Response of a Human Lens Epithelial Cell Line to Hyperglycemic and Oxidative Stress: The Role of Aldose Reductase.

A common feature of different types of diabetes is the high blood glucose levels, which are known to induce a series of metabolic alterations, leading to damaging events in different tissues. Among these alterations, both increased polyol pathway flux and oxidative stress are considered to play relevant roles in the response of different cells. In this work, the effect on a human lens epithelial cell line of stress conditions, consisting of exposure to either high glucose levels or to the lipid peroxidation product 4-hydroxy-2-nonenal, is reported. The occurrence of osmotic imbalance, alterations of glutathione levels, and expression of inflammatory markers was monitored. A common feature of the two stress conditions was the expression of COX-2, which, only in the case of hyperglycemic stress, occurred through NF-κB activation. In our cell model, aldose reductase activity, which is confirmed as the only activity responsible for the osmotic imbalance occurring in hyperglycemic conditions, seemed to have no role in controlling the onset of the inflammatory phenomena. However, it played a relevant role in cellular detoxification against lipid peroxidation products. These results, in confirming the multifactorial nature of the inflammatory phenomena, highlight the dual role of aldose reductase as having both damaging but also protecting activity, depending on stress conditions.

Designed multiple ligands for the treatment of type 2 diabetes mellitus and its complications: Discovery of (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)alkanoic acids active as novel dual-targeted PTP1B/AKR1B1 inhibitors.

Type 2 diabetes mellitus (T2DM) is a serious chronic disease with an alarmingly growing worldwide prevalence. Current treatment of T2DM mainly relies on drug combinations in order to control blood glucose levels and consequently prevent the onset of hyperglycaemia-related complications. The development of multiple-targeted drugs recently emerged as an attractive alternative to drug combinations for the treatment of complex diseases with multifactorial pathogenesis, such as T2DM. Protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AKR1B1) are two enzymes crucially involved in the development of T2DM and its chronic complications and, therefore, dual inhibitors targeted to both these enzymes could provide novel agents for the treatment of this complex pathological condition. In continuing our search for dual-targeted PTP1B/AKR1B1 inhibitors, we designed new (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)alkanoic acids. Among them, 3-(4-phenylbutoxy)benzylidene derivatives 6f and 7f, endowed with interesting inhibitory activity against both targets, proved to control specific cellular pathways implicated in the development of T2DM and related complications.

Evidence of Insulin-Sensitizing and Mimetic Activity of the Sesquiterpene Quinone Avarone, a Protein Tyrosine Phosphatase 1B and Aldose Reductase Dual Targeting Agent from the Marine Sponge Dysidea avara.

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by impaired glucose homeostasis and serious long-term complications. First-line therapeutic options for T2DM treatment are monodrug therapies, often replaced by multidrug therapies to ensure that non-responding patients maintain target glycemia levels. The use of multitarget drugs instead of mono- or multidrug therapies has been emerging as a main strategy to treat multifactorial diseases, including T2DM. Therefore, modern drug discovery in its early stages aims to identify potential modulators for multiple targets; for this purpose, exploration of the chemical space of natural products represents a powerful tool. Our study demonstrates that avarone, a sesquiterpene quinone obtained from the sponge Dysidea avara, is capable of inhibiting in vitro PTP1B, the main negative regulator of the insulin receptor, while it improves insulin sensitivity, and mitochondria activity in C2C12 cells. We observe that when avarone is administered alone, it acts as an insulin-mimetic agent. In addition, we show that avarone acts as a tight binding inhibitor of aldose reductase (AKR1B1), the enzyme involved in the development of diabetic complications. Overall, avarone could be proposed as a novel natural hit to be developed as a multitarget drug for diabetes and its pathological complications.

Ribose Intake as Food Integrator: Is It a Really Convenient Practice?

