Glutathione-Hemin/Hematin Adduct Formation to Disintegrate Cytotoxic Oxidant Hemin/Hematin in Human K562 Cells and Red Blood Cells' Hemolysates: Impact of Glutathione on the Hemolytic Disorders and Homeostasis.

Hemin, an oxidized form of heme, acts as potent oxidant to regulate glutathione (GSH) content in pro-erythroid K562 nucleated cells, via activation of the KEAP1/NRF2 defensive signaling pathway. Moreover, GSH, as an essential metabolite, is involved in the regulation of cell-redox homeostasis and proposed to scavenge cytotoxic free heme, which is released from hemoglobin of damaged red blood cells (RBCs) during different hemolytic disorders. In the present study, we aimed to uncover the molecular mechanism by which GSH inhibits hemin-induced cytotoxicity (HIC) by affecting hemin's structural integrity in K562 cells and in RBC hemolysates. GSH, along with other thiols (cysteine, thioglycolic acid, and mercaptoethanol) altered the spectrum of hemin, while each of them co-added with hemin in cultures of K562 cells prevented HIC and growth arrest and markedly reduced the intracellular level of hemin. In addition, GSH endogenous levels served as a barrier to HIC in K562 cells, as shown by the depletion in GSH. LC-MS/MS analysis of the in vitro reaction between hemin and GSH revealed at least five different isomers of GSH-hemin adducts, as well as hydroxy derivatives as reaction products, which are characterized by unique mass spectra (MS). The latter allowed the detection of adducts in human RBC hemolysates. Based on these findings, we proposed a molecular mechanism via which GSH prevents HIC and structurally disintegrates heme. An analogous reaction was observed in RBC hemolysates via direct inter-reaction between hematin (ferric and hydroxide heme) released from hemoglobin and GSH. Overall, GSH-hematin adducts could be considered as novel entities of the human metabolome of RBCs in hemolytic disorders.

Drug-like Properties and Fraction Lipophilicity Index as a combined metric.

Fraction Lipophicity Index (FLI) has been developed as a composite drug-like metric combining log P and log D in a weighted manner. In the present study, an extended data set confirmed the previously established drug-like FLI range 0-8 using two calculation systems for log P/log D assessment, the freeware MedChem Designer and ClogP. The dataset was split into two classes according to the percentage of fraction absorbed (%FA) - class 1 including drugs with high to medium absorption levels and class 2 including poorly absorbed drugs. The FLI and FLI-C (ClogP based FLI) drug-like range covers 92 % and 91 % of class 1 drugs, respectively. Using MlogP, a narrower drug-like FLI-M range 0-7 was established, covering 91 % of class 1 drugs. The dependence of the degree of ionization to intrinsic lipophilicity within the FLI (FLI-C, FLI-M) drug-like range as well as the inter-relation between the other Ro5 properties (Mw, HD, HA) was explored to define drug-like / non-drug-like combinations as a safer alternative to single properties for drug candidates' prioritization. In this sense, we propose a combined metric of Mw and the number of polar atoms (Mw/NO) to account for both size and polarity. Setting the value 50 as cutoff, a distinct differentiation between class 1 and class 2 drugs was obtained with Mw/NO>50 for more than 70 % of class 1 drugs, while the opposite was observed for class 2 drugs.

Formation of novel N-acetylcysteine-hemin adducts abrogates hemin-induced cytotoxicity and suppresses the NRF2-driven stress response in human pro-erythroid K562 cells.

