Identification of new potent inhibitor of aldose reductase from Ocimum basilicum

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6′-hydroxyhex-3′-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC₅₀ value of 2.095 ± 0.77 µM compare to standard sorbinil (IC₅₀ = 3.14 ± 0.02 µM). Moreover, the compound (1) also showed multifolds higher activity (IC₅₀ = 0.783 ± 0.07 µM) against AKR1A1 as compared to standard valproic acid (IC₅₀ = 57.4 ± 0.89 µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC₅₀ = 4.324 ± 1.25 µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.     

Sequential production of pectinases byPenicillium frequentans

The pectinases produced byPenicillium frequentans were subjected to pectin- or sodium polypectate-PAGE. The fungus secreted one endo- and one exo-polygalacturonase during the first 10 h of incubation, either in media supplemented with pectin or in the absence of carbohydrate source. After 17 h of cultivation, another two exo-and three endo-polygalacturonases were detected in cultures supplemented with pectin. The results indicate that two constitutive polygalacturonases are secreted initially, followed by the synthesis of other inducible pectinases, in a sequential production of the pectinolytic complex. Monogalacturonic acid and sodium polypectate were better inducers for exo- than for endo-polygalacturonases, indicating that these enzymes are independently controlled.The authors are with the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Avenida do Café s/n^o, 14040-903 Ribeirão Preto, São Paulo, Brazil     

Pectinase production by Penicillium frequentans

High activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 35°C, in agitated cultures supplemented with 0.5% citrus pectin and initial pH of 2.5. Under these conditions the fungus also produced high activity of pectinesterase. At an initial pH of 7.0 or 8.0, pectin lyase activity was also detected. Enzyme activity releasing reducing sugars was more stable at 50°C than viscosity-diminishing activity. Both activities were maximal at pH 2.5 to 5.2 and at 55°C.The authors are from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café, s/n^o, Bairro Monte Alegre, 14.049 Ribeirão Preto, S.P., Brazil.     

Enzymatic synthesis of (+)- and (-)-bisdechlorogeodin with sulochrin oxidase from Penicillium frequentans and Oospora sulphurea-ochracea

Sulochrin oxidase is a blue copper-containing glycoenzyme that catalyzes a stereospecific formation of bisdechlorogeodin from sulochrin. The enzyme has been isolated from Penicillium frequentans and Oospora sulphureaochracea which catalyzes the formation of (+)-form and (-)-form of bisdechlorogeodin respectively. The Penicillium enzyme has a molecular weight of 157,000 and contains 19.5% of carbohydrates. Amino acid and carbohydrate compositions are given. The enzyme has probably a dimeric structure containing 6 Cu-atoms. Apparent K _m-values of various substrates are presented. The Oospora enzyme has a molecular weight of 128,000 and except for its stereospecificity its properties are very similar to those of the Penicillium enzyme.     

Inhibitors of aldose reductase and advanced glycation end-products formation from the leaves of Stelechocarpus cauliflorus R.E. Fr.

Two dihydroflavonol glycosides, engeletin and astilbin, were isolated from an EtOAc extract of the leaves of Stelechocarpus cauliflorus R.E. Fr. (Annonaceae). The inhibitory activity of engeletin against a recombinant human aldose reductase (IC₅₀ value=1.16 μM) was twice that of quercetin as a positive control (2.48 μM), and 23 times greater than that of astilbin (26.7 μM). Engeletin inhibited the enzyme uncompetitively. Astilbin was about as potent as the positive control, quercetin, in its inhibition of advanced glycation end-products formation. These flavonoids displayed therapeutic potential in the prevention and treatment of diabetic complications.     

The “flip-flop” of aldose reductase gene in diabetic retinopathy and nephropathy

Retinopathy and nephropathy - current view

The Kumamoto and UKPDS studies of type-2 diabetes demonstrated that high glucose mean faster worsening for both diabetic retinopathy and nephropathy. However, why so many type-2 diabetics develop nephropathy but no retinopathy? Why so many type-2 diabetics have severe retinopathy and little or no nephropathy? The answer may be genetic susceptibility.

Hypothesis: the “flip-flop” effect of aldose reductase gene

When the CC genotype (high aldose reductase expression) of the (−106) polymorphism of the aldose reductase gene was linked to enhanced retinal susceptibility to hyperglycemia, it was expected that it would also accelerate nephropathy. As the case was the opposite, we now hypothesize that in the kidney, higher aldose reductase activity reduces susceptibility to hyperglycaemia by means of shifting glucose away from the synthesis of Transforming Growth Factor-Beta (TGF-β), a stimulator of mesangial expansion - the landmark of diabetic nephropathy. As the CT & TT genotypes (lower expression of aldose reductase) have effects that are the opposite of those of CC genotype, i.e. retinopathy-protection & nephropathy proneness, we have coined the term “flip-flop”, an acronym taken from electronics, meaning a system that is bi-stable.

