RESUMEN
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.
Asunto(s)
Agonismo Inverso de Drogas , Antagonistas de los Receptores H2 de la Histamina/metabolismo , Receptores Histamínicos H2/metabolismo , Transducción de Señal/fisiología , Línea Celular Tumoral , Células HEK293 , Antagonistas de los Receptores H2 de la Histamina/farmacología , Humanos , Ligandos , Ranitidina/metabolismo , Ranitidina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Línea Celular Tumoral , Desarrollo de Medicamentos , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Fosforilación , Dominios Proteicos/efectos de los fármacos , Proteínas RGS/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Antihistamines and glucocorticoids (GCs) are often used together in the clinic to treat several inflammation-related situations. Although there is no rationale for this association, clinical practice has assumed that, due to their concomitant anti-inflammatory effects, there should be an intrinsic benefit to their co-administration. In this work, we evaluated the effects of the co-treatment of several antihistamines on dexamethasone-induced glucocorticoid receptor transcriptional activity on the expression of various inflammation-related genes in A549 and U937 cell lines. Our results show that all antihistamines potentiate GCs' anti-inflammatory effects, presenting ligand-, cell- and gene-dependent effects. Given that treatment with GCs has strong adverse effects, particularly on bone metabolism, we also examined the impact of antihistamine co-treatment on the expression of bone metabolism markers. Using MC3T3-E1 pre-osteoblastic cells, we observed that, though the antihistamine azelastine reduces the expression of dexamethasone-induced bone loss molecular markers, it potentiates osteoblast apoptosis. Our results suggest that the synergistic effect could contribute to reducing GC clinical doses, ineffective by itself but effective in combination with an antihistamine. This could result in a therapeutic advantage, as the addition of an antihistamine may reinforce the wanted effects of GCs, while related adverse effects could be diminished or at least mitigated. By modulating the patterns of gene activation/repression mediated by GR, antihistamines could enhance only the desired effects of GCs, allowing their effective dose to be reduced. Further research is needed to correctly determine the clinical scope, benefits, and potential risks of this therapeutic strategy.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Regulación de la Expresión Génica , Antagonistas de los Receptores Histamínicos/farmacología , Inflamación/genética , Receptores de Glucocorticoides/metabolismo , Animales , Biomarcadores/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Ratones , FN-kappa B/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Ftalazinas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
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.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Receptores Histamínicos H1/efectos de los fármacos , Células A549 , Señalización del Calcio/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Agonismo Inverso de Drogas , Activación Enzimática , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ligandos , Fosforilación , Transporte de Proteínas , Receptores Histamínicos H1/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
RESUMEN
The MasR receptor (MasR) is an orphan G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang-(1-7) protective axis of renin-angiotensin system. This receptor has been suggested to participate in several physiological processes including cardio- and reno-protection and regulation of the central nervous system function. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, these are still poorly understood. Accordingly, in the current study we aimed to characterize the signaling pathways triggered by the MasR. To do that, we measured cAMP and Ca2+ levels in both naïve and MasR transfected cells in basal conditions and upon incubation with putative MasR ligands. Besides, we evaluated activation of ERK1/2 by Ang-(1-7) in MasR transfected cells. Results indicated the existence of a high degree of MasR constitutive activity toward cAMP modulation. This effect was not mediated by the PDZ-binding motif of the MasR but by receptor coupling to Gαi-adenylyl cyclase signaling pathway. Incubation of MasR transfected cells with Ang-(1-7) or the synthetic ligand AVE 0991 amplified MasR negative modulation of cAMP levels. On the other hand, we provided evidence for lack of MasR-associated modulation of Ca2+ levels by Ang-(1-7). Finally, it was determined that the MasR attenuated Ang-(1-7)-induced ERK1/2 phosphorylation mediated by AT1R. We provided further characterization of MasR signaling mechanisms regarding its constitutive activity and response to putative ligands. This information could prove useful to better describe MasR physiological role and development of therapeutic agents that could modulate its action.
RESUMEN
AIMS: G protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. MATERIALS AND METHODS: We performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549â¯cells. GRK2(45-178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. KEY FINDINGS: We identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SIGNIFICANCE: We obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Células A549 , Simulación por Computador , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HeLa , Humanos , Tamizaje Masivo , Simulación del Acoplamiento Molecular/métodos , Fosforilación , Unión Proteica , Dominios Proteicos , Inhibidores de Proteínas Quinasas/química , Receptores Histamínicos H1/metabolismo , Transducción de SeñalRESUMEN
Rac1 GTPase has long been recognized as a critical regulatory protein in different cellular and molecular processes involved in cancer progression, including acute myeloid leukemia. Here we show the antitumoral activity of ZINC69391 and 1A-116, two chemically-related Rac1 pharmacological inhibitors, on a panel of four leukemic cell lines representing different levels of maturation. Importantly, we show that the main mechanism involved in the antitumoral effect triggered by the Rac1 inhibitors comprises the induction of the mitochondrial or intrinsic apoptotic pathway. Interestingly, Rac1 inhibition selectively induced apoptosis on patient-derived leukemia cells but not on normal mononuclear cells. These results show the potential therapeutic benefits of targeting Rac1 pathway in hematopoietic malignancies.
RESUMEN
Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4'-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies.