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.     

The molecular basis for inhibition of sulindac and its metabolites towards human aldose reductase

Sulindac (SLD) exhibits both the highest inhibitory activity towards human aldose reductase (AR) among popular non-steroidal anti-inflammatory drugs and clear beneficial clinical effects on Type 2 diabetes. However, the molecular basis for these properties is unclear. Here, we report that SLD and its pharmacologically active/inactive metabolites, SLD sulfide and SLD sulfone, are equally effective as un-competitive inhibitors of AR in vitro. Crystallographic analysis reveals that π-π stacking favored by the distinct scaffold of SLDs is pivotal to their high AR inhibitory activities. These results also suggest that SLD sulfone could be a potent lead compound for AR inhibition in vivo.     

In vitro aldose reductase inhibitory activity of some flavonyl-2,4-thiazolidinediones

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. AR enzyme appears to be the key factor in the reduction of glucose to sorbitol. Synthesis and accumulation of sorbitol in cells due to AR activity is the main cause of diabetic complications, such as diabetic cataract, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors have been found to prevent sorbitol accumulation in tissues. Numerous compounds have been prepared in order to improve the pharmacological prophile of inhibition of aldose reductase enzyme. In this study, seventeen flavonyl-2,4-thiazolidinediones (flavonyl-2,4-TZD) (Ia-e, IIa-e and IIIa-g) were tested for their ability to inhibit rat kidney AR. Compound Ib showed the highest inhibitory activity (88.69 +/- 1.46%) whereas Ia, IIa, IIIa, IIIb also showed significant inhibitory activity (49.26 +/- 2.85, 67.29 +/- 1.09, 71.11 +/- 1.95, 64.86 +/- 1.21%, respectively).     

Substrate interaction with 5alpha-reductase enzyme: influence of the 17beta-chain chirality in the mechanism of action of 4-azasteroid inhibitors

A series of steroidal compounds were synthesized in order to evaluate the possible influence of the configuration of a stereocenter in the 17beta-side chain on the inhibitory activity on the enzyme 5alpha-reductase (5AR). For this purpose diastereomerically pure 4-azasteroids epimers at C-22 were prepared (compounds 1-11) and tested as inhibitors of 5AR in "in vitro" tests. The obtained data showed that in most cases the couples of epimers possess a significant difference in their biological activity. We also considered, for the tested molecules, a series of chemico-physical parameters in order to find a possible correlation with their biological activity. The findings allowed us to propose a model of the binding site of 5AR which comprises also, for 4-azasteroid inhibitors, the configurational aspect of the 17beta-side chain.     

Novel oxazolidinone based compounds as inhibitors of aromatase and the use of the substrate-heme complex approach in the rationalisation of these compounds

The synthesis, biochemical evaluation and molecular modelling of a series of N-alkylated 4-(4(')-aminobenzyl)-2-oxazolidinones is described involving the derivatisation of the starting R- or S-enantiomer of 4-benzyl-2-oxazolidinones. The compounds were tested for human placental aromatase (AR) inhibition in vitro and were found, in general, to be more potent than the standard compound, aminoglutethimide (AG). The inhibitory activity of the compounds was rationalised through the use of the novel substrate-heme complex (SHC) approach and suggests that the S-enantiomer based compounds protrude beyond the C(13), C(17), and C(16) area of the steroid backbone, resulting in steric hindrance with the active site of AR and thus reduced inhibitory activity. The R-enantiomer based compounds do not protrude in the same area and as such are not thought to undergo any steric hindrance and in comparison to the S-enantiomer, possess greater inhibitory activity.     

Green tea extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro

In this study, we aimed to evaluate in vitro the inhibitory activity of a green tea extract (AR25 standardized at 25% catechins) on gastric and pancreatic lipase activities. We first used tributyrin as a substrate to evaluate the capability of AR25 to induce digestive lipase inhibition. Gastric lipase was totally inhibited by 40 mg AR25/g tributyrin whereas pancreatic lipase inhibition was maximum (78.8 +/- 0.7%) with 80 mg AR25/g tributyrin. We then used triolein, a long-chain triglyceride, to check whether AR25 could alter lipase activities on a physiologic substrate. AR25 60 mg/g triolein induced a dramatic inhibition of gastric lipase (96.8 +/- 0.4%) whereas pancreatic lipase activity was partially reduced (66.50 +/- 0.92%). Finally, the concerted action of gastric and pancreatic lipases was studied with an excess of enzymes to mimic the physiologic conditions observed in vivo. Incubation of AR25 with an excess of digestive lipases resulted in a drastic decrease in gastric lipolysis but the inhibitory effect on pancreatic lipase was less marked. On the whole, as compared to the control, lipolysis of triolein under the successive action of the two digestive lipases was reduced by 37 +/- 0.6% in the presence of AR25. Because a lipid/water interface is necessary for lipolysis to occur, lipid emulsification and emulsion droplet size were measured in gastric and duodenal media in the presence of AR25. In gastric and duodenal conditions, AR25 inhibited the lipid emulsification process. From these data we conclude that (1) in vitro, fat digestion is significantly inhibited by 60 mg AR25/g triolein, and (2) gastric as well as pancreatic lipase inhibition could be related to altered lipid emulsification in gastric or duodenal media. The green tea extract AR25 exhibiting marked inhibition of digestive lipases in vitro is likely to reduce fat digestion in humans.     