If the “flip-flop” was confirmed - what then?

Those diabetics who are retinopathy-protected & nephropathy-prone (CT & TT), should not be given aldose reductase inhibitors (which could worsen nephropathy) but the new breed of TGF-β inhibitors. This might be a first step towards “genetic individualization of diabetes therapy”.     

20(S)-Ginsenoside Rh2 as aldose reductase inhibitor from Panax ginseng

The root of Panax ginseng C. A. Meyer (Araliaceae) is a well-known herbal medicine in East Asia. The major bioactive metabolites in this root are commonly identified as ginsenosides. A series of ginsenosides were determined for in vitro human recombinant aldose reductase. This Letter aims to clarify the structural requirement for aldose reductase inhibition. We discovered that only ginsenoside 20(S)-Rh2 showed potent against aldose reductase, with an IC₅₀ of 147.3 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in aldose reductase inhibition. An understanding of these requirements is considered necessary in order to develop a new type of aldose reductase inhibitor. Furthermore, P. ginseng might be an important herbal medicine in preventing diabetic complications.     

Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors

Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC₅₀ values ranging from (0.099 ± 0.008) μM to (2.833 ± 0.102) μM. O⁷-Nitrooxyethyl-O^3′,O^4′-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC₅₀ = (0.099 ± 0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure–activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications.     

Induction of aldose reductase activity inCandida guilliermondii by pentose sugars

Summary Cell extracts ofCandida guilliermondii grown ind-xylose,l-arabinose,d-galactose,d-glucose,d-mannose and glycerol as sole carbon sources possessed NADPH-dependent aldose reductase activity, but no NADH-dependent activity was detected.d-xylose andl-arabinose were the best inducers of aldose reductase activity. The highest enzyme activity ind-xylose orl-arabinose-grown cells was observed first withl-arabinose followed byd-xylose as substrates of the enzymatic reaction. However, only low activity was found ind-glucose,d-mannose andd-galactose-grown cells, indicating that these carbon sources cause catabolite repression. Enzyme activities induced ind-xylose-grown cells were twice as high as those obtained from the cells under resting conditions. Furthermore, the level of induction of aldose reductase activity depended on the initial concentration ofd-xylose. The present study shows that aldose reductase activity may be efficiently induced by pentose sugars of hemicellulosic hydrolysates and weakly by hemicellulosic hexoses.     

Structure-activity relationships of ganoderma acids from Ganoderma lucidum as aldose reductase inhibitors

A series of lanostane-type triterpenoids, known as ganoderma acids were isolated from the fruiting body of Ganoderma lucidum. Some of these compounds were identified as active inhibitors of the in vitro human recombinant aldose reductase. To clarify the structural requirement for inhibition, some structure–activity relationships were determined. Our structure–activity studies of ganoderma acids revealed that the OH substituent at C-11 is an important feature and the carboxylic group in the side chain is essential for the recognition of aldose reductase inhibitory activity. Moreover, double bond moiety at C-20 and C-22 in the side chain contributes to improving aldose reductase inhibitory activity. In the case of ganoderic acid C2, all of OH substituent at C-3, C-7 and C-15 is important for potent aldose reductase inhibition. These results provide an approach to understanding the structural requirements of ganoderma acids from G. lucidum for aldose reductase inhibitor. This understanding is necessary to design a new-type of aldose reductase inhibitor.     

Bioassay-guided isolation of aldose reductase inhibitors from Artemisia dracunculus

An ethanolic extract of Artemisia dracunculus L. having antidiabetic activity was examined as a possible aldose reductase (ALR2) inhibitor, a key enzyme involved in diabetic complications. At 3.75 microg/mL, the total extract inhibited ALR2 activity by 40%, while quercitrin, a known ALR2 inhibitor, inhibited its activity by 54%. Bioactivity guided fractionation and isolation of the compounds that inhibit ALR2 activity was carried out with the total ethanolic extract yielding four bioactive compounds with ALR2 inhibitory activity ranging from 58% to 77% at 3.75 microg/mL. Using LC/MS, (1)H NMR, (13)C NMR and 2D NMR spectroscopic analyses, the four compounds were identified as 4,5-di-O-caffeoylquinic acid, davidigenin, 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone. This is the first report on their isolation from A. dracunculus and the ALR2 inhibitory activity of 4,5-di-O-caffeoylquinic acid, 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone. These results suggest a use of the extract of A. dracunculus for ameliorating diabetic complications.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

