Angiotensin II type 2 receptor promotes adipocyte differentiation and restores adipocyte size in high-fat/high-fructose diet-induced insulin resistance in rats.

This study was aimed at establishing whether specific activation of angiotensin II (ANG II) type 2 receptor (AT2R) modulates adipocyte differentiation and function. In primary cultures of subcutaneous (SC) and retroperitoneal (RET) preadipocytes, both AT2R and AT1R were expressed at the mRNA and protein level. Cells were stimulated with ANG II or the AT2R agonist C21/M24, alone or in the presence of the AT1R antagonist losartan or the AT2R antagonist PD123,319. During differentiation, C21/M24 increased PPARγ expression in both RET and SC preadipocytes while the number of small lipid droplets and lipid accumulation solely increased in SC preadipocytes. In mature adipocytes, C21/M24 decreased the mean size of large lipid droplets. Upon abolishment of AT2R expression using AT2R-targeted shRNAs, expressions of AT2R, aP2, and PPARγ remained very low, and cells were unable to differentiate. In Wistar rats fed a 6-wk high-fat/high-fructose (HFHF) diet, a significant shift toward larger adipocytes was observed in RET and SC adipose tissue depots. C21/M24 treatments for 6 wk restored normal adipocyte size distribution in both these tissue depots. Moreover, C21/M24 and losartan decreased hyperinsulinemia and improved insulin sensitivity impaired by HFHF diet. A strong correlation between adipocyte size area and glucose infusion rate during euglycemic-hyperinsulinemic clamp was observed. These results indicate that AT2R is involved in early adipocyte differentiation, while in mature adipocytes and in a model of insulin resistance AT2R activation restores normal adipocyte morphology and improves insulin sensitivity.

Selective angiotensin II AT(2) receptor agonists with reduced CYP 450 inhibition.

Structural alterations to the benzylic position of the first drug-like selective angiotensin II AT(2) receptor agonist (1) have been performed, with the emphasis to reduce the CYP 450 inhibitory property of the initial structure. The imidazole moiety, responsible for the CYP 450 inhibitory effect in 1, was replaced with various heterocycles. In addition, the modes of attachment of the heterocycles, that is, carbon versus nitrogen attachment, and introduction of carbonyl functionalities to the benzylic position have been evaluated. In all the three series, AT(2) receptor ligands with affinity in the lower nanomolar range were identified. None of the analogues, regardless of the substituents, exhibited any affinity for the AT(1) receptor. Compounds with substantially reduced inhibition of the CYP 450 enzymes were obtained. Among them the compound 60 was found to induce neurite elongation in NG 108-15 cells and served as potent AT(2) selective agonist.

Angiotensin II type 2 receptor stimulation: a novel option of therapeutic interference with the renin-angiotensin system in myocardial infarction?

BACKGROUND: This study is the first to examine the effect of direct angiotensin II type 2 (AT(2)) receptor stimulation on postinfarct cardiac function with the use of the novel nonpeptide AT(2) receptor agonist compound 21 (C21). METHODS AND RESULTS: Myocardial infarction (MI) was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.01, 0.03, 0.3 mg/kg per day IP) was started 24 hours after MI and was continued until euthanasia (7 days after MI). Infarct size was assessed by magnetic resonance imaging, and hemodynamic measurements were performed via transthoracic Doppler echocardiography and intracardiac Millar catheter. Cardiac tissues were analyzed for inflammation and apoptosis markers with immunoblotting and real-time reverse transcription polymerase chain reaction. C21 significantly improved systolic and diastolic ventricular function. Scar size was smallest in the C21-treated rats. In regard to underlying mechanisms, C21 diminished MI-induced Fas-ligand and caspase-3 expression in the peri-infarct zone, indicating an antiapoptotic effect. Phosphorylation of the p44/42 and p38 mitogen-activated protein kinases, both involved in the regulation of cell survival, was strongly reduced after MI but almost completely rescued by C21 treatment. Furthermore, C21 decreased MI-induced serum monocyte chemoattractant protein-1 and myeloperoxidase as well as cardiac interleukin-6, interleukin-1beta, and interleukin-2 expression, suggesting an antiinflammatory effect. CONCLUSIONS: Direct AT(2) receptor stimulation may be a novel therapeutic approach to improve post-MI systolic and diastolic function by antiapoptotic and antiinflammatory mechanisms.

High affinity rigidified AT2 receptor ligands with indane scaffolds.

Rigidification of the isobutyl side chain of drug-like AT2 receptor agonists and antagonists that are structurally related to the first reported selective AT2 receptor agonist 1 (C21) delivered bioactive indane derivatives. Four enantiomer pairs were synthesized and the enantiomers were isolated in an optical purity >99%. The enantiomers 7a, 7b, 8a, 8b, 9a, 9b, 10a and 10b bind to the AT2 receptor with moderate (K i = 54-223 nM) to high affinity (K i = 2.2-7.0 nM). The enantiomer with positive optical rotation (+) exhibited the highest affinity at the receptor. The indane derivatives 7b and 10a are among the most potent AT2 receptor antagonists reported so far. As illustrated by the enantiomer pairs 7a/b and 10a/b, an alteration at the stereogenic center has a pronounced impact on the activation process of the AT2 receptor, and can convert agonists to antagonists and vice versa.

Selective angiotensin II AT2 receptor agonists: Benzamide structure-activity relationships.

In the investigation of the structure-activity relationship of nonpeptide AT(2) receptor agonists, a series of substituted benzamide analogues of the selective nonpeptide AT(2) receptor agonist M024 have been synthesised. In a second series, the biphenyl scaffold was compared to the thienylphenyl scaffold and the impact of the isobutyl substituent and its position on AT(1)/AT(2) receptor selectivity was also investigated. Both series included several compounds with high affinity and selectivity for the AT(2) receptor. Three of the compounds were also proven to function as agonists at the AT(2) receptor, as deduced from a neurite outgrowth assay, conducted in NG108-15 cells.

Selective angiotensin II AT2 receptor agonists: arylbenzylimidazole structure-activity relationships.

Structural alterations in the 2- and 5-positions of the first drug-like selective angiotensin II AT2 receptor agonist (1) have been performed. The imidazole ring system was proven to be a strong determinant for the AT2 selectivity, and with few exceptions all variations gave good AT2 receptor affinities and with retained high AT2/AT1 selectivities. On the contrary to the findings with AT1 receptor agonists, the impact of structural modifications in the 5-position of the AT2 selective compounds were less pronounced regarding activation of the AT2 receptor. The butyloxyphenyl (56) and the propylthienyl (50) derivatives were found to exert a high agonistic effect as deduced from their capacity to induce neurite elongation in neuronal cells, as does angiotensin II.

Interconversion of Functional Activity by Minor Structural Alterations in Nonpeptide AT2 Receptor Ligands.

Migration of the methylene imidazole side chain in the first reported selective drug-like AT2 receptor agonist C21/M024 (1) delivered the AT2 receptor antagonist C38/M132 (2). We now report that the AT2 receptor antagonist compound 4, a biphenyl derivative that is structurally related to 2, is transformed to the agonist 6 by migration of the isobutyl group. The importance of the relative position of the methylene imidazole and the isobutyl substituent is highlighted herein.

First reported nonpeptide AT1 receptor agonist (L-162,313) acts as an AT2 receptor agonist in vivo.

In this investigation, it is demonstrated that the first nonpeptide AT(1) receptor agonist L-162,313 (1), disclosed in 1994, also acts as an agonist at the AT(2) receptor. In anesthetized rats, administration of compound 1 intravenously or locally in the duodenum increased duodenal mucosal alkaline secretion, effects that were sensitive to the selective AT(2) receptor antagonist PD-123,319. The data strongly suggest that 1 is an AT(2) receptor agonist in vivo. To the best of our knowledge, this substance is the first nonpeptidic low-molecular weight compound with an agonistic effect mediated through the AT(2) receptor.

Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist.

The first druglike selective angiotensin II AT(2) receptor agonist (21) with a K(i) value of 0.4 nM for the AT(2) receptor and a K(i) > 10 microM for the AT(1) receptor is reported. Compound 21, with a bioavailability of 20-30% after oral administration and a half-life estimated to 4 h in rat, induces outgrowth of neurite cells, stimulates p42/p44(mapk), enhances in vivo duodenal alkaline secretion in Sprague-Dawley rats, and lowers the mean arterial blood pressure in anesthetized, spontaneously hypertensive rats. Thus, the peptidomimetic 21 exerts a similar biological response as the endogenous peptide angiotensin II after selective activation of the AT(2) receptor. Compound 21, derived from the prototype nonselective AT(1)/AT(2) receptor agonist L-162,313 will serve as a valuable research tool, enabling studies of the function of the AT(2) receptor in more detail.

Direct angiotensin AT2 receptor stimulation using a novel AT2 receptor agonist, compound 21, evokes neuroprotection in conscious hypertensive rats.

BACKGROUND: In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury. CONCLUSION: These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.

Comparative functional properties of two structurally similar selective nonpeptide drug-like ligands for the angiotensin II type-2 (AT(2)) receptor. Effects on neurite outgrowth in NG108-15 cells.

There is increasing evidence that angiotensin II (Ang II), through binding to the type 2 (AT(2)) receptor may have beneficial effects in various physiological and pathological situations. However, specific action presumably mediated by the angiotensin AT(2) receptor has been hampered by the absence of appropriate selective ligands. The aim of this study was to compare the biological properties of two related and selective drug-like nonpeptide AT(2) ligands, namely an agonist called M024 (also known as Compound 21) and a new ligand, presumably an antagonist, C38/M132, (originally called C38). Properties of the compounds were investigated in NG108-15 cells expressing angiotensin AT(2) receptor and known to develop neurite outgrowth upon Ang II stimulation. NG108-15 cells stimulated for three days with C21/M024 (0.1 or 100nM) exhibited the same neurite outgrowth as cells stimulated with Ang II (100nM) while co-incubation of Ang II or C21/M024 with C38/M132 (10 or 100nM) inhibited their effects, similarly to the angiotensin AT(2) receptor antagonist, PD123319 (10µM). As Ang II, C21/M024 induced a Rap1-dependent activation of p42/p44(mapk) whereas preincubation of cells with C38/M132 inhibited p42/p44(mapk) and Rap1 activation induced by Ang II. Three-day treatment with C21/M024 or Ang II decreased cell number in culture, an effect that was rescued by preincubation with C38/M132. Taken together, these results indicate that the nonpeptide ligand C21/M024 is a potent angiotensin AT(2) receptor agonist while C38/M132 acts as an antagonist. These selective nonpeptide angiotensin AT(2) ligands may represent unique and long-awaited tools for the pursuit of in vivo studies.

From the first selective non-peptide AT(2) receptor agonist to structurally related antagonists.

A para substitution pattern of the phenyl ring is a characteristic feature of the first reported selective AT(2) receptor agonist M024/C21 (1) and all the nonpeptidic AT(2) receptor agonists described so far. Two series of compounds structurally related to 1 but with a meta substitution pattern have now been synthesized and biologically evaluated for their affinity to the AT(1) and AT(2) receptors. A high AT(2)/AT(1) receptor selectivity was obtained with all 41 compounds synthesized, and the majority exhibited K(i) ranging from 2 to 100 nM. Five compounds were evaluated for their functional activity at the AT(2) receptor, applying a neurite outgrowth assay in NG108-15 cells. Notably, four of the five compounds, with representatives from both series, acted as potent AT(2) receptor antagonists. These compounds were found to be considerably more effective than PD 123,319, the standard AT(2) receptor antagonist used in most laboratories. No AT(2) receptor antagonists were previously reported among the derivatives with a para substitution pattern. Hence, by a minor modification of the agonist 1 it could be transformed into the antagonist, compound 38. These compounds should serve as valuable tools in the assessment of the role of the AT(2) receptor in more complex physiological models.

A facilitated approach to evaluate the inhibitor mode and potency of compounds targeting microsomal prostaglandin e synthase-1.

Microsomal prostaglandin E(2) synthase-1 (MPGES1) catalyzes the formation of prostaglandin E(2) from the endoperoxide prostaglandin H(2). MPGES1 expression is induced in inflammatory diseases, and this enzyme is regarded as a potential drug target. To aid in the drug discovery effort, a simple method for determination of inhibition mechanism and potency toward both prostaglandin H(2) and glutathione (GSH) has been developed. Using an assay with thiobarbituric acid-based detection, the inhibitory effects of six MPGES1 inhibitors were evaluated. The IC(50) values obtained at three substrate (S) concentrations ([S]K(M)) were used to estimate inhibition modality and inhibition constant values. This facilitated strategy is a useful and general screening method to evaluate the inhibitory effects of new drug compounds.

Angiotensin II, a Neuropeptide at the Frontier between Endocrinology and Neuroscience: Is There a Link between the Angiotensin II Type 2 Receptor and Alzheimer's Disease?

