<p>The G protein-coupled receptors (GPCRs) are important targets in drug discovery. In several cases, the endogenous ligands that activate the GPCRs of pharmaceutical interest are peptides. Unfortunately, peptides are in general not suitable as drugs, since the peptide structure is associated with several disadvantages, such as low oral bioavailability, rapid degradation and low receptor subtype selectivity. Thus, there is a strong need for drug-like nonpeptide ligands to peptide-activated GPCRs. However, to discover nonpeptide ligands that mimic the effect of the endogenous peptide, i.e. peptidomimetics, is a tremendous challenge. In fact, morphine and the related opioids were the only known examples of peptidomimetics before 1995 and these ligands were known long before the native endogenous peptide ligands were discovered. </p><p>The main objective of the work described in this thesis was to design, synthesize and biologically evaluate selective nonpeptide agonists to the peptide-activated GPCR AT₂. The AT₂ receptor belongs to the renin–angiotensin system, where the octapeptide angiotensin II (Ang II) is the major effector peptide. Ang II mediates its effects through the two GPCRs AT₁ and AT₂. The AT₁ receptor is already an established target in the treatment of hypertension. The physiological role of the AT₂ receptor, which is up-regulated in certain pathological conditions, is not fully understood but it seems to include positive effects such as vasodilatation, tissue repair, tissue regeneration and neuronal differentiation. </p><p>In the current investigation we started from the nonpeptide and nonselective (AT₁/ AT₂) compound L-162,313. This ligand is a known AT₁ receptor agonist but its effect on the AT₂ receptor was unknown at the start of this project. We were able to show that it acts as an agonist also at the AT₂ receptor. Furthermore, stepwise synthetic modifications of L-162,313 led to the identification of the first selective nonpeptide AT₂ receptor agonist. Following the discovery of this compound several selective nonpeptide AT₂ receptor agonists were identified. It was also revealed that a minor structural alteration of one of these compounds interconverted the functional activity from agonism to antagonism. The structural requirement for agonism vs antagonism was therefore studied. The functionality switch was suggested, at least partly, to be due to the spatial relationship between the methyleneimidazole group and the isobutyl side chain of the compounds. To further investigate the bioactive conformation(s) of this series of compounds enantiomerically pure analogues with conformationally constrained isobutyl chains were prepared. This study revealed that the direction of the isobutyl side chain determine whether the compounds act as agonists or antagonists at the AT₂ receptor. Further investigations are required to fully elucidate the bioactive conformation(s) of these nonpeptide AT₂ receptor agonists.</p><p>We believe that the selective nonpeptide AT₂ receptor agonists and antagonists identified in this thesis will serve as important research tools in the continuing investigation of the physiological role of the AT₂ receptor. We also believe that these drug-like compounds might provide potential leads in drug discovery processes.</p>

The G protein-coupled receptors (GPCRs) are important targets in drug discovery. In several cases, the endogenous ligands that activate the GPCRs of pharmaceutical interest are peptides. Unfortunately, peptides are in general not suitable as drugs, since the peptide structure is associated with several disadvantages, such as low oral bioavailability, rapid degradation and low receptor subtype selectivity. Thus, there is a strong need for drug-like nonpeptide ligands to peptide-activated GPCRs. However, to discover nonpeptide ligands that mimic the effect of the endogenous peptide, i.e. peptidomimetics, is a tremendous challenge. In fact, morphine and the related opioids were the only known examples of peptidomimetics before 1995 and these ligands were known long before the native endogenous peptide ligands were discovered. The main objective of the work described in this thesis was to design, synthesize and biologically evaluate selective nonpeptide agonists to the peptide-activated GPCR AT2. The AT2 receptor belongs to the renin–angiotensin system, where the octapeptide angiotensin II (Ang II) is the major effector peptide. Ang II mediates its effects through the two GPCRs AT1 and AT2. The AT1 receptor is already an established target in the treatment of hypertension. The physiological role of the AT2 receptor, which is up-regulated in certain pathological conditions, is not fully understood but it seems to include positive effects such as vasodilatation, tissue repair, tissue regeneration and neuronal differentiation. In the current investigation we started from the nonpeptide and nonselective (AT1/ AT2) compound L-162,313. This ligand is a known AT1 receptor agonist but its effect on the AT2 receptor was unknown at the start of this project. We were able to show that it acts as an agonist also at the AT2 receptor. Furthermore, stepwise synthetic modifications of L-162,313 led to the identification of the first selective nonpeptide AT2 receptor agonist. Following the discovery of this compound several selective nonpeptide AT2 receptor agonists were identified. It was also revealed that a minor structural alteration of one of these compounds interconverted the functional activity from agonism to antagonism. The structural requirement for agonism vs antagonism was therefore studied. The functionality switch was suggested, at least partly, to be due to the spatial relationship between the methyleneimidazole group and the isobutyl side chain of the compounds. To further investigate the bioactive conformation(s) of this series of compounds enantiomerically pure analogues with conformationally constrained isobutyl chains were prepared. This study revealed that the direction of the isobutyl side chain determine whether the compounds act as agonists or antagonists at the AT2 receptor. Further investigations are required to fully elucidate the bioactive conformation(s) of these nonpeptide AT2 receptor agonists. We believe that the selective nonpeptide AT2 receptor agonists and antagonists identified in this thesis will serve as important research tools in the continuing investigation of the physiological role of the AT2 receptor. We also believe that these drug-like compounds might provide potential leads in drug discovery processes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

No description available.

Uma estreita relação entre a ação dos hormônios tireoidianos (HT) no sistema cardiovascular sendo mediada por componentes do sistema renina-angiotensina (SRA) tem sido descrita na literatura. Já foi demonstrado que o processo de isquemia/reperfusão (I/R) promove diminuição na expressão do receptor de angiotensina II do tipo 2 (AT2) no miocárdio, com consequente prejuízo funcional, enquanto o aumento de sua expressão melhorou a recuperação da função cardíaca. A hipótese do presente estudo é que o papel cardioprotetor exercido pelos HT ocorre com a participação do receptor AT2. Esta hipótese foi testada utilizando o modelo de I/R com a perfusão de coração isolado de camundongos selvagens e nocautes para o AT2, submetidos a tratamento por 14 dias com T3. Ainda foi avaliado o SRA mitocondrial, assim como o papel do óxido nítrico (NO) na recuperação pós-isquêmica. Os resultados apontam que a cardioproteção induzida pelo T3 é mediada pelo AT2, com consequente aumento na produção de NO e modulação de parâmetros do metabolismo mitocondrial. / Some authors have shown a close relationship between the action of thyroid hormone (TH) on cardiovascular system and renin angiotensin system (RAS) activation. Have been shown that ischemia/reperfusion (I/R) promotes decrease on angiotensin II type 2 receptor (AT2) expression in the myocardium, with functional worsening, on the other hand the AT2R increased improves the cardiac function after ischemia episodes. So, we have hypothesized that the cardioprotective effect of TH in I/R model may occur with the participation of AT2. This hypothesis was tested using I/R model in isolated hearts AT2 knockout and wild-type mice submitted to high levels of T3 by 14 days. The mitochondrial RAS was evaluated, as well as the nitric oxide (NO) role in post-ischemic recovery. The results show that TH induces cardioprotection through AT2 receptor, some mitochondrial metabolism parameters were modulated by TH and the NO production was increased.