Identification of MRP4/ABCC4 as a Target for Reducing the Proliferation of Pancreatic Ductal Adenocarcinoma Cells by Modulating the cAMP Efflux.

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P < 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P < 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENT: ABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.

PI3K pathway is involved in ERK signaling cascade activation by histamine H2R agonist in HEK293T cells.

BACKGROUND: Histamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. METHODS: Phosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. RESULTS: H2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. CONCLUSIONS: These results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. GENERAL SIGNIFICANCE: This work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.

Histamine H2 Receptor in Blood Cells: A Suitable Target for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) consists in a cancer of early hematopoietic cells arising in the bone marrow, most often of those cells that would turn into white blood cells (except lymphocytes). Chemotherapy is the treatment of choice for AML but one of the major complications is that current drugs are highly toxic and poorly tolerated. In general, treatment for AML consists of induction chemotherapy and post-remission therapy. If no further post-remission is given, almost all patients will eventually relapse. Histamine, acting at histamine type-2 (H2) receptors on phagocytes and AML blast cells, helps prevent the production and release of oxygen-free radicals, thereby protecting NK and cytotoxic T cells. This protection allows immune-stimulating agents, such as interleukin-2 (IL-2), to activate cytotoxic cells more effectively, enhancing the killing of tumor cells. Based on this mechanism, post-remission therapy with histamine and IL-2 was found to significantly prevent relapse of AML. Alternatively, another potentially less toxic approach to treat AML employs drugs to induce differentiation of malignant cells. It is based on the assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment results in tumor reprogramming and the induction of terminal differentiation. There are promissory results showing that an elevated and sustained signaling through H2 receptors is able to differentiate leukemia-derived cell lines, opening the door for the use of H2 agonists for specific differentiation therapies. In both situations, histamine acting through H2 receptors constitutes an eligible treatment to induce leukemic cell differentiation, improving combined therapies.

Signal transduction mechanism of biased ligands at histamine H2 receptors.

7TMRs (seven-transmembrane receptors) exist as conformational collections in which different conformations would lead to differential downstream behaviours such as receptor phosphorylation, G-protein activation and receptor internalization. In this context, a ligand may cause differential activation of some, but not all, of the signalling events, which are associated to a particular receptor, and it would lead to biased agonism. The aim of the present study was to investigate whether H2R (histamine H2 receptor) ligands, described as inverse agonists because of their negative efficacy at modulating adenylate cyclase, could display some positive efficacy concerning receptor desensitization, internalization or even signalling through an adenylate-cyclase-independent pathway. Our present findings indicate that treatment with H2R inverse agonists leads to receptor internalization in HEK (human embryonic kidney)-293T transfected cells, by a mechanism mediated by arrestin and dynamin, but independent of GRK2 (G-protein-coupled receptor kinase 2)-mediated phosphorylation. On the other hand, we prove that two of the H2R inverse agonists tested, ranitidine and tiotidine, also induce receptor desensitization. Finally, we show that these ligands are able to display positive efficacy towards the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by a mechanism that involves Gßγ and PI3K (phosphoinositide 3-kinase)-mediated signalling in both transfected HEK-293T cells and human gastric adenocarcinoma cells. These results point to the aspect of pluridimensional efficacy at H2R as a phenomenon that could be extended to naïve cells, and challenge previous classification of pharmacologically relevant histaminergic ligands.

Cross-desensitization and cointernalization of H1 and H2 histamine receptors reveal new insights into histamine signal integration.

G protein-coupled receptor signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, i.e., signaling networks. In this work we present an exhaustive study of the cross-talk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and Chinese hamster ovary-transfected cells. By desensitization assays we demonstrated the existence of a crossdesensitization between both receptors independent of protein kinase A or C. H1R-agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment of 48 hours. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the cross-talk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. To evaluate potential heterodimerization of the receptors, sensitized emission fluorescence resonance energy transfer experiments were performed in human embryonic kidney 293T cells using H1R-cyan fluorescent protein and H2R-yellow fluorescent protein. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor crossdesensitization, which was mediated by G protein-coupled receptor kinase 2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine.

BACKGROUND: Ceefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. METHODS: U937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. RESULTS: Ceefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. CONCLUSIONS: These results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine. GENERAL SIGNIFICANCE: This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2.

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by ß-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.

Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation.

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling.

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

Expression of a G protein-coupled receptor (GPCR) leads to attenuation of signaling by other GPCRs: experimental evidence for a spontaneous GPCR constitutive inactive form.

