The C-terminal peptide of H-Ras as a target for the covalent binding of drugs modulating ras-dependent pathways

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Detoxifying enzymes at the cross-roads of inflammation, oxidative stress, and drug hypersensitivity: role of glutathione transferase P1-1 and aldose reductase

9 p.-2 figPhase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivityThis work has been supported by grants SAF2012-36519 from MINECO and SAF-2015-68590-R from MINECO/FEDER and ISCIII RETIC RIRAAF RD12/0013/0008 to DP,and RD12/0013/0002 to J A.Peer reviewe

Caracterización de la modificación de proteínas con interés fisiopatológico por prostaglandinas ciclopentenonas: aldo-ceto reductasas

Las prostaglandinas ciclopentenonas (cyPG), como PGA1 y 15d-PGJ2, son mediadores lípidos endógenos que proceden de la deshidratación espontánea de otras prostaglandinas (PG). Estos compuestos ejercen efectos antiinflamatorios y antitumorales. Debido a su elevada reactividad, el principal mecanismo de acción de estos compuestos es la unión covalente a proteínas celulares mediante la adición de Michael. Mediante el uso de abordajes proteómicos, en nuestro grupo de investigación se identificaron proteínas de la superfamilia de las AKR como dianas selectivas de la PGA1. Las AKR son enzimas de metabolismo de fase I que catalizan la oxidorreducción de compuestos tanto endógenos como xenobióticos. En este trabajo hemos caracterizado la modificación por PGA1 de dos proteínas AKR con una elevada importancia fisiopatológica, la aldosa reductasa o AKR1B1 y la AKR1B10, y la repercusión biológica de esta modificación. La aldosa reductasa está implicada en las complicaciones diabéticas secundarias, y la AKR1B10 está implicada en el desarrollo de tumores relacionados con el consumo de tabaco. Hemos demostrado que la PGA1 se une a la proteína recombinante AKR1B1 e inhibe su actividad enzimática. Además, hemos comprobado que la PGA1 se une covalentemente a la proteína AKR1B10 a través del residuo de Cys299 presente en el centro activo de la enzima, e inhibe su actividad enzimática en células. También hemos observado que la PGA1 inhibe la migración in vitro y el crecimiento de colonias en agar de las células A549, y potencia los efectos inducidos por la doxorrubicina (DOX) en estas células. Estos efectos podrían estar potencialmente mediados por la inhibición de la proteína AKR1B10. Los resultados obtenidos en este trabajo aportan nuevas evidencias que ayudan a la comprensión de los mecanismos de acción de las cyPG, confirman la importancia de sus propiedades antitumorales y antiproliferativas, y apoyan su uso como potenciales fármacos para el tratamiento de diferentes patologías en humanos

Anti-Inflammatory Prostanoids: Focus on the Interactions between Electrophile Signaling and Resolution of Inflammation

Prostanoids are products of cyclooxygenase biosynthetic pathways and constitute a family of lipidic mediators of widely diverse structures and biological actions. Besides their known proinflammatory role, numerous works have revealed the anti-inflammatory effects of various prostanoids and established their role in the resolution of inflammation. Among these, prostaglandins with cyclopentenone structure (cyPG) are electrophilic lipids that may act through various mechanisms, including the activation of nuclear and membrane receptors and, importantly, direct addition to protein cysteine residues and modification of protein function. Due to their ability to influence cysteine modification–mediated signaling, cyPG may play a critical role in the interplay between redox and inflammatory signaling pathways. Moreover, cellular redox status modulates cyPG addition to proteins; thus, a reciprocal regulation exists between these two factors. After initial controversy, it is becoming clear that endogenous cyPG are generated at concentrations sufficient to promote inflammatory resolution. As for other prostanoids, cyPG effects are highly dependent on context factors and they may exert pro- or anti-inflammatory actions in a cell type–dependent manner, or even biphasic or dual actions in a given cell type or tissue. In light of the growing number of cyPG protein targets identified, cyPG resemble other pleiotropic mediators acting through protein modification. However, their complex structure results in an inter- and intramolecular selectivity of the residues being modified, thus opening the way for structure-activity and drug discovery studies. Detailed characterization of cyPG interactions with cellular proteins will help us to understand their mechanism of action fully and establish their therapeutic potential in inflammation

Anti-inflammatory prostanoids: focus on the interactions between electrophile signaling and resolution of inflammation