Aldose reductase inhibitory effect by tectorigenin derivatives from Viola hondoensis

Aldose reductase (AR), the key enzyme of the polyol pathway, is known to play important roles in the diabetic complication. The inhibitors of AR, therefore, would be potential agents for the prevention of diabetic complication. The AR inhibition activity of several isoflavonoids was evaluated in vitro against rat lens. Tectoridin-4'-O-beta-D-glucoside exhibited strong AR inhibition activity on rat lens with an IC50 of 0.54 microM. Similar activities were recorded for the natural tectorigenin and tectoridin. In contrast, tectoridin-4'-O-beta-D-glucoside showed a stronger inhibitory activity than tectorigenin and tectoridin. Our results indicate that glucose conjugation position in this type of isoflavonoids may be required for the activity.     

Inhibitory effect of ursolic acid derivatives on recombinant human aldose reductase

Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives with ursane skeleton were tested for recombinant human aldose reductase (rhAR) inhibitory activity for development of diabetic complications. Among them, N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) showed most potent rhAR inhibitory activity in vitro. Inhibition mode of N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) was tested uncompetitively by kinetic analysis using the Lineweaver-Burk plots. Ursolic acid derivative N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid is able to inhibit rhAR uncompetitively and could be offered as a lead compound for AR inhibition.     

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.     

Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles

We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2-adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns.     

Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition

Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.     

Design, synthesis, and biological evaluation of novel 4-alkylamino-1-hydroxymethylimidazo[1,2-a]quinoxalines as adenosine A(1) receptor antagonists

A series of 4-alkylamino-1-hydroxymethylimidazo[1,2-a]quinoxalines have been synthesized and evaluated for their adenosine A(1) receptor inhibitory activity in the radioligand binding assays. The compounds were tested for the inhibition percent (IP) and the affinity toward A(1)AR (K(i)) that IP were more than 90% in the nanomolar range. 4-Cyclopentylamino-7,8-dichloro-1-hydroxymethylimidazo[1,2-a]quinoxaline 18 is the most potent compound in this series, having K(i)=7nM, which is remarkably higher than that of IRFI-165 (K(i)=48). 1-Hydroxymethyl groups of the tricyclic heteroarmatic compounds displayed the potent affinities toward A(1)AR.     

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.     

槲皮素对前列腺癌细胞增殖及转录因子Sp1功能的抑制作用

雄激素受体(androgenreceptor,AR)作为核转录因子,其高表达、基因突变以及AR辅激活因子的过表达等造成AR的异常激活与前列腺癌细胞的增殖、恶化转移、多药耐药等密切相关.天然黄酮槲皮素(quercetin),是一很有潜力的预防和治疗前列腺肿瘤的化合物.槲皮素不仅抑制前列腺癌细胞LNCaP的增殖,并呈剂量依赖性,而且下调前列腺癌中AR的表达、抑制AR的转录激活功能.GCbox是AR核心启动子的主要正调控元件,是转录因子Sp1的结合位点.细胞转染结果表明,槲皮素能抑制Sp1蛋白对AR启动子的激活作用,可能是槲皮素下调AR表达的机理之一.进一步研究显示,槲皮素还能明显抑制Sp1蛋白对AR转录激活功能的增强作用.Western印迹结果显示,槲皮素对Sp1蛋白表达无明显影响,但能够诱导c-Jun的高表达,而高表达的c-Jun蛋白能逆转Sp1蛋白对AR的转录激活作用,由此推测,槲皮素可能通过介导c-Jun与Sp1的蛋白质相互作用,抑制Sp1的功能,进而起到抑制AR表达和功能的作用.免疫沉淀结果又进一步证实了Sp1与c-Jun二者的相互作用.因此,槲皮素可能通过抑制前列腺癌细胞中AR的表达和功能抑制了细胞的增殖,其分子机理可能与槲皮素诱导的c-Jun与Sp1蛋白相互作用、降低Sp1对AR的转录激活作用有关.