The idea of G protein-coupled receptors (GPCRs) coupling to G protein solely in their active form was abolished when it was found that certain ligands induce a G protein-coupled but inactive receptor form. This receptor form interferes with signaling of other receptors by sequestering G protein. However, the spontaneous existence of this receptor species has never been established. The aim of the present work was to evaluate the existence of the spontaneous conformation of the receptor inactively coupled to G protein able to interfere with the response of other GPCRs. According to the law of mass action, receptor overexpression should lead to increased amounts of all spontaneously occurring species. Based on this, we generated Chinese hamster ovary (CHO-K1)-derived cell lines expressing various amounts of the human histamine H2 receptor. In these systems, the signaling of other endogenously and transiently expressed GPCRs was attenuated proportionally to human H2 receptor expression levels. G protein transfection specifically reverted this attenuation, strongly suggesting hijacking of the G protein from a common pool. Similar attenuation effects were observed when the beta(2)- adrenergic receptor was overexpressed, suggesting that this is a more general phenomenon. Moreover, in human mammary MDA-MB-231 cells, a consistent increase in the response of other GPCRs was observed when endogenous expression of beta(2)-adrenergic receptor was knocked down using specific small interfering RNAs. Our findings show that GPCRs may interact with the signaling of other receptors by modulating the availability of the G protein and suggest the existence of GPCR spontaneous coupling to G proteins in an inactive form.

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways.

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G(11)-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP beta2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or beta2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [(3)H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

Biased agonism at histamine H1 receptor: Desensitization, internalization and MAPK activation triggered by antihistamines.

Histamine H1 receptor ligands used clinically as antiallergics rank among the most widely prescribed and over-the-counter drugs in the world. They exert the therapeutic actions by blocking the effects of histamine, due to null or negative efficacy towards Gαq-phospholipase C (PLC)-inositol triphosphates (IP3)-Ca2+ and nuclear factor-kappa B cascades. However, there is no information regarding their ability to modulate other receptor responses. The aim of the present study was to investigate whether histamine H1 receptor ligands could display positive efficacy concerning receptor desensitization, internalization, signaling through Gαq independent pathways or even transcriptional regulation of proinflammatory genes. While diphenhydramine, triprolidine and chlorpheniramine activate ERK1/2 (extracellular signal-regulated kinase 1/2) pathway in A549 cells, pre-treatment with chlorpheniramine or triprolidine completely desensitize histamine H1 receptor mediated Ca2+ response, and both diphenhydramine and triprolidine lead to receptor internalization. Unlike histamine, histamine H1 receptor desensitization and internalization induced by antihistamines prove to be independent of G protein-coupled receptor kinase 2 (GRK2) phosphorylation. Also, unlike the reference agonist, the recovery of the number of cell-surface histamine H1 receptors is a consequence of de novo synthesis. On the other hand, all of the ligands lack efficacy regarding cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA regulation. However, a prolonged exposure with each of the antihistamines impaires the increase in COX-2 and IL-8 mRNA levels induced by histamine, even after ligand removal. Altogether, these findings demonstrate the biased nature of histamine H1 receptor ligands contributing to a more accurate classification, and providing evidence for a more rational and safe use of them.

MRP4/ABCC4 expression is regulated by histamine in acute myeloid leukemia cells, determining cAMP efflux.

Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of ß3-Adrenergic Receptor.

G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although ß3-adrenergic receptor (ß3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce ß3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of ß3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human ß3AR presented a short-term desensitization of cAMP response stimulated by the ß3AR agonist, BRL37344, and not by forskolin. We found that ß3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased ß3AR desensitization. Consistently, stimulation of ß3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing ß3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the ß3AR in heart function and disease.

Imiquimod suppresses respiratory syncytial virus (RSV) replication via PKA pathway and reduces RSV induced-inflammation and viral load in mice lungs.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children, as well as an important cause of morbidity and mortality in elderly and immunocompromised individuals. However, there is no safe and efficacious RSV vaccine or antiviral treatment. Toll Like Receptors (TLR) are important molecular mediators linking innate and adaptive immunity, and their stimulation by cognate agonists has been explored as antiviral agents. Imiquimod is known as a TLR7 agonist, but additionally acts as an antagonist for adenosine receptors. In this study, we demonstrate that imiquimod, but not resiquimod, has direct anti-RSV activity via PKA pathway in HEp-2 and A549 cells, independently of an innate response. Imiquimod restricts RSV infection after viral entry into the host cell, interfering with viral RNA and protein synthesis. Probably as a consequence of these anti-RSV properties, imiquimod displays cytokine modulating activity in RSV infected epithelial cells. Moreover, in a murine model of RSV infection, imiquimod treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. Consequently, imiquimod represents a promising therapeutic alternative against RSV infection and may inform the development of novel therapeutic targets to control RSV pathogenesis.

Toward establishing structure-activity relationships for oxygenated coumarins as differentiation inducers of promonocytic leukemic cells.