Prostanoids are products of cyclooxygenase biosynthetic pathways and constitute a family of lipidic mediators of widely diverse structures and biological actions. Besides their known proinflammatory role, numerous works have revealed the anti-inflammatory effects of various prostanoids and established their role in the resolution of inflammation. Among these, prostaglandins with cyclopentenone structure (cyPG) are electrophilic lipids that may act through various mechanisms, including the activation of nuclear and membrane receptors and, importantly, direct addition to protein cysteine residues and modification of protein function. Due to their ability to influence cysteine modification-mediated signaling, cyPG may play a critical role in the interplay between redox and inflammatory signaling pathways. Moreover, cellular redox status modulates cyPG addition to proteins; thus, a reciprocal regulation exists between these two factors. After initial controversy, it is becoming clear that endogenous cyPG are generated at concentrations sufficient to promote inflammatory resolution. As for other prostanoids, cyPG effects are highly dependent on context factors and they may exert pro-or antiinflammatory actions in a cell type-dependent manner, or even biphasic or dual actions in a given cell type or tissue. In light of the growing number of cyPG protein targets identified, cyPG resemble other pleiotropic mediators acting through protein modification. However, their complex structure results in an inter-and intramolecular selectivity of the residues being modified, thus opening the way for structure-activity and drug discovery studies. Detailed characterization of cyPG interactions with cellular proteins will help us to understand their mechanism of action fully and establish their therapeutic potential in inflammation. KEYWORDS: cyclopentenone prostaglandins, 15d-PGJ 2 , PPAR, proteomic studies, cysteine modification, electrophilic lipids, redox regulation GENERAL ASPECTS OF ANTI-INFLAMMATORY PROSTANOIDS The term prostanoids refers to lipids derived from 20 carbon fatty acids by the action of the enzymes called cyclooxygenases (COX), namely prostaglandins (PG) and thromboxane (TX). Prostanoids are Díez-Dacal and Pérez-Sala: Electrophilic Anti-Inflammatory Prostanoids TheScientificWorldJOURNAL (2010) Díez 657 It is important to note that in addition to enzymatic transformations, PG can be further transformed through nonenzymatic reactions, originating a wide variety of structurally and biologically diverse prostanoids. For instance, levuglandins, which are highly reactive lipids able to induce protein modification and aggregation, can be formed from PGH 2 by nonenzymatic rearrangement Prostanoids play a critical role in inflammation and for many years, COX enzymes and their products have been considered mainly proinflammatory agents. Evidence from the use of anti-inflammatory drugs and from genetically modified animal models deficient in certain PG synthases or PG receptors have evidenced positive roles of prostanoids in the inflammatory response In a work that contributed to changing the view on COX enzymes, Gilroy et al. proposed a role of COX-2 in inflammatory resolution PGD 2 , in turn, has been reported to facilitate allergic reactions. However, PGD 2 or its derivatives, in particular 15d-PGJ 2 , have been reported to contribute to the resolution of Th1-driven delayed-type hypersensitivity reactions Regarding cyPG, recent evidence indicates they may also play opposing roles in different settings. Thus, whereas anti-inflammatory effects have been reported in most experimental systems, cyPG may elicit proinflammatory effects in precise situations. In summary, given their varied effects, a detailed knowledge of the factors that influence PG actions is very important in order to predict potential undesirable effects both of PG and of the pharmacological inhibition of their synthesis. In this line, the development of novel therapeutics based on the endogenous mechanisms of inflammatory resolution and on the identification of novel functions of lipid mediators is a thriving field of research. ELECTROPHILIC PROSTANOIDS: THE CYCLOPENTENONE PROSTAGLANDINS Formation of cyPG cyPG are generated by nonenzymatic dehydration of their parent PG. A-series cyPG arise from the dehydration of PGE-type PG, whereas cyPG of the J series are generated by dehydration of PGD 2 Biological Actions As stated above, the effects of cyPG that were pursued earlier were the antiproliferative effects, for which they were envisaged as potential anticancer agents. cyPG were found to increase the life span of tumor-bearing mice and to inhibit the growth of several transformed cell lines whereas both positive and negative effects of cyPG on the proapoptotic factor p53 have been reported [38,39,40]. In addition, cytoskeletal disruption [41,42,43], inhibition of protein synthesis [44], or down-regulation of telomerase reverse transcriptase (hTERT) could play an important role in the antiproliferative effects of cyPG [43,45]. Nowadays, the effect of cyPG on cell proliferation has been exhaustively studied in a wide variety of experimental models. Although an antiproliferative effect of cyPG has been described in most settings, it should be noted that cyPG may also induce cell proliferation and/or protection from apoptosis through several mechanisms under various experimental conditions. Protection of keratinocytes from apoptosis induced by the carcinogen DMBA has been proposed to be at the basis of an increase