sorbinil and zopolrestat

Microglia-mediated neuroinflammation is a key risk factor to the development of Alzheimer' disease (AD). Aldose reductase (AR) has been found to be widely involved in inflammation-related diseases; however, whether aldose reductase inhibitors (ARIs) could be used to treat neuroinflammation is rarely reported. This study aims to evaluate the anti-neuroinflammatory effects of two major ARIs of Sorbinil (Sor) and Zopolrestat (Zol) in β-amyloid protein (Aβ)-induced microglia (BV-2). We find that Sor and Zol significantly inhibit TNF-α, IL-1β, IL-6 production from microglia in response to Aβ stimulation. Mechanism study showed that Sor and Zol decreased the production of intracellular ROS which resulted in an effective inhibition on the phosphorylation of several protein kinase C (PKC) isoforms including PKCα/β, δ, ζ/λ and mu. Moreover, Sor and Zol inactivated PCK-associated IKKβ-IκB-NF-κB and mitogen-activated protein kinase (JNK, p38, ERK) inflammation pathways. In summary, our findings suggest that Sor and Zol could inhibit Aβ-induced neuroinflammation by regulating ROS/PKC-dependent NF-κB and MAPK signaling pathways, indicating that ARIs could be promising agents for treating inflammation-related neurodegenerative diseases such as AD.

Topics: Aldehyde Reductase; Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzothiazoles; Cell Line; Cytokines; Imidazolidines; Inflammation Mediators; MAP Kinase Signaling System; Mice; Microglia; Neurogenic Inflammation; NF-kappa B; Phthalazines; Protein Kinase C; Reactive Oxygen Species; Risk Factors

Chronic inflammatory diseases such as autoimmune and bacterial infections are associated with an elevated risk of ocular inflammation. Ciliary epithelial cells that play an important role in maintaining aqueous humor dynamics and homeostasis of anterior segment of eye are continuously exposed to inflammatory markers during infections and injury. Lipopolysacchharide (LPS), a Gram-negative bacterial endotoxin, dysregulates aqueous humor (AqH) homeostasis by inducing inflammatory changes. We have investigated how inhibition of a polyol pathway enzyme, aldose reductase (AR), alters LPS-induced inflammatory changes in human non-pigmented ciliary epithelial cells (hNPECs). The stimulation of hNPECs with LPS (1 microg/ml) caused increased secretion of inflammatory markers such as PGE(2) and NO in the culture medium as well as increased expression of COX-2 and iNOS proteins in cell extracts. LPS also increased phosphorylation of MAPKs (ERK1/2) and SAPK/JNK and activation of redox-sensitive transcription factors NF-kappaB and AP-1 in hNPECs and inhibition of AR by zopolrestat and sorbinil ameliorated these changes. Further, LPS-induced decrease in the expression of Na/K-ATPase in hNPECs was restored by AR inhibitors. Similar results were observed in ciliary bodies of LPS-injected rats. Taken together, our results suggest that AR plays an important role in the LPS-induced inflammatory changes in hNPECs and that inhibition of AR could be a novel therapeutic approach for ocular inflammation.

Topics: Aldehyde Reductase; Alkaline Phosphatase; Animals; Benzothiazoles; Blotting, Western; Cells, Cultured; Ciliary Body; Cyclooxygenase 2; Dinoprostone; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Epithelial Cells; Fluorescent Antibody Technique, Indirect; Humans; Imidazolidines; Lipopolysaccharides; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Phosphorylation; Phthalazines; Rats; Rats, Inbred Lew; Signal Transduction; Transcription Factor AP-1; Transfection

Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; DNA, Neoplasm; E2F1 Transcription Factor; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Imidazolidines; Intercellular Signaling Peptides and Proteins; Mice; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Phthalazines; Protein Binding; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; RNA, Messenger; S Phase; Signal Transduction; Xenograft Model Antitumor Assays

Endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, is upregulated in pulmonary tissue during endotoxemia and contributes markedly to endotoxin-induced pulmonary hypertension. It is, however, unknown whether the ET receptors, ET(A) and ET(B), are differentially regulated in endotoxemic pulmonary vasculature and how this may impact on pulmonary vascular tone. To investigate this topic, we used isolated perfused lungs, pulmonary endothelial cells (ECs), and pulmonary vascular smooth muscle cells (SMCs) of the rat. During a 6-h endotoxin exposure, isolated perfused lungs developed significant pulmonary hypertension that was markedly attenuated by antagonizing ET(A) or ET(B) receptors using subtype-selective or a mixed ET(A/B) receptor antagonist. Peptide levels of big ET-1 and ET-1 and gene expression of prepro-ET-1 were increased after endotoxin challenge in all tissues. In endotoxemic isolated perfused lungs and ECs, the significant rise of mature ET-1 seen in controls after ET(B) receptor or mixed antagonism disappeared completely. However, this effect was preserved in endotoxemic SMCs. In ECs, endotoxin markedly downregulated maximum ET(B) receptor sites and ET(B) mRNA levels, whereas in SMCs, it generated substantial ET(B) receptor upregulation and moderate ET(A) receptor downregulation. The aldose reductase inhibitors sorbinil and zopolrestat mitigated endotoxin-induced pulmonary hypertension, ET-1 stimulation, and differential ET(B) receptor regulation. We conclude that endotoxin-induced pulmonary hypertension in the rat results from a loss of endothelial and concomitant gain of vascular smooth muscle ET(B) receptors. These changes are at least partly mediated by aldose reductase.

Topics: Acute Disease; Aldehyde Reductase; Animals; Benzothiazoles; Cells, Cultured; Endotoxins; Hypertension, Pulmonary; Imidazolidines; Male; Muscle, Smooth; Perfusion; Phthalazines; Pulmonary Wedge Pressure; Rats; Rats, Wistar; Receptor, Endothelin B; Up-Regulation

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzothiazoles; Clofibric Acid; Cysteine; Daunorubicin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glyceraldehyde; Humans; Imidazolidines; Kinetics; Mutation; Neoplasms; Oxidation-Reduction; Phthalazines; Pyrimidines; Recombinant Proteins; Serine

To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC).. The vector, pcDNA3-AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA).. The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC.. AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Cells, Cultured; Collagen Type IV; Fibronectins; Imidazolidines; MAP Kinase Signaling System; Mesangial Cells; Phthalazines; Rats; Signal Transduction; Smad2 Protein; Thiazoles; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Aldose reductase (AR; AKR1B1) a member of aldo-keto reductase super family, that we had shown earlier mediates cytotoxic signals induced by high glucose, cytokines and growth factors, also mediates the inflammatory signals induced by Gram-negative bacterial endotoxin, lipopolysaccharide (LPS). Inhibition of AR by three distinct AR inhibitors sorbinil, tolrestat or zopolrestat suppressed the LPS-induced production of inflammatory cytokines such as TNF-alpha, IL-6, IL-1beta, IFN-gamma, and chemokine MCP-1 in murine peritoneal macrophages. Inhibition of AR also prevented the production of nitric oxide, and prostaglandin E2 and expression of iNOS and Cox-2 proteins. The LPS-induced DNA binding activity of NF-kappaB and AP1 were significantly inhibited by AR inhibitors, and this effect was mediated through the inhibition of phosphorylation of IkappaB-alpha, IKK alpha/beta and PKC. These results suggest the therapeutic use of AR inhibitors as anti-inflammatory drugs.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Chemokine CCL2; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; I-kappa B Kinase; Imidazolidines; Inflammation Mediators; Interferon-gamma; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Naphthalenes; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; Phthalazines; Protein Kinase C; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