Heme (iron protoporphyrin IX), as the prosthetic group in hemoproteins, regulates vital cellular functions in human tissues. However, free heme released during hemolysis events promotes severe complications to millions of people worldwide. Over the years, thiols like glutathione (GSH) were known to antagonize heme toxicity. In this study, we have uncovered the underlying molecular mechanism by which N-acetylcysteine (NAC), a well-known thiol prevents hemin-induced cytotoxicity (HIC). Hemin-responsive human pro-erythroid K562 cells were employed to assess hemin intracellular accumulation and cytotoxicity at concentrations ≥50 µΜ, in cultures exposed only to hemin and/or both hemin and NAC. NAC inhibited the intracellular accumulation of hemin and prevented hemin-induced cell growth inhibition, cell death, oxidative stress, and accumulation of ubiquitinated proteins. Meanwhile, the activation of the NF-E2-related factor-2 (NRF2)-driven stress gene activation, a key element involved in HIC, was suppressed by NAC. A refined mechanism of the chemical reaction between NAC and hemin leading to adduct formation via a nucleophilic attack on hemin was uncovered for the first time by tandem mass spectrometry analysis (LC-MS/MS). Such thiol-hemin adducts acted as intermediates to mitigate HIC and to suppress hemin-induced NRF2-driven gene activation. Our findings support the concept that NAC-hemin adduct formation is the major novel molecular mechanism rather than the reactive oxygen species-scavenging capacity of thiols to protect cells from HIC. Our results imply that thiols and their derivatives can be of potential therapeutic value in hemolytic disorders.

Decreasing acidity in a series of aldose reductase inhibitors: 2-Fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for improved membrane permeation.

Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhibitors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with putative pKa≥7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluorophenol group proved the most promising moiety, and further modifications were explored. The most active compound (31), identified as a submicromolar inhibitor (IC50=0.443µM), was also selective against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a pKa of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable membrane permeation. The latter was supported from the successful inhibition of sorbitol formation in rat lenses and the ability to permeate rat jejunum.

Development of aldose reductase inhibitors for the treatment of inflammatory disorders.

INTRODUCTION: Accumulating evidence attributes a significant role to aldose reductase (ALR2) in the pathogenesis of several inflammatory pathologies. Aldose reductase inhibitors (ARIs) were found to attenuate reactive oxygen species (ROS) production both in vitro and in vivo. Thus, they disrupt signaling cascades that lead to the production of cytokines/chemokines, which induce and exacerbate inflammation. As a result, ARIs might hold a significant therapeutic potential as alternate anti-inflammatory drugs. AREAS COVERED: The authors present a comprehensive review of the current data that support the central role of ALR2 in several inflammatory pathologies (i.e., diabetes, cancer, sepsis, asthma and ocular inflammation). Further, the authors describe the potential underlying molecular mechanisms and provide a commentary on the status of ARIs in this field. EXPERT OPINION: It is important that future efforts focus on delineating all the steps of the molecular mechanism that implicates ALR2 in inflammatory pathologies. At the same time, utilizing the previous efforts in the field of ARIs, several candidates that have been proven safe in the clinic may be evaluated for their clinical significance as anti-inflammatory medication. Finally, structurally novel ARIs, designed to target specifically the proinflammatory subpocket of ALR2, should be pursued.

Synthesis of derivatives of the keto-pyrrolyl-difluorophenol scaffold: some structural aspects for aldose reductase inhibitory activity and selectivity.

Seven novel ARIs (3a-c, 4a-c and 5) were synthesized with the implementation of an optimized and, partially, selective synthetic procedure, via a Friedel-Crafts acylation reaction. The synthesized ARIs have values of IC(50)(ALR2) ranging from 0.19µM (in case of compound 3b) to 2.3µM (in case of compound 4a), while the values of selectivity index towards ALR1 range from 1 (in case of compound 3b) to 238 (in case of compound 3a). Finally, we found out that the presence of an additional (secondary) aromatic area is not a prerequisite feature for ARI activity.

Novel aldose reductase inhibitors: a patent survey (2006--present).

INTRODUCTION: Initially studied for its central role in the pathogenesis of chronic diabetic complications, aldose reductase (ALR2) gains more attention over the years as its implication in inflammatory diseases is being established, along with the therapeutic potential of its inhibitors. AREAS COVERED: Reviewing the patents that were published since 2006, it is getting clear that the search for new chemical entities has subsided, giving rise to natural products and plant extracts with ALR2 inhibitory activity. Other aspects that were prominent were the search for proper forms of known inhibitors, in a way to improve their impaired physicochemical profile, as well as potential combination therapies with other compounds of pharmaceutical interest. On the spotlight were patents enhancing the therapeutic usage of aldose reductase inhibitors (ARIs) to various pathological conditions including cancer and inflammation-mediated diseases such as sepsis, asthma, and cancer. EXPERT OPINION: Although new chemical entities are scarcely registered and patented after many years of inconclusive clinical trials, the involvement of ALR2 to inflammatory pathologies might renew the interest in the field of ARIs.