Phenolic compounds inhibit the aldose reductase enzyme from the sheep kidney

Aldose reductase (AR) is the key enzyme for the polyol pathway and responsible for sorbitol accumulation during the hyperglycemia. The present article focuses on the role of phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol in in vitro inhibition of AR. For this purpose, AR was purified from the sheep kidney with 5.33 EU mg^?1 specific activity and 0.64% yield using several chromatographic methods. Various concentrations of the compounds were tested on in vitro AR activity. IC₅₀ values were found for phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol as 6.5, 1.13, 5.45, 2.21, 1.8, and 2.09 mM, respectively, and their K_i constant was calculated as 3.45 ± 0.92, 0.96 ± 0.28, 3.07 ± 0.46, 1.59 ± 0.43, 2.5 ± 0.35, and 2.54 ± 0.45 mM, respectively. Pyrogallol showed better inhibitory effect compared to the other compounds. The inhibition mechanisms of all compounds were noncompetitive. In the presents study, in vitro AR inhibition was examined by the phenolic compounds.     

Novel synthesis of nitro-quinoxalinone derivatives as aldose reductase inhibitors

A novel, non-acid series of nitroquinoxalinone derivatives was synthesized and tested for their inhibitory activity against aldose reductase as targeting enzyme. All active compounds displayed an 8-nitro group, and showed significant activity in IC₅₀ values ranging from 1.54 to 18.17 μM. Among them 6,7-dichloro-5,8-dinitro-3-phenoxyquinoxalin-2(1H)-one (7e), exhibited the strongest aldose reductase activity with an IC₅₀ value of 1.54 μM and a good SAR (structure–activity relationship) profile.     

Benzoxazinone-thiosemicarbazones as antidiabetic leads via aldose reductase inhibition: Synthesis,biological screening and molecular docking study

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).     

Exocellular hydrolases from Penicillium ulaiense

Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layer-chromatography.The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina     

Carboxymethylated pyridoindole antioxidants as aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling

Starting from the efficient hexahydropyridoindole antioxidant stobadine, a series of carboxymethylated tetrahydro- and hexahydropyridoindole derivatives was synthesized and tested for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibiton of rat lens aldose reductase was determined by a conventional method. Kinetic analysis of (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (5b) and (2-phenethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (5c), the most potent compounds in this series with activities in micromolar range, showed uncompetitive inhibition. In addition to the importance of the acidic function, the inhibition efficacy was highly influenced by the steric conformation of the lipophilic aromatic backbone when comparing tetrahydro- and hexahydropyridoindole congeners. Selectivity with respect to the closely related aldehyde reductase was determined by measuring the corresponding inhibitory activities. Antioxidant action of the novel compounds was documented in a DPPH test and in a liposomal membrane model, oxidatively stressed by peroxyl radicals. The presence of a basicity center at the tertiary nitrogen, in addition to the acidic carboxylic function, predisposes these compounds to form double charged zwitterionic species, a characteristic which may remarkably affect their pH-lipophilicity profile. For compounds 5b and 5c, a maximal distribution ratio in a system comprised of 1-octanol/phosphate buffer was recorded near the neutral physiological pH, the region where the isoelectric point lies. Molecular docking simulations into the ALR2 active site performed for the zwitterionic species provided an explanation for the observed structure–activity relationships and the calculated parameters were in agreement with characteristic differences in the stereoelectronic profiles of the tetrahydro- versus hexahydropyridoindoles. ‘Drug-likeness’ of the novel aldose reductase inhibitors was assessed by applying the criteria of Lipinski’s ‘rule of five’.     

Upregulation of aldose reductase by homocysteine in type II alveolar epithelial cells

Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type II alveolar epithelial cells (A549 cells) treated by Hcy. We found that aldose reductase (AR) showed more abundant expression in the cells. Further, Hcy (100-500microM) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hcy. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hcy might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy.     

Early identification of promiscuous attributes of aldose reductase inhibitors using a DMSO-perturbation assay

Aldose reductase (AR) inhibitors are used clinically to treat long-term diabetic complications. Previous studies reported a series of AR inhibitory candidates, but unfortunately the mode of inhibition was poorly described due mainly to the lack of readily available methods for evaluating the specificity. The present study examined the AR inhibitory effects of novel synthetic hydantoins and their structural relatives, some of which were obtained from chemically engineered extracts of natural plants, and discovered several novel AR inhibitors with moderate inhibitory activity. The identified inhibitors were then subjected to a two-step mechanistic characterization using a detergent-addition assay and our novel dimethyl sulfoxide (DMSO)-perturbation assay. The detergent-addition assay revealed aggregation-based inhibitors, and the subsequent DMSO-perturbation assay identified nonspecific binding inhibitors. Thus, the present study demonstrates the usefulness of the DMSO-perturbation screen for identifying nonspecific binding characteristics of AR inhibitors.