Electronic sculpting of ligand-GPCR subtype selectivity:the case of angiotensin II

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 (<i>K</i>_i = 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

Isoform-Specific Biased Agonism of Histamine H 3 Receptor Agonists s

ABSTRACT The human histamine H 3 receptor (hH 3 R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H 3 R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH 3 R splice variants (hH 3 R 445 and hH 3 R 365 ) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3b (GSK3b) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former&apos;s designation as a &quot;protean&quot; agonist. The 80 amino acid IL3 deleted isoform hH 3 R 365 is more permissive in its signaling than hH 3 R 445 : 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH 3 R 365 was completely unable to stimulate GSK3b phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G proteincoupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants

Cardiovascular studies of AT2 receptor in various pathophysiological settings

The renin angiotensin system (RAS) plays an important role in blood pressure regulation and its importance in cardiovascular disease has been well researched. Hypertension is a major risk factor for the development of cardiovascular disease and the RAS is a chief regulator of arterial pressure. The main hormone of RAS, the octapeptide angiotensin II (Ang II), exerts its actions through stimulation of two major receptor subtypes, the angiotensin type-1 receptor (AT1R) and the angiotensin type-2 receptor (AT2R). The focus of current literature has been on the activation of the Ang II-AT1R axis and the unfavourable effects in cardiovascular pathology. However, shorter peptide fragments of Ang II are thought to have biological activity in their own right and elicit effects that oppose those mediated by Ang II by stimulating other RAS receptors such as AT2R. Given that hypertension is well established in aged populations, it is of particular interest to determine role of the AT2R in this setting. Additionally, it has been hypothesized that females have a greater vasodilator component of the RAS than males, which may contribute to sex differences in the rates of cardiovascular disease. Therefore, aspects of this thesis explored the function of AT2R stimulation with particular attention directed towards the influence of age and sex. In Chapter 2, HEK-293 cells stably transfected with either the AT1R or the AT2R were used to perform a systematic analysis of binding affinities of all the major Ang peptides. Additionally, the novel AT2R agonist, Compound 21, as well as the MasR agonist and antagonist, AVE0991 and A779 respectively, were tested for their ability to bind to the AT1R or the AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using [125I]- Sar1Ile8Ang II in AT1R transfected HEK-293 cells revealed only Ang II, Ang III and candesartan to have high affinity for AT1R. In AT2R transfected HEK-293 cells, blood pressure in adult normotensive rats via AT2R. However, in aged rats, the depressor effect of Ang (1-7) was maintained but also inhibited by either AT2R blockade or MasR blockade. At the same time, AT2R, MasR and ACE2 immunoreactivity was markedly elevated in aortic sections from aged animals. These results indicate that the Ang (1-7)-mediated depressor effect was preserved in aged animals and involved both AT2R and MasR, whereas Ang (1-7) effects were mediated exclusively via stimulation of AT2R in adult rats. Chapters 5 and 6 examined the effect of both chronic and acute treatment with the AT2R selective nonpeptide agonist, Compound 21. In Chapter 5, chronic treatment with Compound 21 failed to lower blood pressure in normotensive female mice or rats. However, in Chapter 6, the effect of acute administration of Compound 21 was examined in an hypertensive model in which β-5 Ile Ang II had already been shown to exert a vasodepressor effect (Chapter 3). In this case, against a background of AT1R blockade, Compound 21 reduced blood pressure in adult hypertensive rats, an effect that was reversed by AT2R blockade. Additionally, Compound 21 evoked vasorelaxation in both mouse and rat vessels via AT2R stimulation. The development of Compound 21 has resulted in a useful tool for further investigation of AT2R function but this thesis has also highlighted the inability of this compound to lower blood pressure under chronic conditions. In conclusion, this thesis provides evidence for the hypothesis that shorter Ang peptide fragments may act as endogenous AT2R ligands. Taken collectively, these results emphasise the importance of understanding the complex relationships that exist within the RAS. In particular, this thesis highlights that the AT2R axis as well as the Ang (1-7)/MasR axis (at least in aging) may act as counter-regulatory mechanisms to the Ang II/AT1R axis. Moreover, the development of additional AT2R ligands utilising relatively simple synthetic methods as shown in this thesis is warranted as these receptors are potential therapeutic targets for the treatment of hypertension and related cardiovascular remodelling