Bis-pyrrolyl-tetrazolyl derivatives as hybrid polar compounds: A case of lipophilic functional bioisosterism with bis-acetamides.

Based on previous studies on bis-acetamides that act as hybrid polar compounds to induce leukemia cell differentiation, an attempt was made to bioisosterically replace the amide moiety with the lipophilic non-classical bioisostere tetrazole. A pyrrole group was also included in the molecule in order to retain the hydrogen bond donor capability. Thus, by linking the two polar ring systems with a highly lipophilic methylene chain compounds 2-4 were synthesized and assessed for their anti-proliferative activity in combination with their ability to induce murine erythroleukemia (MEL) cell differentiation. Furthermore, an initial investigation of the structure-activity relation points for the active compound 3 was undertaken by synthesizing compound 5 (a p-xylene analog) and compound 8 (a methylamidopyrrolyl analog). All compounds caused a dose-dependent inhibition of MEL cell growth but to a different extent. Compound 3 (1,6-bis[5-(1H-pyrrol-1-yl)-2H-tetrazol-2-yl]hexane) promoted erythroid differentiation in a fifty-fold lower concentration than hexamethylenebisacetamide (HMBA). Though induction of differentiation was to a lesser extent than HMBA, it caused accumulation of 80% Hb-producing cells as compared to that produced by HMBA, leading to differentiation-depended cell growth inhibition equal to that of HMBA after 96 h in culture. Compound 3 represents a potent inducer of hemoglobin gene activation in leukemic cells.

Structure-activity relations on [1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanone. The effect of methoxy substitution on aldose reductase inhibitory activity and selectivity.

Based on our previous work, we studied the effect of methoxy-substitution as well as the regioposition of the benzoyl-moiety of 4a [(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone]. On this basis, compounds 4b-c and 5a-c were synthesized and assayed for aldose and aldehyde reductase inhibitory activity. Furthermore, a 4,6-difluoro-5-hydroxyphenyl pattern (9) was studied, in order to verify the optimum position of the phenol-moiety. Compound 5b emerged as the most potent and selective inhibitor. Moreover, further assays proved 5b as a potent antioxidant and an inhibitor of sorbitol accumulation in isolated rat lenses. Combining the above attributes, 5b could serve as a lead compound targeted at long-term diabetes complications.

Nutritional overview on the management of type 2 diabetes and the prevention of its complications.

Diabetes mellitus is an increasing world health problem; particularly the prevalence of type 2 diabetes has assumed epidemic dimensions in Western industrialized societies. It is mainly the environmental, dietary and lifestyle behavioral factors that are the control keys in the progress of this disease. Several epidemiological studies have linked over nutrition and lack of physical activity with type 2 diabetes. Indeed, the excessive consumption of energy dense foods as source of carbohydrates and fats along with ineffective medical management has negative impact on controlling blood glucose levels and on insulin response. This usually leads to a hyperglycemic state, which is associated with the development of the devastating secondary complications. Dietary guidelines have always been important for people with diabetes mellitus. Nutrition management aims to improve health quality maintaining blood glucose levels in normal range so as to reduce the risk for diabetes complications. A well-balanced diet that provides the essential macro- and micro-nutrients is always an impaired need for a patient with diabetes. In this article nutrition recommendations will be displayed for the management of diabetes type 2 and the prevention of its complications. Particular emphasis will be given to the important role of micronutrients such as trace elements and vitamins as well as to the potentiality of some dietary agents to inhibit aldose reductase enzyme, implicated in the etiology of diabetes complications.

A diverse series of substituted benzenesulfonamides as aldose reductase inhibitors with antioxidant activity: design, synthesis, and in vitro activity.

