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1.
Inhibition of human aldose reductase (ALR2) evolved as a promising therapeutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein-ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermodynamics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding.  相似文献   

2.
We report the structure–activity relationship of a series of coumarins as aldose reductase 2 (ALR2) inhibitors and their suppressive effect on the accumulation of galactitol in the rat lens. We evaluated their ALR2 selectivity profile against sorbitol dehydrogenase and aldehyde reductase (ALR1). Our study revealed that substitutions in the C7 OH group enhanced the potency toward ALR2, while the C6 OH group interferes with ALR1 inhibition activity. Having the phenyl moiety at C4 leads to improved potency and improved selectivity. A molecular docking study suggested that 6,7-dihydroxy-4-phenylcoumarin (15) binds to ALR2 in a different manner from epalrestat. Furthermore, compound 15 clearly suppressed galactitol accumulation in a dose-dependent manner. These results provide an insight into the structural requirements of coumarins for developing a new-type of selective ALR2 inhibitor.  相似文献   

3.
Aldose reductase (ALR2) plays a vital role in the etiology of long-term diabetic microvascular complications (DMCs) such as retinopathy, nephropathy and neuropathy. It initializes the polyol pathway and under hyperglycemic conditions, catalyzes the conversion of glucose into sorbitol in the presence of NADPH. Many ALR2 inhibitors have been withdrawn from clinical trial studies due to their cross reactivity with other analogues enzymes or due to impairment with detoxification role of ALR2. To address these issues we characterized the possible rationalities behind the selectivity problem associated with the enzyme-inhibitor interactions. Novel molecules were designed for the induce fit cavity region of ALR2. Docking studies were carried out using Glide to analyze the binding affinity of the designed molecules for ALR2. The analysis showed that the designed ALR2 inhibitors are selective for ALR2 over its close analogs. These inhibitors are also specific for the induced cavity region of ALR2 and do not interfere with the detoxification role of ALR2.  相似文献   

4.
Aldose reductase (ALR2) has been purified to homogeneity from human psoas muscle. From sodium dodecyl sulfate-polyacrylamide electrophoresis the enzyme is monomeric and has a molecular weight of 37,000. ALR2 catalyzes the primarily NADPH-dependent reduction of a wide variety of aldehydes, although the enzyme can also utilize NADH. The best substrates for ALR2 are aromatic aldehydes (e.g. pyridine-3-aldehyde; Km = 9 microM; kcat/Km = 150,000 s-1 M-1), while among aldoses DL-glyceraldehyde is the preferred substrate (Km = 72 microM; kcat/Km = 17,250). Low (100 microM) concentrations of CaCl2 and CaSO4 cause a marked inhibition (90%) of ALR2 as do higher concentrations (0.2 M) of MgCl2. (NH4)2SO4 caused a 2-fold activation of ALR2. The enzyme is also inhibited by quercetin and the commercially developed aldose reductase inhibitors alrestatin and sorbinil. ALR2 is inhibited only very slightly by sodium valproate and barbiturates. ALR2 cross-reacts immunologically with human brain and human placental aldose reductase and with ALR2 from monkey tissue. There is no precipitin cross-reaction of ALR2 with aldose reductases from other species nor with human aldehyde reductase 1 (ALR1) or with ALR1 from other species. The data show that human muscle is a new and relatively rich source of a monomeric NADPH/NADH reductase which is clearly identifiable as aldose reductase.  相似文献   

5.
Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).  相似文献   

6.
Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.  相似文献   

7.
Dihydrobenzoxazinone based design and synthesis produced two series of compounds as aldose reductase (ALR2) inhibitor candidates. In particular, phenolic residues were embodied into the compounds for the combination of strengthening the inhibitory acitvity and antioxidant ability to retard the progression of diabetic complications. Most of the derivatives with styryl side chains exhibited excellent activities on selective ALR2 inhibition with IC50 values ranging from 0.082 to 0.308 μM, and {8-[2-(4-hydroxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl}-acetic acid (3a) was the most potent. More significantly, most of dihydrobenzoxazinone compounds revealed not only good inhibitory effect on ALR2, but also showed powerful antioxidant activity. Notably, phenolic compound 3a was even comparable to the well-known antioxidant Trolox, confirming that the C8 p-hydroxystyryl substitution was key structure of lowering oxidative stress. Therefore, these results provided an achievement of multifunctional ALR2 inhibitors possessing capacities for both ALR2 inhibition and as antioxidants.  相似文献   