Cardiovascular studies of AT2 receptor in various pathophysiological settings

The renin angiotensin system (RAS) plays an important role in blood pressure regulation and its importance in cardiovascular disease has been well researched. Hypertension is a major risk factor for the development of cardiovascular disease and the RAS is a chief regulator of arterial pressure. The main hormone of RAS, the octapeptide angiotensin II (Ang II), exerts its actions through stimulation of two major receptor subtypes, the angiotensin type-1 receptor (AT1R) and the angiotensin type-2 receptor (AT2R). The focus of current literature has been on the activation of the Ang II-AT1R axis and the unfavourable effects in cardiovascular pathology. However, shorter peptide fragments of Ang II are thought to have biological activity in their own right and elicit effects that oppose those mediated by Ang II by stimulating other RAS receptors such as AT2R. Given that hypertension is well established in aged populations, it is of particular interest to determine role of the AT2R in this setting. Additionally, it has been hypothesized that females have a greater vasodilator component of the RAS than males, which may contribute to sex differences in the rates of cardiovascular disease. Therefore, aspects of this thesis explored the function of AT2R stimulation with particular attention directed towards the influence of age and sex. In Chapter 2, HEK-293 cells stably transfected with either the AT1R or the AT2R were used to perform a systematic analysis of binding affinities of all the major Ang peptides. Additionally, the novel AT2R agonist, Compound 21, as well as the MasR agonist and antagonist, AVE0991 and A779 respectively, were tested for their ability to bind to the AT1R or the AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using [125I]- Sar1Ile8Ang II in AT1R transfected HEK-293 cells revealed only Ang II, Ang III and candesartan to have high affinity for AT1R. In AT2R transfected HEK-293 cells, blood pressure in adult normotensive rats via AT2R. However, in aged rats, the depressor effect of Ang (1-7) was maintained but also inhibited by either AT2R blockade or MasR blockade. At the same time, AT2R, MasR and ACE2 immunoreactivity was markedly elevated in aortic sections from aged animals. These results indicate that the Ang (1-7)-mediated depressor effect was preserved in aged animals and involved both AT2R and MasR, whereas Ang (1-7) effects were mediated exclusively via stimulation of AT2R in adult rats. Chapters 5 and 6 examined the effect of both chronic and acute treatment with the AT2R selective nonpeptide agonist, Compound 21. In Chapter 5, chronic treatment with Compound 21 failed to lower blood pressure in normotensive female mice or rats. However, in Chapter 6, the effect of acute administration of Compound 21 was examined in an hypertensive model in which β-5 Ile Ang II had already been shown to exert a vasodepressor effect (Chapter 3). In this case, against a background of AT1R blockade, Compound 21 reduced blood pressure in adult hypertensive rats, an effect that was reversed by AT2R blockade. Additionally, Compound 21 evoked vasorelaxation in both mouse and rat vessels via AT2R stimulation. The development of Compound 21 has resulted in a useful tool for further investigation of AT2R function but this thesis has also highlighted the inability of this compound to lower blood pressure under chronic conditions. In conclusion, this thesis provides evidence for the hypothesis that shorter Ang peptide fragments may act as endogenous AT2R ligands. Taken collectively, these results emphasise the importance of understanding the complex relationships that exist within the RAS. In particular, this thesis highlights that the AT2R axis as well as the Ang (1-7)/MasR axis (at least in aging) may act as counter-regulatory mechanisms to the Ang II/AT1R axis. Moreover, the development of additional AT2R ligands utilising relatively simple synthetic methods as shown in this thesis is warranted as these receptors are potential therapeutic targets for the treatment of hypertension and related cardiovascular remodelling

A single beta-amino acid substitution to angiotensin II confers AT(2) receptor selectivity and vascular function

Novel AT2R ligands were designed by substituting individual β-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT2R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT1R- or AT2R- transfected HEK-293 cells, only β-Asp-Ang II and Ang II fully displaced [I]-Ang II from AT1R. In contrast, β-substitutions at each position of Ang II exhibited AT2R affinity, with β-Tyr-Ang II and β-Ile-Ang II exhibiting 1000-fold AT2R selectivity. In mouse aortic rings, β-Tyr-Ang II and β-Ile-Ang II evoked vasorelaxation that was sensitive to blockade by the AT2R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT1R blockade, β-Ile-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (β-Ile-Ang II plus candesartan, -24±4 mm Hg) to a greater extent than candesartan alone (-11±3 mm Hg, n=7,

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms

International audienceIntroduction: A potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21.Research design and methods: We hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target.Results: High-fat feeding studies in Gpr21-/- mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21-/- monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels.Conclusions: Collectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21-/- bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis