RESUMO
The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Assuntos
Modelos Moleculares , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/química , Bloqueadores do Receptor Tipo 2 de Angiotensina II/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Desenho de Fármacos , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/genética , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato/genética , beta-Arrestinas/metabolismoRESUMO
Substantial evidence supports the involvement of the renin-angiotensin system in pulmonary hypertension (PH), and the angiotensin II type 2 receptor (AT2R) is known to exert tissue protective actions. The effect of the selective AT2R agonist C21 (also known as Compound 21 or buloxibutid) was evaluated in the rat Sugen-hypoxia PH model. After a single injection of Sugen 5416 and hypoxia for 21 days, C21 (2 or 20 mg/kg) or vehicle was administered perorally twice daily from Day 21 to Day 55. On Day 56, hemodynamic assessments were performed, and lung and heart tissue were prepared for quantification of cardiac and vascular remodeling and fibrosis. Treatment with C21 20 mg/kg improved cardiac output and stroke volume and decreased right ventricular hypertrophy (all p < 0.05). Treatment with C21 2 mg/kg significantly decreased vessel wall and muscular layer thickness and increased the luminal opening in vessels >100 µm (all p < 0.05). There were no significant differences between the two C21 doses on any parameter, and post hoc analyses comparing the merged C21 groups with the vehicle group showed that C21 treatment reduced vascular remodeling (reduced endothelial proliferation and thickening of the vascular wall) in vessels of all sizes; moreover, the diastolic pulmonary artery pressure and right ventricular pressure were reduced along with reduction of right ventricular hypertrophy. Sugen 5416 and hypoxia increased pulmonary collagen deposition, which was counteracted by C21 20 mg/kg. In conclusion, the effects of C21 on vascular remodeling, hemodynamic alterations, and fibrosis suggest that AT2R agonists may have a role in Group 1 and 3 PH treatment.
Assuntos
Hipertensão Pulmonar , Ratos , Animais , Hipertensão Pulmonar/tratamento farmacológico , Hipertrofia Ventricular Direita , Receptor Tipo 2 de Angiotensina/agonistas , Remodelação Vascular , Fibrose , Hipóxia/complicações , Hipóxia/tratamento farmacológicoRESUMO
For years, the AT2R-selective ligand CGP42112 has been erroneously characterized as a partial agonist, partly due to its ability to also interact with the AT1R at high concentrations. As late as 2009, it was still being characterized as an antagonist as well. In this perspective/opinion piece, we try to resolve the ambiguity that surrounds the efficacy of this compound by extensively reviewing the literature, tracing its beginnings to 1989, showing that CGP42112 has never been convincingly shown to be a partial agonist or an antagonist at the AT2R. While CGP42112 is now routinely characterized as an AT2R agonist, regrettably, there is a paucity of studies that can validate its efficacy as a full agonist at the AT2R, leaving the door open for continuing speculation regarding the extent of its efficacy. Hopefully, the information presented in this perspective/opinion piece will firmly establish CGP42112 as a full agonist at the AT2R such that it can once again be used as a tool to study the AT2R.
Assuntos
Receptor Tipo 2 de Angiotensina , Sistema Renina-Angiotensina , Receptor Tipo 2 de Angiotensina/agonistas , Oligopeptídeos , LigantesRESUMO
Ligands comprising a benzimidazole rather than the imidazole ring that is common in AT2R ligands e.g. in the AT2R agonist C21, can provide both high affinity and receptor selectivity. In particular, compounds encompassing benzimidazoles, substituted in the 2-position with small bulky groups such as an isopropyl (Ki = 4.0 nM) or a tert-butyl (Ki = 5.3 nM) or alternatively a thiazole heterocycle (Ki = 5.1 nM) demonstrate high affinity and AT2R selectivity. An n-butyl chain, as found in the AT1R selective sartans, makes the ligand less receptor selective. The isobutyl group on the biaryl scaffold present in most AT2R selective ligands reported so far was originally derived from the nonselective potent AT1R/AT2R ligand L-162,313. Notably, in all ligands discussed herein, the isobutyl group was substituted by an n-propyl group and ligands with high affinity to AT2R were provided and in addition the majority of them demonstrate a favorable AT2R/AT1R selectivity. The introduction of fluoro atoms in various positions had no pronounced effect on the affinity data. Ligands with a thiazole or a tert-butyl group attached to the 2-position and with a terminal trifluoromethyl butoxycarbonyl sidechain exhibited a similar stability as C21 in human liver microsomes, while other ligands examined were less stable in the microsome assay.