Reports concerning the beneficial effects of D-ribose administration in cardiovascular and muscle stressful conditions has led to suggestions for the use of ribose as an energizing food supplement for healthy people. However, this practice still presents too many critical issues, suggesting that caution is needed. In fact, there are many possible negative effects of this sugar that we believe are underestimated, if not neglected, by the literature supporting the presentation of the product to the market. Here, the risks deriving from the use of free ribose as ATP source, forcing ribose-5-phosphate to enter into the pentose phosphate pathway, is emphasized. On the basis of the remarkable glycation capacity of ribose, the easily predictable cytotoxic effect of the molecule is also highlighted.

Dissecting the Activity of Catechins as Incomplete Aldose Reductase Differential Inhibitors through Kinetic and Computational Approaches.

The inhibition of aldose reductase is considered as a strategy to counteract the onset of both diabetic complications, upon the block of glucose conversion in the polyol pathway, and inflammation, upon the block of 3-glutathionyl-4-hydroxynonenal reduction. To ameliorate the outcome of aldose reductase inhibition, minimizing the interference with the detoxifying role of the enzyme when acting on toxic aldehydes, "differential inhibitors", i.e., molecules able to inhibit the enzyme depending on the substrate the enzyme is working on, has been proposed. Here we report the characterization of different catechin derivatives as aldose reductase differential inhibitors. The study, conducted through both a kinetic and a computational approach, highlights structural constraints of catechin derivatives relevant in order to affect aldose reductase activity. Gallocatechin gallate and catechin gallate emerged as differential inhibitors of aldose reductase able to preferentially affect aldoses and 3-glutathionyl-4-hydroxynonenal reduction with respect to 4-hydroxynonenal reduction. Moreover, the results highlight how, in the case of aldose reductase, a substrate may affect not only the model of action of an inhibitor, but also the degree of incompleteness of the inhibitory action, thus contributing to differential inhibitory phenomena.

Models of enzyme inhibition and apparent dissociation constants from kinetic analysis to study the differential inhibition of aldose reductase.

In order to explain the negative slope of KappM/kappcat versus inhibitor concentration observed in the study of epigallocatechin gallate acting as an inhibitor of aldose reductase, a kinetic analysis was performed to rationalise the phenomenon. Classical and non-classical models of complete and incomplete enzyme inhibition were devised and analysed to obtain rate equations suitable for the interpretation of experimental data. The results obtained from the different approaches were discussed in terms of the meaning of the emerging kinetic constants. A decrease of KappM/kappcat versus the inhibitor concentration was revealed to be a valuable indication of the occurrence of an incomplete inhibition. This indication, which is univocal in the case of an uncompetitive inhibition, may be especially useful when the residual activity resulting from inhibition is rather low.

In Search of Differential Inhibitors of Aldose Reductase.

Aldose reductase, classified within the aldo-keto reductase family as AKR1B1, is an NADPH dependent enzyme that catalyzes the reduction of hydrophilic as well as hydrophobic aldehydes. AKR1B1 is the first enzyme of the so-called polyol pathway that allows the conversion of glucose into sorbitol, which in turn is oxidized to fructose by sorbitol dehydrogenase. The activation of the polyol pathway in hyperglycemic conditions is generally accepted as the event that is responsible for a series of long-term complications of diabetes such as retinopathy, cataract, nephropathy and neuropathy. The role of AKR1B1 in the onset of diabetic complications has made this enzyme the target for the development of molecules capable of inhibiting its activity. Virtually all synthesized compounds have so far failed as drugs for the treatment of diabetic complications. This failure may be partly due to the ability of AKR1B1 to reduce alkenals and alkanals, produced in oxidative stress conditions, thus acting as a detoxifying agent. In recent years we have proposed an alternative approach to the inhibition of AKR1B1, suggesting the possibility of a differential inhibition of the enzyme through molecules able to preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. The rationale and examples of this new generation of aldose reductase differential inhibitors (ARDIs) are presented.

Identifying Human PTP1B Enzyme Inhibitors from Marine Natural Products: Perspectives for Developing of Novel Insulin-Mimetic Drugs.

Diabetes mellitus (DM) represents a complex and multifactorial disease that causes metabolic disorders with acute and long-term serious complications. The onset of DM, with over 90% of cases of diabetes classified as type 2, implies several metabolic dysfunctions leading to consider DM a worldwide health problem. In this frame, protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AR) are two emerging targets involved in the development of type 2 diabetes mellitus (T2DM) and its chronic complications. Herein, we employed a marine-derived dual type inhibitor of these enzymes, phosphoeleganin, as chemical starting point to perform a fragment-based process in search for new inhibitors. Phosphoeleganin was both disassembled by its oxidative cleavage and used as model structure for the synthesis of a small library of functionalized derivatives as rationally designed analogues. Pharmacological screening supported by in silico docking analysis outlined the mechanism of action against PTP1B exerted by a phosphorylated fragment and a synthetic simplified analogue, which represent the most potent inhibitors in the library.

Dual Targeting of PTP1B and Aldose Reductase with Marine Drug Phosphoeleganin: A Promising Strategy for Treatment of Type 2 Diabetes.

An in-depth study on the inhibitory mechanism on protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AR) enzymes, including analysis of the insulin signalling pathway, of phosphoeleganin, a marine-derived phosphorylated polyketide, was achieved. Phosphoeleganin was demonstrated to inhibit both enzymes, acting respectively as a pure non-competitive inhibitor of PTP1B and a mixed-type inhibitor of AR. In addition, in silico docking analyses to evaluate the interaction mode of phosphoeleganin with both enzymes were performed. Interestingly, this study showed that phosphoeleganin is the first example of a dual inhibitor polyketide extracted from a marine invertebrate, and it could be used as a versatile scaffold structure for the synthesis of new designed multiple ligands.

In Search for Multi-Target Ligands as Potential Agents for Diabetes Mellitus and Its Complications-A Structure-Activity Relationship Study on Inhibitors of Aldose Reductase and Protein Tyrosine Phosphatase 1B.

Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates.

Aldose Reductase Differential Inhibitors in Green Tea.

Aldose reductase (AKR1B1), the first enzyme in the polyol pathway, is likely involved in the onset of diabetic complications. Differential inhibition of AKR1B1 has been proposed to counteract the damaging effects linked to the activity of the enzyme while preserving its detoxifying ability. Here, we show that epigallocatechin gallate (EGCG), one of the most representative catechins present in green tea, acts as a differential inhibitor of human recombinant AKR1B1. A kinetic analysis of EGCG, and of its components, gallic acid (GA) and epigallocatechin (EGC) as inhibitors of the reduction of L-idose, 4-hydroxy2,3-nonenal (HNE), and 3-glutathionyl l-4-dihydroxynonanal (GSHNE) revealed for the compounds a different model of inhibition toward the different substrates. While EGCG preferentially inhibited L-idose and GSHNE reduction with respect to HNE, gallic acid, which was still active in inhibiting the reduction of the sugar, was less active in inhibiting HNE and GSHNE reduction. EGC was found to be less efficient as an inhibitor of AKR1B1 and devoid of any differential inhibitory action. A computational study defined different interactive modes for the three substrates on the AKR1B1 active site and suggested a rationale for the observed differential inhibition. A chromatographic fractionation of an alcoholic green tea extract revealed that, besides EGCG and GA, other components may exhibit the differential inhibition of AKR1B1.

Pathways of 4-Hydroxy-2-Nonenal Detoxification in a Human Astrocytoma Cell Line.

One of the consequences of the increased level of oxidative stress that often characterizes the cancer cell environment is the abnormal generation of lipid peroxidation products, above all 4-hydroxynonenal. The contribution of this aldehyde to the pathogenesis of several diseases is well known. In this study, we characterized the ADF astrocytoma cell line both in terms of its pattern of enzymatic activities devoted to 4-hydroxynonenal removal and its resistance to oxidative stress induced by exposure to hydrogen peroxide. A comparison with lens cell lines, which, due to the ocular function, are normally exposed to oxidative conditions is reported. Our results show that, overall, ADF cells counteract oxidative stress conditions better than normal cells, thus confirming the redox adaptation demonstrated for several cancer cells. In addition, the markedly high level of NADP+-dependent dehydrogenase activity acting on the glutahionyl-hydroxynonanal adduct detected in ADF cells may promote, at the same time, the detoxification and recovery of cell-reducing power in these cells.