We have previously reported the successful replacement of a carboxylic acid functionality with that of a difluorophenolic group on the known aldose reductase inhibitors (ARIs) of 2-(phenylsulfonamido)acetic acid chemotype. In the present work, based on bioisosteric principles, additional 2,6-difluorophenol and tetrazole, methylsulfonylamide, and isoxazolidin-3-one phenylsulfonamide derivatives were synthesized and tested in vitro in protocols primarily related to the long-term diabetic complications. Most of the compounds were found as ARIs at IC(50) < 100 µM, while the introduction of the 4-bromo-2-fluorobenzyl group in a phenylsulfonamidodifluorophenol structure resulted in a compound (4c) presenting a submicromolar inhibitory profile. However, the derivatives of tetrazole, methylsulfonylamine, and the (R)-enantiomer of isoxazolidin-3-one did not exhibit appreciable ARI activity. The selectivity of the active ARIs is also discussed. Furthermore, the synthesized compounds exhibited potent antioxidant potential (homogeneous and heterogeneous systems).

Design and synthesis of novel series of pyrrole based chemotypes and their evaluation as selective aldose reductase inhibitors. A case of bioisosterism between a carboxylic acid moiety and that of a tetrazole.

Pyrrolyl-propionic and butyric-acid derivatives 1 and 2 were synthesized in order to study the effect of the variation of the methylene chain in comparison to the previously reported pyrrolyl-acetic acid compound I, which was found as potent aldose reductase inhibitor, while the pyrrolyl-tetrazole derivatives 3-5 were prepared as a non-classical bioisosteres of a carboxylic acid moiety. Also, pyrrolyl-tetrazole isomers 6 and 7 without an alkyl chain between the two aromatic rings were synthesized. The in vitro aldose reductase inhibitory activity of the prepared 1-7 compounds were estimated and compared with that of the initial compound (I). Overall, the data indicate that the presented chemotypes 6 and 7 are a promising lead compounds for the development of selective aldose reductase inhibitors, aiming to the long-term complications of diabetes mellitus.

Evaluation of aldose reductase inhibition and docking studies of 6'-nitro and 6',6''-dinitrorosmarinic acids.

Aldose reductase (ALR2) of the polyol metabolic pathway is a target enzyme for the treatment of diabetic complications. A variety of synthetic and natural compounds have been observed to inhibit aldose reductase. Among them, rosmarinic acid has been shown to be in vitro an aldose reductase inhibitor in a micromolar range. In this study, two nitro derivatives of rosmarinic acid synthesized previously, 6'-nitro and 6',6''-dinitrorosmarinic acids, are proposed as aldose reductase inhibitors. Docking studies of the nitro derivatives have been carried out in the active site of aldose reductase. The theoretical results have shown a higher estimated binding energy of both compounds in comparison to that of rosmarinic acid suggesting a higher ALR2 inhibitory activity. The in vitro biological assays confirmed that these compounds were more potent than the parent rosmarinic acid.

HPLC-based lipophilicity of pyrrolyl-acetic acid ARIs: Relationships with biological activity.

Reversed phase HPLC was used to assess the lipophilicity of a series pyrrolyl-acetic acid derivatives with aldose reductase inhibitory activity. The pH conditions were adjusted at 3.0 to investigate the behavior of the neutral species and at pH 7.4, at which the ionized form predominates, using phosphate and MOPS buffer. Retention was monitored in absence and in presence of different amounts of n-octanol in the mobile phase in order to explore the chromatographic conditions which best reproduce the octanol-water partition or distribution coefficients. The effect of n-octanol in retention was systematically studied and its role in lipophilicity assessment was evaluated. Nevertheless rather moderate regression equations were obtained, which deviated significantly from the ideal 1:1 correlation. No significant effect of buffer was observed. The appropriateness of retention factors to be used in correlation with aldose reductase inhibitory activity was further evaluated and compared to the efficiency of the corresponding octanol-water logP values.

Aldose reductase enzyme and its implication to major health problems of the 21(st) century.