8.
By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high-Km aldehyde reductase, the low-Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues.  相似文献   

9.
Aldose reductase (ALR2) belongs to the aldo–keto reductase (AKR) superfamily of enzymes, is the first enzyme involved in the polyol pathway of glucose metabolism and has been linked to the pathologies associated with diabetes. Molecular modelling studies together with binding constant measurements for the four inhibitors Tolrestat, Minalrestat, quercetin and 3,5-dichlorosalicylic acid (DCL) were used to determine the type of inhibition, and correlate inhibitor potency and binding energies of the complexes with ALR2 and the homologous aldehyde reductase (ALR1), another member of the AKR superfamily. Our results show that the four inhibitors follow either uncompetitive or non-competitive inhibition pattern of substrate reduction for ALR1 and ALR2. Overall, there is correlation between the IC50 (concentration giving 50% inhibition) values of the inhibitors for the two enzymes and the binding energies (ΔH) of the enzyme–inhibitor complexes. Additionally, the results agree with the detailed structural information obtained by X-ray crystallography suggesting that the difference in inhibitor binding for the two enzymes is predominantly mediated by non-conserved residues. In particular, Arg312 in ALR1 (missing in ALR2) contributes favourably to the binding of DCL through an electrostatic interaction with the inhibitor’s electronegative halide atom and undergoes a conformational change upon Tolrestat binding. In ALR2, Thr113 (Tyr116 in ALR1) forms electrostatic interactions with the fluorobenzyl moiety of Minalrestat and the 3- and 4-hydroxy groups on the phenyl ring of quercetin. Our modelling studies suggest that Minalrestat’s binding to ALR1 is accompanied by a conformational change including the side chain of Tyr116 to achieve the selectivity for ALR1 over ALR2.  相似文献   

10.
Novel non-sulfonylureas derivatives bearing an acetamide linker between a spirohydantoin scaffold and a phenyl ring were prepared and their hypoglycemic activity was estimated in vivo. Their abilities to discriminate in vitro between aldehyde reductase (ALR1) and aldose reductase (ALR2) were determined. The molecular docking and the in silico prediction studies were performed to rationalize the obtained biological results and to predict the physicochemical properties and drug-likeness scores of the new compounds. N-(2,4-Dichlorophenyl)-2-(2′,4′-dioxospiro[fluorene-9,5′-imidazolidine]-3′-yl)acetamide (3e) displayed an 84% reduction in blood glucose level superior to that of repaglinide 66% and showed an IC50 value of 0.37 μM against ALR2 that is superior to that of sorbinil 3.14 µM. Compound (3e) was selective 96 fold towards ALR2 which is closely related to serious diabetic complications. Based on the identification of this hit candidate, a new generation of safe and effective antidiabetic agents could be designed.  相似文献   

11.
An NADPH-dependent aldehyde reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The aldehyde reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5' noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor.  相似文献   

12.
Aldose reductase is an important enzyme in the polyol pathway, where glucose is converted to fructose, and sorbitol is released. Aldose reductase activity increases in diabetes as the glucose levels increase, resulting in increased sorbitol production. Sorbitol, being less cell permeable tends to accumulate in tissues such as eye lenses, peripheral nerves and glomerulus that are not insulin sensitive. This excessive build-up of sorbitol is responsible for diabetes associated complications such as retinopathy and neuropathy. In continuation of our interest to design and discover potent inhibitors of aldo-keto reductases (AKRs; aldehyde reductase ALR1 or AKR1A, and aldose reductase ALR2 or AKR1B), herein we designed and investigated a series of new benzoxazinone-thiosemicarbazones (3a-r) as ALR2 and ALR1 inhibitors. Most compounds exhibited excellent inhibitory activities with IC50 values in lower micro-molar range. Compounds 3b and 3l were found to be most active ALR2 inhibitors with IC50 values of 0.52 ± 0.04 and 0.19 ± 0.03 μM, respectively, both compounds were more effective inhibitors as compared to the standard ALR2 inhibitor (sorbinil, with IC50 value of 3.14 ± 0.02 μM).  相似文献   

13.
The role of aldose reductase (ALR2) in diabetes mellitus is well-established. Our interest in finding ALR2 inhibitors led us to explore the inhibitory potential of new thiosemicarbazones. In this study, we have synthesized adamantyl-thiosemicarbazones and screened them as aldehyde reductase (ALR1) and aldose reductase (ALR2) inhibitors. The compounds bearing phenyl 3a, 2-methylphenyl 3g and 2,6-dimethylphenyl 3m have been identified as most potent ALR2 inhibitors with IC50 values of 3.99 ± 0.38, 3.55 ± 0.26 and 1.37 ± 0.92 µM, respectively, compared with sorbinil (IC50 = 3.14 ± 0.02 μM). The compounds 3a, 3g, and 3m also inhibit ALR1 with IC50 value of 7.75 ± 0.28, 7.26 ± 0.39 and 7.04 ± 2.23 µM, respectively. Molecular docking was also performed for putative binding of potent inhibitors with target enzyme ALR2. The most potent 2,6-dimethylphenyl bearing thiosemicarbazone 3m (IC50 = 1.37 ± 0.92 µM for ALR2) and other two compound 3a and 3g could potentially lead for the development of new therapeutic agents.  相似文献   

14.
In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.  相似文献   

15.
In continuation of our previous efforts directed towards the development of potent and selective inhibitors of aldose reductase (ALR2), and to control the diabetes mellitus (DM), a chronic metabolic disease, we synthesized novel coumarin-thiazole 6(a–o) and coumarin-oxadiazole 11(a–h) hybrids and screened for their inhibitory activity against aldose reductase (ALR2), for the selectivity against aldehyde reductase (ALR1). Compounds were also screened against ALR1. Among the newly designed compounds, 6c, 11d, and 11g were selective inhibitors of ALR2. Whereas, (E)-3-(2-(2-(2-bromobenzylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one 6c yielded the lowest IC50 value of 0.16 ± 0.06 μM for ALR2. Moreover, compounds (E)-3-(2-(2-benzylidenehydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6a; IC50 = 2.94 ± 1.23 μM for ARL1 and 0.12 ± 0.05 μM for ARL2) and (E)-3-(2-(2-(1-(4-bromophenyl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6e; IC50 = 1.71 ± 0.01 μM for ARL1 and 0.11 ± 0.001 μM for ARL2) were confirmed as dual inhibitors. Furthermore, compounds 6i, 6k, 6m, and 11b were found to be selective inhibitors for ALR1, among which (E)-3-(2-(2-((2-amino-4-chlorophenyl)(phenyl)methylene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6m) was most potent (IC50 = 0.459 ± 0.001 μM). Docking studies performed using X-ray structures of ALR1 and ALR2 with the given synthesized inhibitors showed that coumarinyl thiazole series lacks the carboxylate function that could interact with the anionic binding site being a common ALR1/ALR2 inhibitors trait. Molecular docking study with dual inhibitor 6e also suggested plausible binding modes for the ALR1 and ALR2 enzymes. Hence, the results of this study revealed that coumarinyl thiazole and oxadiazole derivatives could act as potential ALR1/ALR2 inhibitors.  相似文献   

16.
Aldose reductase 2 (ALR2), which catalyzes the reduction of glucose to sorbitol using NADP as a cofactor, has been implicated in the etiology of secondary complications of diabetes. A pharmacophore model, Hypo1, was built based on 26 compounds with known ALR2-inhibiting activity values. Hypo1 contains important chemical features required for an ALR2 inhibitor, and demonstrates good predictive ability by having a high correlation coefficient (0.95) as well as the highest cost difference (128.44) and the lowest RMS deviation (1.02) among the ten pharmacophore models examined. Hypo1 was further validated by Fisher's randomization method (95%), test set (r = 0.91), and the decoy set shows the goodness of fit (0.70). Furthermore, during virtual screening, Hypo1 was used as a 3D query to screen the NCI database, and the hit leads were sorted by applying Lipinski's rule of five and ADME properties. The best-fitting leads were subjected to docking to identify a suitable orientation at the ALR2 active site. The molecule that showed the strongest interactions with the critical amino acids was used in molecular dynamics simulations to calculate its binding affinity to the candidate molecules. Thus, Hypo1 describes the key structure-activity relationship along with the estimated activities of ALR2 inhibitors. The hit molecules were searched against PubChem to find similar molecules with new scaffolds. Finally, four molecules were found to satisfy all of the chemical features and the geometric constraints of Hypo1, as well as to show good dock scores, PLPs and PMFs. Thus, we believe that Hypo1 facilitates the selection of novel scaffolds for ALR2, allowing new classes of ALR2 inhibitors to be designed.  相似文献   

17.
A series of quinoxalinone scaffold-based acyl sulfonamides were designed as aldose reductase inhibitors and evaluated for aldose reductase (ALR2)/aldehyde reductase (ALR1) inhibition and antioxidation. Compounds 9b-g containing styryl side chains at C3-side exhibited good ALR2 inhibitory activity and selectivity. Of them, 9g demonstrated the most potent inhibitory activity with an IC50 value of 0.100?μM, and also exhibited excellent antioxidant activity, even comparable to the typical antioxidant Trolox. Compounds 9 had higher lipid-water partition coefficients relative to the carboxylic acid compounds 8, indicating that they may have better lipophilicity and membrane permeability. Structure-activity relationship (SAR) studies found that acyl trifluoromethanesulfonamide group at N1 and the C3-dihydroxystyryl side chain were the key structure for improving the aldose reductase inhibitory activity and antioxidant activity.  相似文献   

18.
Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.  相似文献   

19.
Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. In this study we show that curcumin inhibits ALR2 with an IC50 of 10 μM in a non-competitive manner, but is a poor inhibitor of closely-related members of the aldo-keto reductase superfamily, particularly aldehyde reductase. Results from molecular docking studies are consistent with the pattern of inhibition of ALR2 by curcumin and its specificity. Moreover, curcumin is able to suppress sorbitol accumulation in human erythrocytes under high glucose conditions, demonstrating an in vivo potential of curcumin to prevent sorbitol accumulation. These results suggest that curcumin holds promise as an agent to prevent or treat diabetic complications.  相似文献   

20.
Aldose reductase (ALR2) is a target enzyme for the treatment of diabetic complications. Owing to the limited number of currently available drugs for the treatment of diabetic complications, the discovery of new inhibitors of ALR2 that can potentially be optimized as drugs appears highly desirable. In this study, a molecular docking analysis of the structures of more than 127,000 organic compounds contained in the National Cancer Institute database was performed to find and score molecules that are complementary to ALR2. Besides retrieving several carboxylic acid derivatives, which are known to generally inhibit aldose reductase, docking proposed other families of putative inhibitors such as sulfonic acids, nitro-derivatives, sulfonamides and carbonyl derivatives. Twenty-five compounds, chosen as the highest-scoring representatives of each of these families, were tested as aldose reductase inhibitors. Five of them were found to inhibit aldose reductase in the micromolar range. For these active compounds, selectivity with respect to the closely-related aldehyde reductase was determined by measuring the corresponding inhibitory activities. The structures of the complexes between the new lead inhibitors and aldose reductase, here refined with molecular mechanics and molecular dynamics calculations, suggest that new pharmacophoric groups can bind aldose reductase very efficiently. In the case of the family of the nitro-derivative inhibitors, a class of particularly interesting compounds, a round of optimizations was performed with the synthesis and biological evaluation of a series of derivatives aimed at testing the proposed binding mode and at improving interaction with active site residues. Starting from a hit compound having an IC(50) of 42 microM, the most potent compound synthesized showed a 10-fold increase in inhibitory activity and 10-fold selectivity with respect to ALR1, and structure--activity relationships of the designed compounds were in agreement with the proposed mode of binding at the active site.  相似文献   

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