Intra-site differential inhibition of multi-specific enzymes.

The ability to catalyse a reaction acting on different substrates, known as "broad-specificity" or "multi-specificity", and to catalyse different reactions at the same active site ("promiscuity") are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach.

Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group.

Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.

The furanosidic scaffold of d-ribose: a milestone for cell life.

The recruitment of the furanosidic scaffold of ribose as the crucial step for nucleotides and then for nucleic acids synthesis is presented. Based on the view that the selection of molecules to be used for relevant metabolic purposes must favor structurally well-defined molecules, the inadequacy of ribose as a preferential precursor for nucleotides synthesis is discussed. The low reliability of ribose in its furanosidic hemiacetal form must have played ab initio against the choice of d-ribose for the generation of d-ribose-5-phosphate, the fundamental precursor of the ribose moiety of nucleotides. The latter, which is instead generated through the 'pentose phosphate pathway' is strictly linked to the affordable and reliable pyranosidic structure of d-glucose.

Stereoselectivity of Aldose Reductase in the Reduction of Glutathionyl-Hydroxynonanal Adduct.

The formation of the adduct between the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and glutathione, which leads to the generation of 3-glutathionyl-4-hydroxynonane (GSHNE), is one of the main routes of HNE detoxification. The aldo-keto reductase AKR1B1 is involved in the reduction of the aldehydic group of both HNE and GSHNE. In the present study, the effect of chirality on the recognition by aldose reductase of HNE and GSHNE was evaluated. AKR1B1 discriminates very modestly between the two possible enantiomers of HNE as substrates. Conversely, a combined kinetic analysis of the glutathionyl adducts obtained starting from either 4R- or 4S-HNE and mass spectrometry analysis of GSHNE products obtained from racemic HNE revealed that AKR1B1 possesses a marked preference toward the 3S,4R-GSHNE diastereoisomer. Density functional theory and molecular modeling studies revealed that this diastereoisomer, besides having a higher tendency to be in an open aldehydic form (the one recognized by AKR1B1) in solution than other GSHNE diastereoisomers, is further stabilized in its open form by a specific interaction with the enzyme active site. The relevance of this stereospecificity to the final metabolic fate of GSHNE is discussed.

Soyasaponins from Zolfino bean as aldose reductase differential inhibitors.

Seven triterpenoid saponins were identified in methanolic extracts of seeds of the Zolfino bean landrace (Phaseolus vulgaris L.) by HPLC fractionation, revealing their ability to inhibit highly purified human recombinant aldose reductase (hAKR1B1). Six of these compounds were associated by MS analysis with the following saponins already reported in different Phaseolus vulgaris varieties: soyasaponin Ba (V), soyasaponin Bb, soyasaponin Bd (sandosaponin A), soyasaponin αg, 3-O-[R-l-rhamnopyranosyl(1 → 2)-α-d-glucopyranosyl(1 → 2)-α-d-glucuronopyranosyl]olean-12-en-22-oxo-3α,-24-diol, and soyasaponin ßg. The inhibitory activity of the collected fractions containing the above compounds was tested for hAKR1B1-dependent reduction of both l-idose and 4-hydroxynonenal, revealing that some are able to differentially inhibit the enzyme. The present work also highlights the difficulties in the search for aldose reductase differential inhibitors (ARDIs) in mixtures due to the masking effect on ARDIs exerted by the presence of conventional aldose reductase inhibitors. The possibility of differential inhibition generated by a different inhibitory model of action of molecules on different substrates undergoing transformation is also discussed.

An investigation on 4-thiazolidinone derivatives as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, in the search for potential agents for the treatment of type 2 diabetes mellitus and its complications.

Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1-17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes.

Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors.

Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually AKR1B1 is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part in the antioxidant defense mechanism of the body. In this work we report the evidence that a class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and an ionizable fragment, modulates differently the catalytic activity of the enzyme, depending on the presence of specific substrates such as sugar, toxic aldehydes, and glutathione conjugates of toxic aldehydes. The study stands out as a systematic attempt to generate aldose reductase differential inhibitors (ARDIs) intended to target long-term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase.

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.