To study the effect of aldose reductase (AR) on expression of fibronectin and collagen IV in cultured rat renal mesangial cells (MsC).. AR expression plasmid vector (pCDNA3-AR) was constructed by restriction endonuclease digestion and ligation procedures. Stable expression of AR in MsC was established by Lipofectin transfection. Western blot and immunofluorescence analyses were performed to verify the transfection efficiency. Expression of fibronectin and collagen IV proteins were analyzed using Western blot.. Expression of fibronectin and collagen IV in naive MsC treated with TGF-beta1 was upregulated in comparison to that of the untreated naive MsC (P < 0.01). MsC transfected with pCDNA3-AR showed a remarkable increase of expression of fibronectin and collagen IV (P < 0.01). Aldose reductase inhibitors (Sorbinil and Zopolrestat) significantly inhibited the expression of fibronectin and collagen IV in naive MsC (P < 0.05).. Overexpression or inhibition of AR activity significantly alters the expression of fibronectin and collagen IV proteins in cultured rat MsC, suggesting that AR plays a significant role in the pathogenesis of glomerulosclersis.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Cells, Cultured; Collagen Type IV; Fibronectins; Genetic Vectors; Imidazolidines; Mesangial Cells; Phthalazines; Plasmids; Rats; Recombinant Proteins; Transfection; Transforming Growth Factor beta1

To study the effects of aldose reductase (AR) on the proliferation of rat mesangial cells (MsC) in vitro and to investigate its mechanism.. Cell proliferation was assessed by MTT colorimetric assay. Cell cycle and apoptosis were analyzed by flow cytometry. The growth of normal MsC and AR transfected MsC was compared. The proliferation of PDGF-BB and cellular growth stimulation by 10% NBS were investigated using AR inhibitors (ARI) Sorbinil and Zopolrestat. The effects of PDGF-BB on the expression of AR, p65 and c-Jun were assessed by Western blot. Activation of AP-1 was measured by EMSA.. AR expression of transfected MsC was distinctly higher than that of the control. Transfected MsC grew quicker than normal cells. ARI partially inhibited the proliferation of transfected MsC under the stimulation of PDGF-BB and 10% NBS, whereas 10% NBS had no effect on normal MsC. PDGF-BB upregulated the expression of AR and c-Jun, but had no effect on p65. The upregulation of c-Jun and the activation of AP-1 could be attenuated by ARI.. AR may participate in the pathological proliferation of MsC through the pathway related to the activation of AP-1.

Topics: Aldehyde Reductase; Animals; Becaplermin; Benzothiazoles; Cell Cycle; Cell Proliferation; Cells, Cultured; Genetic Vectors; Imidazolidines; Mesangial Cells; Phthalazines; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-sis; Rats; Transcription Factor AP-1; Transfection

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes. This enzyme catalyzes the reduction of glucose to sorbitol using nicotinamide adenine dinucleotide phosphate as an essential cofactor. AR has been localized at the sites of tissue damage, and inhibitors of this enzyme prevent the development of neuropathy, nephropathy, retinopathy, and cataract formation in animal models of diabetes. The crystal structure of AR complexed with zopolrestat, a potent inhibitor of AR, has been described.(1) We have generated a model of the AR-inhibitor complex based on the reported Calpha coordinates of the protein and results of a structure-activity relationship study using four structurally distinct classes of inhibitors, recombinant human AR, and four single-site-directed mutants of this enzyme. The effects of the site-directed mutations on residues within the active site of the enzyme were evaluated by average interaction energy calculations and by calculations of carbon atom surface area changes. These values correlated well with the IC(50) values for zopolrestat with the wild-type and mutant enzymes, validating the model. On the basis of the zopolrestat-binding model, we have proposed binding models for 10 other AR inhibitors. Our models have enabled us to gain a qualitative understanding of the binding domains of the enzyme and how different inhibitors impact the size and shape of the binding site.

Topics: Aldehyde Reductase; Benzothiazoles; Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Models, Molecular; Mutagenesis, Site-Directed; Phthalazines; Recombinant Proteins; Structure-Activity Relationship; Thiazoles

Aldose and aldehyde reductases are monomeric NADPH-dependent oxidoreductases that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The overall three-dimensional structures of the enzymes are composed of similar alpha/beta TIM-barrels, and the active site residues Tyr 50, His 113, and Trp 114 interacting with the hydrophilic heads of inhibitors are conserved. We have used molecular modeling and mass spectrometry to characterize the interactions between the enzymes and three aldose reductase inhibitors: tolrestat, sorbinil, and zopolrestat. Unlike the IC(50) values (concentration of inhibitor giving 50% of inhibition in solution), the Vc(50) values measured by mass spectrometry (accelerating voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the two enzymes are similar, and they correlate with the electrostatic and hydrogen-bonding energies calculated between the conserved Tyr 50, His 113, and Trp 114 and the inhibitors. The results of our comparison agree with detailed structural information obtained by X-ray crystallography, suggesting that nonconserved residues from the C-terminal loop account for differences in IC(50) values for the two enzymes. Additionally, they confirm our previous assumption that the Vc(50) values reflect the enzyme-inhibitor electrostatic and hydrogen-bonding interactions and exclude the hydrophobic interactions.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Inhibitory Concentration 50; Models, Molecular; Naphthalenes; Phthalazines; Spectrometry, Mass, Electrospray Ionization; Swine; Thiazoles

Giant cell arteritis (GCA) is a systemic vasculitis preferentially affecting large and medium-sized arteries. Inflammatory infiltrates in the arterial wall induce luminal occlusion with subsequent ischemia and degradation of the elastic membranes, allowing aneurysm formation. To identify pathways relevant to the disease process, differential display-PCR was used. The enzyme aldose reductase (AR), which is implicated in the regulation of tissue osmolarity, was found to be upregulated in the arteritic lesions. Upregulated AR expression was limited to areas of tissue destruction in inflamed arteries, where it was detected in T cells, macrophages, and smooth muscle cells. The production of AR was highly correlated with the presence of 4-hydroxynonenal (HNE), a toxic aldehyde and downstream product of lipid peroxidation. In vitro exposure of mononuclear cells to HNE was sufficient to induce AR production. The in vivo relationship of AR and HNE was explored by treating human GCA temporal artery-severe combined immunodeficiency (SCID) mouse chimeras with the AR inhibitors Sorbinil and Zopolrestat. Inhibition of AR increased HNE adducts twofold and the number of apoptotic cells in the arterial wall threefold. These data demonstrate that AR has a tissue-protective function by preventing damage from lipid peroxidation. We propose that AR is an oxidative defense mechanism able to neutralize the toxic effects of lipid peroxidation and has a role in limiting the arterial wall injury mediated by reactive oxygen species.

Topics: Aldehyde Reductase; Aldehydes; Animals; Apoptosis; Benzothiazoles; Chimera; Enzyme Inhibitors; Free Radical Scavengers; Giant Cell Arteritis; Humans; Imidazoles; Imidazolidines; Lipid Peroxidation; Mice; Mice, SCID; Phthalazines; RNA, Messenger; Temporal Arteries; Thiazoles; Up-Regulation; Vasculitis

In experimental diabetic neuropathy, defective arachidonic acid metabolism characterized by a decrease in the proportion of glycerophospholipid arachidonoyl-containing molecular species (ACMS) occurs and has been implicated in the pathogenesis of the disorder. In this study, we evaluated the suitability of a tumor-derived human Schwann cell line (NF1T) as a model to investigate the mechanism underlying the loss of ACMS. NF1T cells grown in 30 versus 5.5 mM glucose undergo a marked reduction in ACMS in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, in a manner resembling that of diabetic nerve. The depletion of ACMS can be reversed on transferring the cells from 30 mM glucose to medium containing physiological levels of glucose. Cells maintained in 5.5 mM glucose plus 25 mM mannitol or sorbitol did not exhibit decreased ACMS levels, indicating that osmotic effects were not responsible for ACMS depletion. However, growth in 25 mM fructose elicited a reduction of ACMS similar to that produced by 30 mM glucose. Excessive glucose flux through the polyol pathway has been implicated in the neural and vascular abnormalities associated with diabetes. Therefore, we examined the effects of polyol pathway inhibitors, including two aldose reductase inhibitors, zopolrestat and sorbinil, and a sorbitol dehydrogenase inhibitor (SDI), CP166,572, on ACMS levels in NF1T cells cultured in elevated glucose concentrations. At 200 microM, zopolrestat fully and sorbinil partially corrected ACMS depletion. The SDI at concentrations up to 100 microM failed to affect diminished ACMS levels. Neither zopolrestat nor the SDI restored ACMS levels reduced in the presence of elevated fructose concentrations. These findings suggest that enhanced flux through the polyol pathway and, in particular, elevated aldose reductase activity may play a significant role in the reduction of ACMS levels in the cells brought about by elevated glucose levels.

Topics: Aldehyde Reductase; Arachidonic Acid; Benzothiazoles; Cell Division; Culture Media; Enzyme Inhibitors; Fructose; Glucose; Humans; Imidazoles; Imidazolidines; L-Iditol 2-Dehydrogenase; Mannitol; Neurofibroma; Organic Chemicals; Phospholipids; Phthalazines; Piperazines; Pyrimidines; Schwann Cells; Sorbitol; Thiazoles; Tumor Cells, Cultured

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Topics: Aldehyde Reductase; Benzothiazoles; Binding Sites; Computer Simulation; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Kinetics; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; NADP; Naphthalenes; Phthalazines; Recombinant Proteins; Thiazoles

The effects of two structurally dissimilar aldose reductase inhibitors, Zopolrestat and Sorbinil, were investigated on the sodium-dependent, myo-inositol (MI) cotransporter in rat lenses maintained in either normal (5.5 mmol/l) or high sugar medium (35.5 mmol/l glucose or 30 mmol/l galactose). MI influx was compared to the lens polyol content. The effects of Sorbinil (10, 20 and 40 mumol/l) were determined on normal lens MI influx. At all concentrations, Sorbinil had no effect on normal MI influx; therefore, there was no direct effect on the MI transporter. Acute exposure (4-hour incubation) in either high D- or L-glucose media significantly inhibited lens MI influx, which was attributed to competitive inhibition by either D- or L-glucose with MI cotransporter. Due to the short incubation period and rapid metabolism of D-glucose to fructose, there was a low level of polyol (sorbitol) in these lenses. Thus, concomitant administration of Sorbinil (10, 20 and 40 mumol/l) had no significant effect on MI influx in this short-term experiment. Sorbinil had no effect in the presence of L-glucose because L-glucose was not metabolized; thus the polyol content remained normal. To investigate the effects of large accumulations of polyol, lenses were preincubated for 8, 12 and 16 h in 30 mmol/l galactose medium. Large amounts of polyol (galactitol) rapidly accumulated because galactitol was not metabolized. Galactose served as substrate for aldose reductase, and lens polyol (galactitol) content increased markedly. Inhibition of MI influx directly correlated with the increased lens polyol content. Lens polyol accumulation resulted in noncompetitive inhibition of MI influx. Coadministration of 40 mumol/l Sorbinil inhibited 80% of polyol formation and protected 80% of MI influx. Furthermore, in the presence of Sorbinil, lens galactose increased rapidly and equilibrated with galactose in the medium further indicating that Sorbinil inhibited aldose reductase. The effects of 40 mumol/l Sorbinil were compared to 40 mumol/l Zopolrestat. Zopolrestat was as effective as Sorbinil; both aldose reductase inhibitors maintained MI influx at approximately 80% of control values after 12- and 16-hour incubations in high galactose medium. In conclusion, Sorbinil did not exert a direct effect on the sodium-dependent, MI cotransport system or prevent the direct competitive inhibition of either D- or L-glucose. Sorbinil and Zopolrestat inhibited lens polyol formation, thereby eliminating n

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Carbohydrate Metabolism; Carbohydrates; Culture Media; Enzyme Inhibitors; Galactose; Glucose; Imidazoles; Imidazolidines; In Vitro Techniques; Inositol; Lens, Crystalline; Male; Phthalazines; Polymers; Rats; Rats, Sprague-Dawley; Sorbitol; Thiazoles

Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monosaccharides. Intense interest in the discovery and characterization of inhibitors has developed since the action of this enzyme has been linked to the pathogenesis of some diabetic complications. Since past studies indicated that most inhibitors act noncompetitively or uncompetitively versus substrate in the direction of aldehyde reduction, it was assumed that they bind at one or more sites distinct from the active site. However, the crystal structure of aldose reductase complexed with inhibitor [Wilson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9847-9851] unambiguously revealed the inhibitor bound at the active site. The present study was undertaken to address this apparent discrepancy. Using a fluorometric assay, it was determined that zopolrestat, an acetic acid-type inhibitor, bound to aldose reductase complexed with either NADPH or NADP+. In contrast, the spirohydantoin-type inhibitor sorbinil demonstrated preferential binding to the binary enzyme.NADPH complex. Prior incubation of the enzyme.NADPH complex with zopolrestat prevented subsequent sorbinil binding. These results, together with the published structure of the ternary enzyme.NADPH.zopolrestat complex, are consistent with the conclusion that both sorbinil and zopolrestat bind at the active site. We propose that mixed inhibition patterns previously observed with sorbinil are due to inhibitor binding to both structural isomers of the enzyme.NADPH complex. Similar patterns for inhibition by zopolrestat are due to tight binding of the inhibitor. Substrate inhibition in the direction of aldehyde reduction occurs as a result of substrate binding to the enzyme.NADP+ complex.

Topics: Aldehyde Reductase; Benzothiazoles; Binding Sites; Enzyme Inhibitors; Glyceraldehyde; Humans; Hydantoins; Imidazoles; Imidazolidines; Kinetics; Phthalazines; Protein Binding; Recombinant Proteins; Spectrometry, Fluorescence; Thermodynamics; Thiazoles

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only approximately 7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was approximately 1-5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.

Topics: Aldehyde Reductase; Amino Acid Oxidoreductases; Animals; Arginine; Benzothiazoles; Blood Pressure; Body Weight; Capillary Permeability; Citrulline; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Glycation End Products, Advanced; Guanidines; Imidazoles; Imidazolidines; Inositol; Iodine Radioisotopes; Male; Methylguanidine; Naphthalenes; Nitric Oxide Synthase; omega-N-Methylarginine; Phthalazines; Rats; Rats, Sprague-Dawley; Retina; Sciatic Nerve; Serum Albumin, Bovine; Sorbitol; Thiazoles; Uvea

A new working hypothesis that there is a hitherto unrecognized binding site on the aldose reductase (AR) enzyme with strong affinity for benzothiazoles was pursued for the design of novel, potent aldose reductase inhibitors (ARIs). The first application of this hypothesis led to a novel series of 3,4-dihydro-4-oxo-3-(benzothiazolylmethyl)-1-phthalazineacetic+ + + acids. The parent of this series (207) was a potent inhibitor of AR from human placenta (IC50 = 1.9 x 10(-8) M) and was orally active in preventing sorbitol accumulation in rat sciatic nerve, in an acute test of diabetic complications (ED50 = 18.5 mg/kg). Optimization of this lead through medicinal chemical rationale, including analogy from other drug series, led to more potent congeners of 207 and culminated in the design of 3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl] methyl]-1-phthalazineacetic acid (216, CP-73,850, zopolrestat). Zopolrestat was found to be more potent than 207, both in vitro and in vivo. Its IC50 against AR and ED50 in the acute test were 3.1 x 10(-9)M and 3.6 mg/kg, respectively. Its ED50s in reversing already elevated sorbitol accumulation in rat sciatic nerve, retina, and lens in a chronic test were 1.9, 17.6, and 18.4 mg/kg, respectively. It was well absorbed in diabetic patients, resulting in high blood level, showed a highly favorable plasma half-life (27.5 h), and is undergoing further clinical evaluation. An assortment of synthetic methods used for the construction of benzothiazoles, including an efficient synthesis of zopolrestat, is described. Structure-activity relationships in the new series are discussed.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Enzyme Inhibitors; Female; Humans; Indicators and Reagents; Kinetics; Lens, Crystalline; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Phthalazines; Placenta; Pregnancy; Rats; Structure-Activity Relationship; Thiazoles; X-Ray Diffraction