Aldose reductase enzyme (ALR2) of the polyol metabolic pathway, apart from its role as detoxifying enzyme towards toxic aldehydes, osmoregulator in the kidney and regulator of sperm maturation, was first found to be implicated in the etiology of the long term diabetic complications. However, to date, emerging reports have suggested that under normal glucose concentration, ALR2 may be up-regulated by factors other than hyperglycemia and therefore be involved also in other pathological processes that have become major threats to human health in the 21(st) century. Such pathologies are a number of cardiac disorders, inflammation, mood disorders, renal insufficiency and ovarian abnormalities. In addition, ALR2 was found to be over-expressed in different human cancers such as liver, breast, ovarian, cervical and rectal cancers. Although several aldose reductase inhibitors (ARIs) have progressed to the clinical level, only one is currently on the market. Thus, attention is currently targeted to discover ARIs of distinct chemical structures, being neither hydantoin nor carboxylic acid derivatives. The present review focuses on the molecular mechanisms by which ALR2 is implicated in a number of pathologies, on various aspects concerning its catalytic mechanism and its active site, and on the main classes of ARIs that have been developed to date, as well as on reported (quantitive) structure-activity relationships. The presented data aim to support the notion that ARIs are of pharmacotherapeutic interest for the pharmaceutical community and highlight essential aspects for the development of efficient and potent ARIs.

Design and synthesis of N-(3,5-difluoro-4-hydroxyphenyl)benzenesulfonamides as aldose reductase inhibitors.

N-(3,5-Difluoro-4-hydroxyphenyl)benzenesulfonamide (4) and its derivatives 5-7 were prepared as putative bioisosteres of the previously reported aldose reductase inhibitors, which are the N-benzenesulfonylglycine derivatives I-IV. The in vitro aldose reductase inhibitory activity of the prepared compounds is higher than that of the respective glycine derivatives. Furthermore, the parent compound 4 reveals high antioxidant potential. Additionally, the intestine permeability of 4 is determined, and there is initial evidence that there is an operating influx mechanism. Overall, the data indicate that the presented chemotype could serve as a core structure for the design of putative pharmacotherapeutic agents, aiming to the long-term complications of diabetes mellitus.

Inhibitory effect of polar oregano extracts on aldose reductase and soybean lipoxygenase in vitro.

The effect of methanol and aqueous methanol extract of Origanum vulgare L. ssp. hirtum on aldose reductase and soybean lipoxygenase was investigated. The results revealed a promising potential of oregano for preventing diabetes complications in the long term and an antiinflammatory efficacy by inhibiting soybean lipoxygenase.

Permeability characteristics of novel aldose reductase inhibitors using rat jejunum in vitro.

The aim of this study was to estimate in vivo permeability and bioavailability of epalrestat and newly synthesized compounds with possible therapeutic activity as aldose enzyme inhibitors (ARIs). For this purpose permeability in vitro using rat jejunum mounted in side-by-side diffusion cells was determined. Tested substances were found to be low and moderately permeable and some of them were also substrates for efflux transporters. It was shown, that the higher efflux for some derivatives was due to MRP-2, but not Pgp involvement. Tested ARIs do not share the same efflux transporter with epalrestat, the only ARI currently on the market in Japan. The most permeable compound, a 2,6-difluoro-4-pyrrol-1ylphenol derivative, is not a substrate for efflux transporters and would therefore be the most promising lead compound for further investigation of potent ARIs.

Evaluation of aldose reductase inhibition and docking studies of some secondary metabolites, isolated from Origanum vulgare L. ssp. hirtum.

Five polar constituents of Origanum vulgare L. ssp. hirtum were investigated for their ability to inhibit aldose reductase (ALR2), the first enzyme of the polyol pathway implicated in the secondary complications of diabetes. The most active compound was found to be lithospermic acid B. Caffeic acid was inactive as it showed no inhibitory activity against the enzyme. The order of the inhibitory activity of the remaining compounds was: rosmarinic acid >12-hydroxyjasmonic acid 12-O-beta-glucopyranoside > p-menth-3-ene-1,2-diol 1-O-beta-glucopyranoside. Docking studies have been undertaken to gain insight into the binding mode of the investigated compounds at the active site of ALR2. The predicted hydrogen bonding and hydrophobic interactions may explain the observed inhibitory activity.