in cell survival after mutagenic injury and, therefore, of a potentiation of tumor progression [46]. Moreover, a protective effect of 15d-PGJ 2 on apoptosis induced by H 2 O 2 has been observed in PC12 cells [47], associated with the induction of the antioxidant response. In hepatic cells, both potentially beneficial and adverse effects of cyPG may take place since 15d-PGJ 2 has been found to reduce the fibrogenic response of human hepatoma cells [48], but enhance the toxicity of allyl alcohol and its active metabolite acrolein in isolated hepatocytes [49]. Early studies on the biological activity of cyPG also revealed that they are potent antiviral agents. This effect was attributed to their ability to inhibit NF-B, which is necessary for the replication of several viruses, or to the induction of a heat shock response, which elicits the expression of cytoprotective enzymes [50]. More recently it has been shown that certain cyPG may directly target viral proteins involved in transcription [51]. Thus, the mechanisms involved in the antiviral effects of cyPG may depend on both cellular and viral targets. Díez-Dacal and Pérez-Sala: Electrophilic Anti-Inflammatory Prostanoids TheScientificWorldJOURNAL (2010) 10, 655-675 660 The studies on the antiviral effects of cyPG, and in particular of PGA 1 , led to a wealth of knowledge on the cellular effects of these compounds. Inhibition of NF-B[52], a transcription factor involved in the induction of numerous proinflammatory genes, was noted more than 10 years ago. Since then, the antiinflammatory actions of cyPG have been explored in cellular and animal models of inflammation. 15d-PGJ 2 was shown to inhibit the production of monocyte inflammatory cytokines [53], and to reduce the expression of iNOS in cerebellar granule cells [54] and in glial cells [55]. 15d-PGJ 2 also inhibits COX-2 and ICAM-1 induction by cytokines Mechanisms of Action From the early studies with cyPG, it was recognized that the cyclopentenone structure and the ability of these compounds to modify thiol groups covalently was important for their actions. Binding of radioactive cyPG to intracellular structures, likely proteins, was evidenced in works by Narumiya and Fukushima exploring the subcellular distribution of cyPG Shortly after the connection between cyPG and PPAR activation was proposed, many works addressed the potential PPAR-dependent or -independent nature of the effects of 15d-PGJ 2 . Several approaches have been employed, including comparison of the effects of cyPG with those of other PPAR agonists, such as rosiglitazone or BRL49653, considered highly selective PPAR agonists, assessment of the ability of PPAR antagonists to block cyPG effects, and comparison of cyPG actions with the effects of other compounds with different ability to modify proteins covalently. Several works can be cited that outline this classical approach cyPG may also interact with membrane receptors. The DP2 receptor is a chemotactic receptor present in various types of leukocytes, which has been involved in allergic inflammation As research on cyPG progresses, it becomes clear that these prostanoids may covalently bind to multiple cellular targets. Identification of the targets for protein modification by cyPG, some of which appear in pathways. The NF-B pathway is activated in response to numerous proinflammatory stimuli and mediates the transcriptional induction of genes such as iNOS, COX-2, MMPs, and cytokines. cyPG targets along this pathway include IKK, one of the components of the kinase complex that phosphorylates the NF-B inhibitory subunit IB, triggering its proteasomal degradation, both the p65 and p50 subunits of NF-B, and components of the proteasome 663 Nrf2 In addition to direct modification by cyPG, redox-sensitive cellular proteins and, in particular, transcription factors can be regulated by cyPG through indirect mechanisms. Oxidation-reduction cycles are key for the function of several transcription factors, including NF-B, AP-1, p53, and Nrf2. Regarding NF-B, for instance, oxidation events in the cytoplasm may contribute to activation, whereas DNA binding requires the factor to be in a reduced state. The thioredoxin/thioredoxin reductase (Trx/TR) system plays a key role in the reduction of transcription factors. In the case of NF-B, Trx-1 in the cytosol can interfere with NF-B activation and nuclear translocation, whereas in the nucleus it enhances NF-B activity. The regulation of nuclear redox signaling has been reviewed recently in detail SELECTIVITY OF PROTEIN MODIFICATION BY cyPG, INTERACTIONS WITH GSH AND INDUCTION OF PROTEIN CROSS-LINKING cyPG modify multiple cellular proteins. To date, around 100 potential or confirmed targets for cyPG modification have been identified in various studies [43, One important structural feature of cyPG is the presence of one or more electrophilic carbons, resulting from the presence of one or more double bonds conjugated with the carbonyl group (see In a physiological context, the presence of GSH can be an important factor modulating the selectivity of protein modification. GSH is the main, low-molecular-weight antioxidant molecule in cells and it can be present at millimolar concentrations. cyPG may form adducts with GSH by enzymatic and nonenzymatic mechanisms, and the kinetics and the stability of adduct formation is different depending on the structure of the cyPG GST enzymes catalyze the conjugation of GSH with cyPG, the resulting GSH-cyPG conjugates being exported from cells by multidrug-resistance transporter

Proteomic studies on protein modification by cyclopentenone prostaglandins: expanding our view on electrophile actions

21 páginas, 4 figuras, 2 tablas -- PAGS nros. 2243-2263Cyclopentenone prostaglandins (cyPG) are lipid mediators that participate in the mechanisms regulating inflammation and tumorigenesis. cyPG are electrophilic compounds that act mainly through the covalent modification of cellular proteins. The stability of many cyPG-protein adducts makes them suitable for proteomic analysis. Indeed, methodological advances in recent years have allowed identifying many cyPG targets, including components of pro-inflammatory transcription factors, cytoskeletal proteins, signaling kinases and proteins involved in redox control. Insight into the diversity of cyPG targets is providing a better understanding of their mechanism of action, uncovering novel links between resolution of inflammation, proliferation and redox regulation. Moreover, identification of the target residues has unveiled the selectivity of protein modification by these electrophiles, providing valuable information for potential pharmacological applications. Among the challenges ahead, the detection of proteins modified by endogenous cyPG and the quantitative aspects of the modification require further efforts. Importantly, only a few years after the appearance of the first proteomic studies, research on cyPG targets is yielding new paradigms for redox and electrophilic signalingWork in the authors' laboratory is supported by grants from Ministerio de Ciencia e Innovación, SAF2009-11642, RETIC “Red de Investigación de Reacciones Adversas a Alergenos y Fármacos” from Instituto de Salud Carlos III and COST Action CM1001 from EU. B. G. and C.L. O. are the recipients of fellowships from the FPI Program from Ministerio de Ciencia e InnovaciónPeer reviewe

Use of 5-Carboxymethyl3-Mercapto-1,2,4-Triazino-[5,6-B]Indoles and their pharmaceutical composition

: The present invention relates to the use of 5-carboxymethyl-3- mercapto-1,2,4-triazino-[5,6-b]indoles (the general formula (I)) and their pharmaceutically acceptable salts hydrates and solvates thereof for the use in treatment, control and prevention of human and veterinary diseases in which activities of aldo- keto reductases AKR1B1 and AKR1B10 are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic, endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system (menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases. The present invention relates to pharmaceutical compositions comprising effective amount of 5-carboxymethyl-3-mercapto-1,2,4-triazino-[5,6-b]indoles of the general formula (I) and a pharmaceutically acceptable carrier for the use in treatment, control and prevention of human and veterinary diseases. (Formula (I))Peer reviewedÚstav Experimentálnej Farmakilógie a Toxikológie Sav, Consejo Superior de Investigaciones Científicas, Centrum vedecko-technických informácií SRA1 Solicitud de adición a la patent

Cyclopentenone prostaglandins with dienone structure promote cross-linking of the chemoresistance-inducing enzyme glutathione transferase P1-1

Full article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.110.065391Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in cancer chemoprevention and chemoresistance and is a key target for anticancer drug development. Oxidative stress or inhibitor-induced GSTP1-1 oligomerization leads to the activation of stress cascades and apoptosis in various tumor cells. Therefore, bivalent glutathione transferase (GST) inhibitors with the potential to interact with GST dimers are been sought as pharmacological and/or therapeutic agents. Here we have characterized GSTP1-1 oligomerization in response to various endogenous and exogenous agents. Ethacrynic acid, a classic GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and diamide all induced reversible GSTP1-1 oligomerization in Jurkat leukemia cells through the formation of disulphide bonds involving Cys47 and/or Cys101, as suggested by reducing and nonreducing SDS-polyacrylamide gel electrophoresis analysis of cysteine to serine mutants. Remarkably, the electrophilic prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induced irreversible GSTP1-1 oligomerization, specifically involving Cys101, a residue present in the human but not in the murine enzyme. 15d-PGJ2-induced GSTP1-1 cross-linking required the prostaglandin (PG) dienone structure and was associated with sustained c-Jun NH2-terminal kinase activation and induction of apoptosis. It is noteworthy that 15d-PGJ2 elicited GSTP1-1 cross-linking in vitro, a process that could be mimicked by other dienone cyclopentenone PG, such as Δ12-PGJ2, and by the bifunctional thiol reagent dibromobimane, suggesting that cyclopentenone PG may be directly involved in oligomer formation. Remarkably, Δ12-PGJ2-induced oligomeric species were clearly observed by electron microscopy showing dimensions compatible with GSTP1-1 tetramers. These results provide the first direct visualization of GSTP1-1 oligomeric species. Moreover, they offer novel strategies for the modulation of GSTP1-1 cellular functions, which could be exploited to overcome its role in cancer chemoresistance.This work was supported by the Spanish Ministry of Science and Innovation [Grants SAF-2006-03489, SAF-2009-11642, BFU2008-00666, BFU-2009-08977, SAF-2008-00451]; Instituto de Salud Carlos III [Grants RD07/0064/0007, RD06/0020/1001]; Human Frontiers Science Program [Grant RGP39/2008]; and the Autonomous Region of Madrid [Grant CAM S-BIO-0214-2006].Peer reviewe

Identification of Aldo-keto reductase AKR1B10 as a selective target for modification and inhibition by prostaglandin A1: Implications for anti-tumoral activity

11 páginas, 6 figuras -- PAGS nros. 4161-4171Cyclopentenone prostaglandins (cyPG) are reactive eicosanoids that may display anti-inflammatory and antiproliferative actions, possibly offering therapeutic potential. Here we report the identification of members of the aldo-keto reductase (AKR) family as selective targets of the cyPG prostaglandin A1 (PGA1). AKR enzymes metabolize aldehydes and drugs containing carbonyl groups and are involved in inflammation and tumorigenesis. Thus, these enzymes represent a class of targets to develop small molecule inhibitors with therapeutic activity. Molecular modeling studies pointed to the covalent binding of PGA1 to Cys299, close to the active site of AKR, with His111 and Tyr49, which are highly conserved in the AKR family, playing a role in PGA1 orientation. Among AKR enzymes, AKR1B10 is considered as a tumor marker and contributes to tumor development and chemoresistance. We validated the direct modification of AKR1B10 by biotinylated PGA1 (PGA1-B) in cells, and confirmed that mutation of Cys299 abolishes PGA1-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA1 correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA1 reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this hemotherapeutic agent. Our findings define PGA1 as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistanceThis work was supported by grants SAF2009-11642 from MiCInn and RETICS RD07/0064/0007 from ISCIII to D. Pérez-Sala and SAF2006-12713-C02-02 (CICYT) and S-BIO/0214/2006 (CAM) to F. Gago. J. Gayarre was the recipient of fellowships from CSIC (I3P), EMBO, and FEBS (short term fellowships). C. Coderch was supported by a MiCInn FPU (AP2007-01225) fellowship. Part of this work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. Feedback from COST Action CM1001 is appreciated. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this factPeer reviewe

[5-(Benzyloxy)-1H-indol-1-yl]acetic acid, an aldose reductase inhibitor and PPARγ ligand

Based on overlapping structural requirements for both efficient aldose reductase inhibitors and PPAR ligands, [5-(benzyloxy)-1H-indol-1-yl]acetic acid (compound 1) was assessed for inhibition of aldose reductase and ability to interfere with PPARγ. Aldose reductase inhibition by 1 was characterized by IC 50 in submicromolar and low micromolar range, for rat and human enzyme, respectively. Selectivity in relation to the closely related rat kidney aldehyde reductase was characterized by approx. factor 50. At organ level in isolated rat lenses, compound 1 significantly inhibited accumulation of sorbitol in a concentration-dependent manner. To identify crucial interactions within the enzyme binding site, molecular docking simulations were performed. Based on luciferase reporter assays, compound 1 was found to act as a ligand for PPARγ, yet with rather low activity. On balance, compound 1 is suggested as a promising lead-like scaffold for agents with the potential to interfere with multiple targets in diabetes