Beatriz Díez-Dacal

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.

Identification of Aldo-Keto Reductase AKR1B10 as a Selective Target for Modification and Inhibition by Prostaglandin A1: Implications for Antitumoral Activity

Cyclopentenone 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 A(1) (PGA(1)). 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 PGA(1) 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 PGA(1) 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 PGA(1) (PGA(1)-B) in cells, and confirmed that mutation of Cys299 abolishes PGA(1)-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA(1) correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA(1) reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this chemotherapeutic agent. Our findings define PGA(1) as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistance.

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

Cyclopentenone 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 signaling.

Cyclopentenone Prostaglandins with Dienone Structure Promote Cross-Linking of the Chemoresistance-Inducing Enzyme Glutathione Transferase P1-1

Glutathione 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.

The C-terminus of H-ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and Δ12-PGJ2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

A-class prostaglandins: Early findings and new perspectives for overcoming tumor chemoresistance

The antiproliferative properties of cyclopentenone prostaglandins of the A-class have long been known. Considerable research has led to the elucidation of some of the mechanisms of action of these pleiotropic compounds. A-class prostaglandins or derived molecules (A-PG) may block the cell cycle, inhibit anti-apoptotic transcription factors, activate apoptotic cascades, induce a stress response and inhibit protein synthesis in a cell type-dependent manner. In addition, recent reports indicate that A-class PG may interact with various cellular detoxification systems and drug metabolizing enzymes used by cancer cells as mechanisms of chemoresistance. Some of these findings may open new perspectives for the development of strategies aimed at overcoming cancer resistance to widely used antitumor drugs. Here we outline the mechanisms of action for the antitumoral effects of PGA and related compounds, emphasizing those with impact on cellular defence systems which may contribute to cancer chemoresistance. The ability of A-PG to form covalent adducts with thiol groups in proteins and in glutathione is essential for their biological actions. Therefore, identification of the protein targets and elucidation of the interactions of A-PG with the glutathione biotransformation system will be critical for understanding the antitumoral effects of these compounds per se or through their ability to sensitize cancer cells towards other drugs.

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA(1) displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA(1) derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA(1) (PGA(1)-biotinamide, PGA(1)-B), to establish its validity to identify cyPG-protein interactions of potential therapeutic interest. PGA(1) and PGA(1)-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA(1)-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA(1)-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA(1)-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA(1) that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.

15-Deoxy-Δ 12,14-Prostaglandin J2 Exerts Pro- and Anti-Inflammatory Effects in Mesangial Cells in a Concentration-Dependent Manner

Cyclopentenone prostaglandins play a modulatory role in inflammation, in part through their ability to covalently modify key proinflammatory proteins. Using mesangial cells as a cellular model of inflammation we have observed that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a biphasic effect on cell activation by cytokines, with nanomolar concentrations eliciting an amplification of nitric oxide (NO) production and iNOS and COX-2 levels, and concentrations of 5 μM and higher inhibiting proinflammatory gene expression. An analog of 15d-PGJ(2) lacking the cyclopentenone structure (9,10-dihydro-15d-PGJ(2)) showed reduced ability to elicit both types of effects, suggesting that the electrophilic nature of 15d-PGJ(2) is important for its biphasic action. Interestingly, the switch from stimulatory to inhibitory actions occurred within a narrow concentration range and correlated with the ability of 15d-PGJ(2) to induce heme oxygenase 1 and γ-GCSm expression. These events are highly dependent on the triggering of the antioxidant response, which is considered as a sensor of thiol group modification. Indeed, the levels of the master regulator of the antioxidant response Nrf2 increased upon treatment with concentrations of 15d-PGJ(2) above 5 μM, an effect that could not be mimicked by 9,10-dihydro-15d-PGJ(2). Thus, an interplay of redox and electrophilic signalling mechanisms can be envisaged by which 15d-PGJ(2), as several other redox mediators, could contribute both to the onset and to the resolution of inflammation in a context or concentration-dependent manner.

Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase

Phase 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 hypersensitivity.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.