioxidant properties at lower concentration (50 µg/mL), and slight prooxidant properties at higher concentration (100 µg/mL). The prooxidant properties could contribute to cytotoxicity when the cells incubated with the extracts of concentration 100 µg/mL reduced 10% of MTT of control cells. The biological activities of two Cornus species were quite similar in all our tests. Low concentrations of both water leaves extracts could be perspective agents for potential treatment of chronic diabetic complications, inflammation, and another diseases connected with aldose reductase enzyme.     

The protein composition of ferret parotid saliva as revealed by high-resolution electrophoretic methods.

Ferret parotid saliva has been analysed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) to determine, for the first time, its protein composition. SDS-PAGE, in combination with Coomassie Brilliant Blue (CBB) staining, revealed up to 20 bands and the patterns were characterised by major protein constituents of Mr 105000, 51000, 47000, 33000, 22000 and 16 400 common to all samples from all animals. Sequential samples collected from the same animal during prolonged stimulation of the parasympathetic nerve (40 min at 40 Hz) showed subtle but reproducible protein changes. Saliva collected from different animals varied widely in the amount of a protein Mr 66000. 2-DE, in combination with silver staining, revealed up to 300 spots and the patterns were characterised by major protein constituents of Mr 105000 (pI 6.3-7.2), Mr 66000 (pI 4.7-5.3), Mr 51000 (pI 5.0-5.7), Mr 47000 (pI 6.0-7.5), and Mr 33000 (pI 4.7-6.0). Many of the polypeptide spot clusters consisted of one or more horizontal strings of spots suggesting extensive microheterogeneity. Both SDS-PAGE and 2-DE indicated that the protein patterns of ferret parotid saliva evoked by electrical stimulation of the parasympathetic nerve in the absence or presence of atropine are similar, i.e., the protein composition of the atropine-resistant nonadrenergic, noncholinergic (NANC) secretion is similar to that of saliva evoked in the absence of muscarinic receptor blockade.     

Calculation of the isoelectric point of tryptic peptides in the pH 3.5-4.5 range based on adjacent amino acid effects

Current algorithms for the calculation of peptide or protein pI, based on the charge associated with individual amino acids, can calculate pI values to within +/-0.2 pI units. Here, we present a new pI calculation algorithm that takes into account the effect of adjacent amino acids on the pI value. The algorithm accounts for the effect of adjacent amino acids+/-3 residues away from a charged aspartic or glutamic acid, as well as effects on the free C terminus, and applies a correction term to the corresponding pK values. The correction increments are derived from a 5000-peptide training set using a genetic optimization approach. The accuracy of the new pI values obtained with this method approaches the error associated with the manufacture of the IPG strip (<+/-0.03 pI units). The approach is demonstrated for cytosolic cell extracts derived from the breast-cancer cell line DU4475, and from membrane preparations from human lung-tissue samples. One potential application of a more highly accurate pI calculation is data filtering of MS/MS outputs that will allow for more complex database searches including gene finding, and validation, and detection of coding single-nucleotide polymorphisms in their expressed form.     

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.