RESUMO
GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.
Assuntos
Angiotensina II/análogos & derivados , Antineoplásicos/química , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Angiotensina II/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Ligantes , Simulação de Acoplamento Molecular , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/químicaRESUMO
Infiltration of macrophages into the artery wall plays detrimental roles during hypertension by promoting vascular inflammation and endothelial dysfunction, and it occurs via a chemo-attractant action of chemokines on macrophage cytokine receptors. We sought to identify the key chemokine receptors associated with macrophage infiltration into the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure and leukocyte accumulation. Mice treated with DOCA/salt for 21 days displayed markedly elevated systolic blood pressure (158 ± 2 versus 114 ± 5 mm Hg in sham group; P<0.0001). Polymerase chain reaction screening via a gene array of 20 chemokine receptors indicated an increased expression of CCR2 in aortas of DOCA/salt-treated mice. Real-time polymerase chain reaction confirmed mRNA upregulation of CCR2 in aortas from DOCA/salt-treated animals and of the CCR2 ligands CCL2, CCL7, CCL8, and CCL12 (all >2-fold versus sham; P<0.05). Flow cytometry revealed 2.9-fold higher macrophage numbers (ie, CD45(+) CD11b(+) F4/80(+) cells) in the aortic wall of DOCA/salt versus sham-treated mice. Intervention with a CCR2 antagonist, INCB3344 (30 mg/kg per day, IP), 10 days after the induction of hypertension with DOCA/salt treatment, reduced the aortic expression of CCR2 mRNA and completely reversed the DOCA/salt-induced influx of macrophages. Importantly, INCB3344 substantially reduced the elevated blood pressure in DOCA/salt-treated mice. Hence, our findings highlight CCR2 as a promising therapeutic target to reduce both macrophage accumulation in the vascular wall and blood pressure in hypertension.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hipertensão/prevenção & controle , Macrófagos/efeitos dos fármacos , Pirrolidinas/farmacologia , Receptores CCR2/antagonistas & inibidores , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Pressão Sanguínea/genética , Quimiocina CCL2/genética , Quimiocina CCL7/genética , Quimiocina CCL8/genética , Desoxicorticosterona , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Hipertensão/induzido quimicamente , Hipertensão/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio , Regulação para Cima/efeitos dos fármacosRESUMO
AT1R (angiotensin type 1 receptor) and AT2R (angiotensin type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (angiotensin II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT1R or AT2R to perform a systematic analysis of binding affinities of all the major angiotensin peptides. Additionally, we tested the novel AT2R agonist Compound 21, as well as the MasR (Mas receptor) agonist and antagonist AVE0991 and A-779 respectively, for their ability to bind to AT1R or AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using 125I-[Sar1Ile8]AngII on the AT1R-transfected HEK-293 cells revealed only AngII, AngIII [angiotensin III; angiotensin-(2-8)] and candesartan to have high affinity for AT1R. In the AT2R-transfected HEK-293 cells, competition for 125I-[Sar1Ile8]AngII binding was observed for all ligands except candesartan, AVE0991 and A-779, the latter two compounds having negligible affinity at either AT1R or AT2R. The rank order of affinity of ligands at AT2R was CGP42112>AngII≥AngIII>Compound 21≥PD123319â«AngIV [angiotensin IV; angiotensin-(3-8)]>Ang-(1-7) [angiotensin-(1-7)]. Of note, although AngIV and Ang-(1-7) exhibited only modest affinity at AT2R compared with AngII, these two angiotensin peptides, together with AngIII, had substantial AT2R selectivity over AT1R. Collectively, our results suggest that shorter angiotensin peptides can act as endogenous ligands at AT2R.