Assuntos
Benzimidazóis , Receptor Tipo 2 de Angiotensina , Benzimidazóis/farmacologia , Humanos , Imidazóis , Ligantes , Receptor Tipo 2 de Angiotensina/agonistas , Sulfonamidas , Tiazóis , TiofenosRESUMO
The syntheses and the AT1R and AT2R binding data of a series of new small molecule ligands are reported. These ligands comprise a phenylthiazole scaffold rather than the biphenyl or phenylthiophene scaffolds found in essentially all of the previously described ligands originating from the nonselective AT1R/AT2R ligand L-162,313 and the AT2R selective agonist C21, the latter now in Phase II/III clinical trials. A phenylthiazole rather than the phenylthiophene scaffold that is present in the AT2R selective agonist C21 and in the AT2R selective antagonist C38 had a deleterious effect on the affinity to AT2R. Nevertheless, a significant improvement could be accomplished by introduction of a small bulky alkyl group in the 2-position of the imidazole ring attached through a methylene group bridge to the phenylthiazole scaffold. Hence, a combination of a 2-tert-butyl or a 2-isopropyl group and a butoxycarbonyl furnished potent AT2R selective ligands. Furthermore, a high affinity ligand derived from L-162,313 and exhibiting a > 35 fold selectivity for AT1R was identified (10). The ligand 21 with the 2-tert-butyl group and â¼ 35 fold selectivity for AT2R, demonstrated high stability in human, rat and mouse liver microsomes and a very attractive profile with regard to the inhibition of common drug-metabolizing CYP enzymes. Thus, very low levels of inhibition of CYP 3A (5%), 2D6 (12%), 2C8 (26%), 2C9 (23%) and 2B6 (24%) were observed with the 2-tert-butyl derivative comprising the methoxycarbonyl sulfonamide function, levels that are significantly lower than those obtained with C21 under the same experimental conditions.
Assuntos
Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina , Angiotensina II/química , Angiotensina II/farmacologia , Animais , Humanos , Imidazóis , Ligantes , Camundongos , Ratos , Receptor Tipo 2 de Angiotensina/agonistas , Sulfonamidas , TiofenosRESUMO
Angiotensin II (Ang II) is the most dominant effector component of the renin-angiotensin system (RAS) that generally acts through binding to two main classes of G protein-coupled receptors, namely Ang II subtype 1 receptor (AT1R) and angiotensin II subtype 2 receptor (AT2R). Despite some controversial reports, the activation of AT2R generally antagonizes the effects of Ang II binding on AT1R. Studying AT2R signaling, function, and its specific ligands in cell culture or animal studies has confirmed its beneficial effects throughout the body. These characteristics classify AT2R as part of the protective arm of the RAS that, along with functions of Ang (1-7) through Mas receptor signaling, modulates the harmful effects of Ang II on AT1R in the activated classic arm of the RAS. Although Ang II is the primary ligand for AT2R, we have summarized other natural or synthetic peptide and nonpeptide agonists with critical evaluation of their structure, mechanism of action, and biologic activity. SIGNIFICANCE STATEMENT: AT2R is one of the main components of the RAS and has a significant prospective for mediating the beneficial action of the RAS through its protective arm on the body's homeostasis. Targeting AT2R offers substantial clinical application possibilities for modulating various pathological conditions. This review provided concise information regarding the AT2R peptide and nonpeptide agonists and their potential clinical applications for various diseases.