The presumption that some coumarins might be lead compounds in the search for new differentiation agents against leukemia is based on the fact that natural coumarins, 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin (C-2) and 5-methoxy-6,7-methylenedioxycoumarin (C-1) inhibit proliferation and induce differentiation in U-937 cells [Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.; De Kimpe, N.; Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett.2004, 210, 179-188]. These promising findings prompted us to investigate the anti-leukemia activity of a broader range of related polyoxygenated coumarins. Twenty related natural or synthetically prepared coumarins, including a range of 5-substituted ayapin derivatives which have become easy accessible via newly developed synthesis methods, were evaluated, where treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-3) and 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were able to inhibit the cell growth and induce the differentiation of U-937 cells after 48 h treatment. These results provide insight into the correlation between some structural properties of polyoxygenated coumarins and their in vitro leukemic differentiation activity.

MRP4/ABCC4 As a New Therapeutic Target: Meta-Analysis to Determine cAMP Binding Sites as a Tool for Drug Design.

MRP4 transports multiple endogenous and exogenous substances and is critical not only for detoxification but also in the homeostasis of several signaling molecules. Its dysregulation has been reported in numerous pathological disorders, thus MRP4 appears as an attractive therapeutic target. However, the efficacy of MRP4 inhibitors is still controversial. The design of specific pharmacological agents with the ability to selectively modulate the activity of this transporter or modify its affinity to certain substrates represents a challenge in current medicine and chemical biology. The first step in the long process of drug rational design is to identify the therapeutic target and characterize the mechanism by which it affects the given pathology. In order to develop a pharmacological agent with high specific activity, the second step is to systematically study the structure of the target and identify all the possible binding sites. Using available homology models and mutagenesis assays, in this review we recapitulate the up-to-date knowledge about MRP structure and aligned amino acid sequences to identify the candidate MRP4 residues where cyclic nucleotides bind. We have also listed the most relevant MRP inhibitors studied to date, considering drug safety and specificity for MRP4 in particular. This meta-analysis platform may serve as a basis for the future development of inhibitors of MRP4 cAMP specific transport.

Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model.

Glucocorticoids are among the most effective drugs to treat asthma. However, the severe adverse effects associated generate the need for its therapeutic optimization. Conversely, though histamine is undoubtedly related to asthma development, there is a lack of efficacy of antihistamines in controlling its symptoms, which prevents their clinical application. We have reported that antihistamines potentiate glucocorticoids' responses in vitro and recent observations have indicated that the coadministration of an antihistamine and a synthetic glucocorticoid has synergistic effects on a murine model of allergic rhinitis. Here, the aim of this work is to establish if this therapeutic combination could be beneficial in a murine model of asthma. We used an allergen-induced model of asthma (employing ovalbumin) to evaluate the effects of the synthetic glucocorticoid dexamethasone combined with the antihistamine azelastine. Our results indicate that the cotreatment with azelastine and a suboptimal dose of dexamethasone can improve allergic lung inflammation as shown by a decrease in eosinophils in bronchoalveolar lavage, fewer peribronchial and perivascular infiltrates, and mucin-producing cells. In addition, serum levels of allergen-specific IgE and IgG1 were also reduced, as well as the expression of lung inflammatory-related genes IL-4, IL-5, Muc5AC, and Arginase I. The potentiation of dexamethasone effects by azelastine could allow to reduce the effective glucocorticoid dose needed to achieve a therapeutic effect. These findings provide first new insights into the potential benefits of glucocorticoids and antihistamines combination for the treatment of asthma and grants further research to evaluate this approach in other related inflammatory conditions.

Involvement of histamine H1 and H2 receptor inverse agonists in receptor's crossregulation.

Histamine [2-(4-Imidazolyl)-ethylamine] modulates different biological processes, through histamine H1 and H2 receptors, and their respective blockers are widely used in treating allergic and gastric acid-related disorders. Histamine H1 and H2 receptor crossdesensitization and cointernalization induced by its agonists have been previously described. In this study, we show how this crosstalk determines the response to histamine H1 and H2 receptor inverse agonists and how histamine H1 and H2 receptor inverse agonists interfere with the other receptor's response to agonists. By desensitization assays we demonstrate that histamine H1 and H2 receptor inverse agonists induce a crossregulation between both receptors. In this sense, the histamine H1 receptor inverse agonists desensitize the cAMP response to amthamine, a histamine H2 receptor agonist. In turn, histamine H2 receptor inverse agonists interfere with histamine H1 receptor signaling. We also determine that the crossdesensitization induced by histamine H1 or H2 receptor agonists alters the histamine inverse agonists receptor response: activation of histamine H1 receptor affects cAMP response induced by histamine H2 receptor inverse agonists, whereas histamine H2 receptor agonist induces a negative regulation on the anti-inflammatory response of histamine H1 receptor inverse agonists. Binding studies revealed that histamine H1 and H2 receptors cointernalize after stimulus with histamine receptor inverse agonists. In addition, the inhibition of the internalization process prevents receptor crossregulation. Our study provides new insights in the mechanisms of action of histamine H1 and H2 receptors that explain the effect of histamine H1 and H2 receptor inverse agonists and opens up new venues for novel therapeutic applications.