Amyloid-ß peptide deposition, abnormal hyperphosphorylation of tau, as well as inflammation and vascular damage, are associated with the development of Alzheimer's disease (AD). Angiotensin II (Ang II) is a peripheral hormone, as well as a neuropeptide, which binds two major receptors, namely the Ang II type 1 receptor (AT1R) and the type 2 receptor (AT2R). Activation of the AT2R counteracts most of the AT1R-mediated actions, promoting vasodilation, decreasing the expression of pro-inflammatory cytokines, both in the brain and in the cardiovascular system. There is evidence that treatment with AT1R blockers (ARBs) attenuates learning and memory deficits. Studies suggest that the therapeutic effects of ARBs may reflect this unopposed activation of the AT2R in addition to the inhibition of the AT1R. Within the context of AD, modulation of AT2R signaling could improve cognitive performance not only through its action on blood flow/brain microcirculation but also through more specific effects on neurons. This review summarizes the current state of knowledge and potential therapeutic relevance of central actions of this enigmatic receptor. In particular, we highlight the possibility that selective AT2R activation by non-peptide and highly selective agonists, acting on neuronal plasticity, could represent new pharmacological tools that may help improve impaired cognitive performance in AD and other neurological cognitive disorders.

Development of selective non-peptide angiotensin II type 2 receptor agonists.

The development of the first drug-like selective angiotensin II type 2 (AT(2)) receptor agonist (22) derived from the non-selective angiotensin II type 1 (AT( 1)) receptor/AT(2) receptor agonist L-162,313 is presented. Compound 22 with a K(i) value of 0.4 nM for the AT( 2) receptor and a K(i) > 10 microM for the AT(1) receptor induces outgrowth of neurite cells, stimulates p42/p44( mapk), enhances in vivo duodenal alkaline secretion in Sprague-Dawley rats and lowers the mean arterial blood pressure in anaesthetised spontaneously hypertensive rats. Thus, the peptidomimetic 22 exerts a similar biological response as the endogenous peptide angiotensin II after selective activation of the AT(2) receptor. In addition, Compound 22 has a bioavailability of 20-30% after oral administration and a half-life estimated to four hours in the rat. Compound 22 will therefore serve as a valuable research tool enabling studies of the function of the AT(2) receptor in more detail.

Direct angiotensin II type 2 receptor stimulation acts anti-inflammatory through epoxyeicosatrienoic acid and inhibition of nuclear factor kappaB.

Angiotensin II type 2 (AT(2)) receptors can be regarded as an endogenous repair system, because the AT(2) receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT(2) receptor agonist, compound 21 (C21; dissociation constant for AT(2) receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10,000 nM). This study tested AT(2) receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor kappaB. C21 dose-dependently (1 nM to 1 micromol/L) reduced tumor necrosis factor-alpha-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT(2) receptor specificity was controlled for by inhibition with the AT(2) receptor antagonist PD123319 and by the absence of effects in AT(2) receptor-deficient cells. AT(2) receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor kappaB, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-alpha in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT(2) receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT(2) receptor agonist C21. These data suggest that pharmacological AT(2) receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor kappaB.

Selective angiotensin II AT(2) receptor agonists devoid of the imidazole ring system.

A versatile parallel synthetic method to obtain three series of non-cyclic analogues of the first drug-like selective angiotensin II AT(2) receptor agonist (1) has been developed. In analogy with the transformation of losartan to valsartan it was demonstrated that a non-cyclic moiety could be employed as an imidazole replacement to obtain AT(2) selective compounds. In all the three series, AT(2) receptor ligands with affinities in the lower nanomolar range were found. None of the analogues exhibited any affinity for the AT(1) receptor. Four compounds, 17, 22, 39 and 51, were examined in a neurite outgrowth cell assay. All four compounds were found to exert a high agonistic effect as deduced from their capacity to induce neurite elongation in neuronal cells, as does angiotensin II.

Dimethylformamide as a carbon monoxide source in fast palladium-catalyzed aminocarbonylations of aryl bromides.

Dimethylformamide (DMF) acts as an efficient source of carbon monoxide and dimethylamine in the palladium-catalyzed aminocarbonylation (Heck carbonylation) of p-tolyl bromide to provide the dimethylamide. Addition of amines to the reaction mixture in excess delivers the corresponding aryl amides in good yields. The amines employed, benzylamine, morpholine, and aniline, all constitute good reaction partners. The reaction proceeds smoothly with bromobenzene and more electron-rich aryl bromides, but electron-deficient aryl bromides fail to undergo aminocarbonylation. The reactions are conducted at 180-190 degrees C for 15-20 min with microwave heating in a reaction mixture containing imidazole and potassium tert-butoxide: the latter is required to promote decomposition of the DMF solvent at a suitable rate. The beneficial effects of controlled microwave irradiation as an energy source for the rapid heating of the carbonylation reaction mixture are demonstrated. The carbonylation procedure reported herein, which relies on the in situ generation of carbon monoxide, serves as a convenient alternative to other carbonylation methods and is particularly applicable to small-scale reactions where short reaction times are desired and the direct use of carbon monoxide gas is impractical.

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.