Assuntos
Peptídeos/farmacologia , Receptor Tipo 2 de Angiotensina/agonistas , Angiotensina II/química , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Humanos , Ligantes , Peptídeos/química , Conformação Proteica , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Human kidney organoid technology holds promise for novel kidney disease treatment strategies and utility in pharmacological and basic science. Given the crucial roles of the intrarenal renin-angiotensin system (RAS) and angiotensin II (ANG II) in the progression of kidney development and injury, we investigated the expression of RAS components and effects of ANG II on cell differentiation in human kidney organoids. Human induced pluripotent stem cell-derived kidney organoids were induced using a modified 18-day Takasato protocol. Gene expression analysis by digital PCR and immunostaining demonstrated the formation of renal compartments and expression of RAS components. The ANG II type 1 receptor (AT1R) was strongly expressed in the early phase of organoid development (around day 0), whereas ANG II type 2 receptor (AT2R) expression levels peaked on day 5. Thus, the organoids were treated with 100 nM ANG II in the early phase on days 0-5 (ANG II-E) or during the middle phase on days 5-10 (ANG II-M). ANG II-E was observed to decrease levels of marker genes for renal tubules and proximal tubules, and the downregulation of renal tubules was inhibited by an AT1R antagonist. In contrast, ANG II-M increased levels of markers for podocytes, the ureteric tip, and the nephrogenic mesenchyme, and an AT2R blocker attenuated the ANG II-M-induced augmentation of podocyte formation. These findings demonstrate RAS expression and ANG II exertion of biphasic effects on cell differentiation through distinct mediatory roles of AT1R and AT2R, providing a novel strategy to establish and further characterize the developmental potential of human induced pluripotent stem cell-derived kidney organoids.NEW & NOTEWORTHY This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.
Assuntos
Angiotensina II/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Rim/efeitos dos fármacos , Organoides/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Rim/metabolismo , Organoides/citologia , Organoides/metabolismo , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Kidney infiltrating immune cells such as monocytes, neutrophils, and T cells play critical roles in renal ischemia-reperfusion (IR) injury and repair. Recently, the angiotensin II type 2 receptor (AT2R) has been implicated in protecting kidneys against injury and monocyte infiltration, particularly in chronic kidney disease. However, the role of AT2R in IR injury and repair phases and T cell modulation is unknown. To address this question, Sprague-Dawley rats were subjected to IR with or without AT2R agonist C21 treatment. IR caused early (2 h postreperfusion) renal functional injury (proteinuria, plasma urea, and creatinine) and enhanced immune cells (T cells and CD4 T cells) infiltration and levels of the proinflammatory cytokines monocyte chemoattractant protein-1, TNF-α, and IL-6. C21 treatment reversed these changes but increased the anti-inflammatory IL-10 level. On day 3, C21 treatment increased CD4+FoxP3+ (regulatory T cells) and CD4+IL-10+ cells and reduced kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the kidney compared with the IR control, suggesting the involvement of AT2R in kidney repair. These data indicate that AT2R activation protects the kidney against IR injury and immune cell infiltration in the early phase and modulates CD4 T cells toward the regulatory T cell phenotype, which may have long-term beneficial effects on kidney function.NEW & NOTEWORTHY The angiotensin II type 2 receptor agonist C21 has been known to have a renoprotective role in various kidney pathologies. C21 treatment (before renal ischemia) attenuated postischemic kidney injury, kidney dysfunction, and immune cell infiltration during the injury phase. Also, C21 treatment modulated the kidney microenvironment by enhancing anti-inflammatory responses mainly mediated by IL-10. During the repair phase, C21 treatment enhanced IL-10-secreting CD4 T cells and FoxP3-secreting regulatory T cells in Sprague-Dawley rats.
Assuntos
Injúria Renal Aguda/prevenção & controle , Anti-Inflamatórios/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Rim/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/agonistas , Traumatismo por Reperfusão/prevenção & controle , Sulfonamidas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Tiofenos/farmacologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Rim/imunologia , Rim/metabolismo , Rim/patologia , Fenótipo , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/metabolismo , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de TempoRESUMO
Diabetes-induced coronary endothelial cell (CEC) dysfunction contributes to diabetic heart diseases. Angiotensin II (Ang II), a vasoactive hormone, is upregulated in diabetes, and is reported to increase oxidative stress in CECs. 4-hydroxy-2-nonenal (4HNE), a key lipid peroxidation product, causes cellular dysfunction by forming adducts with proteins. By detoxifying 4HNE, aldehyde dehydrogenase (ALDH) 2 reduces 4HNE mediated proteotoxicity and confers cytoprotection. Thus, we hypothesize that ALDH2 improves Ang II-mediated defective CEC angiogenesis by decreasing 4HNE-mediated cytotoxicity. To test our hypothesis, we treated the cultured mouse CECs (MCECs) with Ang II (0.1, 1 and 10 µM) for 2, 4 and 6 h. Next, we treated MCECs with Alda-1 (10 µM), an ALDH2 activator or disulfiram (2.5 µM)/ALDH2 siRNA (1.25 nM), the ALDH2 inhibitors, or blockers of angiotensin II type-1 and 2 receptors i.e. Losartan and PD0123319 respectively before challenging MCECs with 10 µM Ang II. We found that 10 µM Ang II decreased tube formation in MCECs with in vitro angiogenesis assay (P < .0005 vs control). 10 µM Ang II downregulated the levels of vascular endothelial growth factor receptor 1 (VEGFR1) (p < .005 for mRNA and P < .05 for protein) and VEGFR2 (p < .05 for mRNA and P < .005 for protein) as well as upregulated the levels of angiotensin II type-2 receptor (AT2R) (p < .05 for mRNA and P < .005 for protein) and 4HNE-adducts (P < .05 for protein) in cultured MCECs, compared to controls. ALDH2 inhibition with disulfiram/ALDH2 siRNA exacerbated 10 µM Ang II-induced decrease in coronary angiogenesis (P < .005) by decreasing the levels of VEGFR1 (P < .005 for mRNA and P < .05 for protein) and VEGFR2 (P < .05 for both mRNA and protein) and increasing the levels of AT2R (P < .05 for both mRNA and protein) and 4HNE-adducts (P < .05 for protein) relative to Ang II alone. AT2R inhibition per se improved angiogenesis in MCECs. Additionally, enhancing ALDH2 activity with Alda 1 rescued Ang II-induced decrease in angiogenesis by increasing the levels of VEGFR1, VEGFR2 and decreasing the levels of AT2R. In summary, ALDH2 can be an important target in reducing 4HNE-induced proteotoxicity and improving angiogenesis in MCECs. Finally, we conclude ALDH2 activation can be a therapeutic strategy to improve coronary angiogenesis to ameliorate cardiometabolic diseases.
Assuntos
Aldeído-Desidrogenase Mitocondrial/metabolismo , Inibidores da Angiogênese/farmacologia , Angiotensina II/farmacologia , Vasos Coronários/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Aldeídos/metabolismo , Linhagem Celular , Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aim of the present study was to evaluate the effect of Compound 21 (C21), a selective AT2R agonist, on the prevention of endothelial dysfunction, extracellular matrix (ECM) remodeling and arterial stiffness associated with diet-induced obesity (DIO). Five-week-old male C57BL/6J mice were fed a standard (Chow) or high-fat diet (HF) for 6 weeks. Half of the animals of each group were simultaneously treated with C21 (1 mg/kg/day, in the drinking water), generating four groups: Chow C, Chow C21, HF C, and HF C21. Vascular function and mechanical properties were determined in the abdominal aorta. To evaluate ECM remodeling, collagen deposition and TGF-ß1 concentrations were determined in the abdominal aorta and the activity of metalloproteinases (MMP) 2 and 9 was analyzed in the plasma. Abdominal aortas from HF C mice showed endothelial dysfunction as well as enhanced contractile but reduced relaxant responses to Ang II. This effect was abrogated with C21 treatment by preserving NO availability. A left-shift in the tension-stretch relationship, paralleled by an augmented ß-index (marker of intrinsic arterial stiffness), and enhanced collagen deposition and MMP-2/-9 activities were also detected in HF mice. However, when treated with C21, HF mice exhibited lower TGF-ß1 levels in abdominal aortas together with reduced MMP activities and collagen deposition compared with HF C mice. In conclusion, these data demonstrate that AT2R stimulation by C21 in obesity preserves NO availability and prevents unhealthy vascular remodeling, thus protecting the abdominal aorta in HF mice against the development of endothelial dysfunction, ECM remodeling and arterial stiffness.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Imidazóis/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Rigidez Vascular/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Colágeno/metabolismo , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Fator de Crescimento Transformador beta1/sangueRESUMO
Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Imidazóis/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor B2 da Bradicinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sulfonamidas/uso terapêutico , Tiofenos/uso terapêutico , Doenças Vasculares/prevenção & controle , Animais , Aorta Torácica/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Cross-Talk , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismoRESUMO
A series of meta-substituted acetophenone derivatives, encompassing N-(alkyloxycarbonyl)thiophene sulfonamide fragments have been synthesized. Several selective AT2 receptor ligands were identified, among those a tert-butylimidazole derivative (20) with a Ki of 9.3 nM, that demonstrates a high stability in human liver microsomes (t½ = 62 min) and in human hepatocytes (t½ = 194 min). This methyloxycarbonylthiophene sulfonamide is a 20-fold more potent binder to the AT2 receptor and is considerably more stable in human liver microsomes, than a previously reported and broadly studied structurally related AT2R prototype antagonist 3 (C38). Ligand 20 acts as an AT2R agonist and caused an AT2R mediated concentration-dependent vasorelaxation of pre-contracted mouse aorta. Furthermore, in contrast to imidazole derivative C38, the tert-butylimidazole derivative 20 is a poor inhibitor of CYP3A4, CYP2D6 and CYP2C9. It is demonstrated herein that smaller alkyloxycarbonyl groups make the ligands in this series of AT2R selective compounds less prone to degradation and that a high AT2 receptor affinity can be retained after truncation of the alkyloxycarbonyl group. Binding modes of the most potent AT2R ligands were explored by docking calculations combined with molecular dynamics simulations.
Assuntos
Receptor Tipo 2 de Angiotensina/agonistas , Medula Espinal/efeitos dos fármacos , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hepatócitos/química , Hepatócitos/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Medula Espinal/patologia , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Tiofenos/síntese química , Tiofenos/químicaRESUMO
About 70% of stroke victims present with comorbid diseases such as diabetes and hypertension. The integration of comorbidities in pre-clinical experimental design is important in understanding the mechanisms involved in the development of stroke injury and recovery. We recently showed that administration of compound C21, an angiotensin II type 2 receptor agonist, at day 3 post-stroke improved sensorimotor outcomes by lowering neuroinflammation in diabetic male animals. In the current study, we hypothesized that a delayed administration of C21 would also lower chronic inflammation post-stroke in diabetic female animals. Young female diabetic rats were subjected to 1 h of middle cerebral artery occlusion (MCAO). Three days post-stroke, rats were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 4 weeks. The impact of C21 on microglial polarization was analyzed by flow cytometry in vivo and in vitro. Compound 21 treatment improved fine motor skills after MCAO through modulation of the microglia/macrophage inflammatory properties. In addition, C21 increased M2 polarization and reduced the M1:M2 ratio in vitro. In conclusion, delayed administration of C21 downregulates post-stroke inflammation in female diabetic animals. C21 may be a useful therapeutic option to lower neuro-inflammation and improve the post-stroke recovery in diabetes.
Assuntos
Diabetes Mellitus Experimental/complicações , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Receptor Tipo 2 de Angiotensina/agonistas , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Cognição/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Feminino , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Camundongos , Microglia/patologia , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 2 de Angiotensina/metabolismo , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP-6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP-6A4-induced AT2R activation could mitigate AngII-induced abdominal aortic aneurism (AAA) using AngII-treated Apoe-/- mice. Male Apoe-/- mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre-treated subcutaneously with NP-6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP-6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP-6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII-induced increases in monocyte chemotactic protein-1, osteopontin and proteolytic activity of the aorta. However, NP-6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/fisiopatologia , Proteólise , Receptor Tipo 2 de Angiotensina/agonistas , Rigidez Vascular , Angiotensina II , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Knockout , Osteopontina/metabolismo , Fenótipo , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
Infection of lung cells by the corona virus results in a loss of the balance between, on the one hand, angiotensin II-mediated stimulation of the angiotensin II type 1 receptor and, on the other hand, stimulation of the angiotensin II type 2 receptor and/or the Mas receptor. The unbalanced enhanced stimulation of the angiotensin II type 1 receptor causes inflammation, edema and contributes to the pathogenesis of severe acute respiratory distress syndrome. Here we hypothesize that stable, receptor-specific agonists of the angiotensin II type 2 receptor and of the Mas receptor are molecular medicines to treat COVID-19 patients. These agonists have therapeutic potential in the acute disease but in addition may reduce COVID-19-associated long-term pulmonary dysfunction and overall end-organ damage of this disease.
Assuntos
Peptidil Dipeptidase A/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Sistema Renina-Angiotensina/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Ensaios Clínicos como Assunto , Infecções por Coronavirus/tratamento farmacológico , Humanos , Imidazóis/farmacologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Proto-Oncogene Mas , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Tratamento Farmacológico da COVID-19RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is responsible for the global corona virus disease 2019 (COVID-19) pandemic enters host cells via a mechanism that includes binding to angiotensin converting enzyme (ACE) 2 (ACE2). Membrane-bound ACE2 is depleted as a result of this entry mechanism. The consequence is that the protective renin-angiotensin system (RAS), of which ACE2 is an essential component, is compromised through lack of production of the protective peptides angiotensin-(1-7) and angiotensin-(1-9), and therefore decreased stimulation of Mas (receptor Mas) and angiotensin AT2-receptors (AT2Rs), while angiotensin AT1-receptors (AT1Rs) are overstimulated due to less degradation of angiotensin II (Ang II) by ACE2. The protective RAS has numerous beneficial actions, including anti-inflammatory, anti-coagulative, anti-fibrotic effects along with endothelial and neural protection; opposite to the deleterious effects caused by heightened stimulation of angiotensin AT1R. Given that patients with severe COVID-19 exhibit an excessive immune response, endothelial dysfunction, increased clotting, thromboses and stroke, enhancing the activity of the protective RAS is likely beneficial. In this article, we discuss the evidence for a dysfunctional protective RAS in COVID and develop a rationale that the protective RAS imbalance in COVID-19 may be corrected by using AT2R agonists. We further review preclinical studies with AT2R agonists which suggest that AT2R stimulation may be therapeutically effective to treat COVID-19-induced disorders of various organ systems such as lung, vasculature, or the brain. Finally, we provide information on the design of a clinical trial in which patients with COVID-19 were treated with the AT2R agonist Compound 21 (C21). This trial has been completed, but results have not yet been reported.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Receptor Tipo 2 de Angiotensina/agonistas , Proteínas ras/metabolismo , Enzima de Conversão de Angiotensina 2 , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , SARS-CoV-2RESUMO
Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding ß-amino acid. ß-Amino acid substitution at the proline residue of Ang III (ß-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of ß-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of ß-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and ß-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of ß-Pro7-Ang III. In both male and female rats, acute systemic administration of ß-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of ß-Pro7-Ang III administered. Moreover, intra-renal administration of ß-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of ß-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function.
Assuntos
Angiotensina III/análogos & derivados , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Natriurese/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/agonistas , Circulação Renal/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Angiotensina III/farmacologia , Animais , Feminino , Rim/metabolismo , Masculino , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/metabolismo , Fatores Sexuais , Transdução de SinaisRESUMO
Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally. Cumulative evidence has implicated renin-angiotensin system (RAS) in the pathogenesis of COPD. This study aimed to investigate potential protective effects of angiotensin II type-2 receptor (AT2R) activation in cigarette smoke (CS)-induced COPD models. Compound 21 (C21), a selective and potent non-peptide small molecule AT2R agonist, was evaluated for anti-inflammatory, anti-oxidative and anti-remodeling activities in a two-week (acute) and an eight-week (chronic) CS-induced COPD models. C21 inhibited CS-induced increases in macrophage and neutrophil counts, pro-inflammatory cytokines and oxidative damage markers in bronchoalveolar lavage (BAL) fluid, and TGF-ß1 in lung tissues, from COPD models. C21 restored phosphatase activities and reduced phospho-p38 MAPK, phospho-ERK and p65 subunit of NF-κB levels in CS-exposed lung tissues. C21 also suppressed CS-induced increases in α-Sma, Mmp9, Mmp12 and hydroxyproline levels in lung tissues, and neutrophil elastase activity in BAL fluid. C21 modulated RAS in CS-exposed lungs by downregulating Ang II but upregulating Ang-(1-7) and Mas receptor levels. C21 prevented CS-induced emphysema and improved lung functions in chronic COPD model. We report here for the first time the protective effects of AT2R agonist C21 against CS-induced COPD, and provide strong evidence for further development of AT2R agonist for the treatment of COPD.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Imidazóis/farmacologia , Pulmão/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Enfisema Pulmonar/prevenção & controle , Receptor Tipo 2 de Angiotensina/agonistas , Sistema Renina-Angiotensina/efeitos dos fármacos , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos Endogâmicos BALB C , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatologia , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G , Transdução de Sinais , Fumaça , Produtos do TabacoRESUMO
BACKGROUND: Abdominal adhesions (AAs) are post-traumatic fibrous bands that connect visceral and/or peritoneal surfaces, leading to possible long-term complications. The effect of a novel antifibrotic selective angiotensin II type 2 receptor agonist, compound 21 (C21) on AA formation was assessed in a murine model. METHODS: Female BALB/c mice were laparotomized and the cecum and overlying parietal peritoneum abraded. C21 (10 µg/kg) or saline (vehicle) were administered orally or intraperitoneally daily. Mice were sacrificed 8 days after surgery, adhesions graded, and peritoneal fluid collected for transforming growth factor (TGF)-ß levels. Laparotomy incisions were excised for immunohistochemistry. In vitro, scratch assays were performed using primary parietal peritoneal fibroblasts and visceral mesothelial cells treated with C21 (10 µM), angiotensin II (1 µM), or both. Western blot analysis of primary cell lysates was performed for total and phosphorylated SMAD 2/3. RESULTS: Oral and intraperitoneal C21 reduced AA formation and TGF-ß levels in peritoneal fluid. Surgical incisions demonstrated decreased α-smooth muscle actin expression in C21-treated animals, but no difference in vascularity, macrophage infiltration, collagen I/III distribution and density, and dermal thickness. Migration and expression of phosphorylated SMAD 2/3 was reduced in parietal peritoneal fibroblasts and visceral mesothelial cells treated with C21. CONCLUSIONS: Local and systemic C21 administration reduced or completely prevented AA formation. These findings may be attributed to decreased intraperitoneal TGF-ß in vivo and decreased migration of peritoneal fibroblasts and visceral mesothelial cells. Importantly, C21 did not have histologically quantifiable effects on laparotomy wounds, suggesting C21 could reduce AA formation without compromising laparotomy healing.
Assuntos
Imidazóis/farmacologia , Peritônio/patologia , Complicações Pós-Operatórias/prevenção & controle , Receptor Tipo 2 de Angiotensina/agonistas , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Aderências Teciduais/prevenção & controle , Administração Oral , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Humanos , Imidazóis/uso terapêutico , Injeções Intraperitoneais , Camundongos , Peritônio/efeitos dos fármacos , Peritônio/cirurgia , Cuidados Pós-Operatórios/métodos , Complicações Pós-Operatórias/patologia , Cultura Primária de Células , Sulfonamidas/uso terapêutico , Tiofenos/uso terapêutico , Aderências Teciduais/etiologia , Aderências Teciduais/patologia , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.