Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist.

As a biased agonist, peptide angiotensin II (Ang II) type 1 (AT1) receptor ligand antagonizes Ang II-stimulated G protein signaling but stimulates several kinase pathways. Here, we developed a non-peptide AT1 receptor compound as a biased ligand. We synthesized three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) as candidates of biased ligands. Extracellular signal-regulated kinase (ERK) 1/2 activation and inositol phosphate (IP) production were measured using a cell system that overexpressed AT1 receptors (wild-type, L112A, Q257A, Y292A, and N295A receptors). We also examined the modes of receptor-ligand binding using a competition binding study. The Kd values of R239470, R781253, and R794847 for the AT1 wild-type receptor were 0.8, 21, and 48 nM, respectively, as assessed in a competition binding study. Those of R239470, R781253, and R794847 for the L112A receptor were 37, 23, and 31 nM, respectively. R781253 and R794847 decreased and increased IP production, respectively, whereas R239470 did not change IP production. R781253 and R794847, but not R239470, activated ERK1/2. In conclusion, R239470, R781253, and R794847 act as a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, R781253 and R794847, but not R239470, are agonists toward ERK1/2 activation. Thus, we developed a non-peptide AT1 receptor compound as a biased ligand.

Ezetimibe Monotherapy Reduces Serum Levels of Platelet-Activating Factor Acetylhydrolase in Patients With Dyslipidemia.

BACKGROUND: The combination of ezetimibe with statin therapy reduced cardiovascular events compared to statin monotherapy in IMPROVEIT study, and ezetimibe monotherapy attenuated atherosclerosis in basic study. We previously showed ezetimibe monotherapy was especially effective for metabolic syndrome (MetS) patients. We investigated the effects of ezetimibe monotherapy for high-density lipoprotein cholesterol (HDL-chol) function and platelet-activating factor acetylhydrolase (PAF-AH) activity. METHODS: Forty-two patients who initially received ezetimibe (10 mg/day) without statin treatment for 16 weeks from January 2009 to August 2011 were enrolled. Patients were divided into MetS and non-MetS groups, and serum levels of lipids, PAF-AH, and HDL-chol efflux capacity (HDL-CEC) at baseline and after 16 weeks of treatment were investigated. Serum PAF-AH, HDL-associated PAF-AH (HDL-PAF-AH), and LDL-associated PAF-AH (LDL-PAF-AH) were measured. RESULTS: In all patients, age, the percentages of males, and body mass index were 61.0 ± 8.8 years, 59.5% and 26.3 ± 3.4 kg/m2, respectively. Total cholesterol and low-density lipoprotein cholesterol (LDL-chol) were significantly decreased by ezetimibe monotherapy. Serum PAF-AH and LDL-PAF-AH were significantly decreased by ezetimibe monotherapy, whereas HDL-PAF-AH and HDL-CEC were not. There was no difference in the results of PAF-AH and HDL-CEC between MetS and non-MetS groups. CONCLUSIONS: Ezetimibe monotherapy might prevent coronary heart disease (CHD) regardless of the presence of MetS, because PAF-AH was independent risk factor for CHD.

Anti-atherosclerotic effects of an improved apolipoprotein A-I mimetic peptide.

BACKGROUND: Apolipoprotein (Apo)A-I is a major protein component of high-density lipoprotein (HDL) that causes cholesterol efflux from peripheral cells through ATP-binding cassette transporter A1 (ABCA1) and the generation of HDL. Furthermore, it has a possible protective function against atherosclerotic cardiovascular disease (ASCVD). We previously developed a novel ApoA-I mimetic peptide without phospholipids (Fukuoka University ApoA-I Mimetic Peptide, FAMP). According to our previous studies, FAMP had an anti-arteriosclerotic effect. Since the required dose and reaction time of conventional FAMP were relatively large and short, respectively, we newly developed an improved FAMP (i-FAMP). METHODS AND RESULTS: We synthesized four candidate i-FAMPs, i-FAMP-D1, -D2, -D3 and -D4, and examined which i-FAMP has greater cholesterol efflux capacity than FAMP in A172 human glioblastoma cells transiently transfected with human ABCA1 cDNA. Only i-FAMP-D1 showed significantly greater cholesterol efflux capacity than conventional FAMP. i-FAMP-D1 formed stronger α-helical conformations than FAMP as assessed by circular dichroism spectra. Thus, we selected i-FAMP-D1 for further experiments. i-FAMP-D1 had a greater atheroprotective effect than FAMP in ApoE knockout mice. In addition, i-FAMP-D1 activated cholesterol efflux from macrophage to HDL more strongly than FAMP and increased cholesterol excretion from liver to feces. CONCLUSION: These results suggest that i-FAMP-D1 has a stronger anti-atherosclerotic effect than conventional FAMP.

Changes in the function of angiotensin II type 1 receptor due to cholesterol depletion from cell membrane.

Blockers of G-protein coupled receptors (GPCRs), angiotensin II (Ang II) type 1 (AT1) receptor and ß1-adrenergic (Ad) receptor, have been shown to improve the prognosis of cardiovascular disease. Cholesterol molecules in the cell membrane are needed to stabilize GPCRs as well as the cell membrane itself. We determined whether the functions of AT1 and ß1-Ad receptors were changed by cholesterol depletion from cardiovascular cell membranes. Ang II-induced inositol phosphate production through AT1 receptor was suppressed by cholesterol depletion from cell membranes using rosuvastatin or methyl-ß-cyclodextrin (MßCD), whereas isoproterenol-induced cyclic AMP production through ß1-Ad receptor did not change after cholesterol depletion. In addition, the binding affinities of Ang II and AT1 receptor blocker after cholesterol depletion were significantly lower than those before depletion. Although AT1 receptor expression levels did not change after cholesterol depletion, the expression levels of AT1 receptor that could bind to Ang II significantly decreased after depletion. The changes in the structure of AT1 receptor due to depletion were confirmed by substituted-cysteine accessibility mapping. In conclusion, Ang II-induced activation of AT1 receptor is reduced without affecting the function of ß1-Ad receptor after cholesterol depletion from cardiovascular cell membranes.

Unique mode of binding between angiotensin II type 1 receptor and its blockers.

BACKGROUND: We hypothesized that the 5-oxo-1,2,4-oxadiazole moiety of azilsartan (AZL), which represents a small difference in the molecular structures of AZL and candesartan (CAN) [angiotensin II type 1 receptor (AT1R) blockers], may be responsible for the molecular effects of AZL. METHODS: We examined the binding affinities of AZL and CAN to AT1R, along with their ability to block receptor activity. A competition binding study, inositol phosphate (IP) production assay and extracellular signal-regulated kinase (ERK) assay were performed using wild-type (WT) and mutants AT1R-transfected cells. RESULTS: The binding affinities of CAN and AZL were reduced by > 5-fold for Y35F, W84F, R167K, K199Q and I288A compared with WT. In addition, AZL showed a > 5-fold reduction in its binding affinity to V108A. CAN and AZL exhibited > 20-fold and > 100-fold reductions in binding affinity to R167K, respectively. The loss of binding affinity of AZL to R167K was greater than that of CAN. CAN-7H exhibited a > 10-fold reduction in binding affinity to R167K compared with CAN. On the other hand, the binding affinity of AZL-7H to R167K was comparable to that of AZL. While 10-6M CAN and CAN-7H partly blocked Ang II-induced IP production in R167K, 10-6M AZL and AZL-7H did not. In addition, 10-6M CAN, but not 10-6M AZL, partly blocked Ang II-induced ERK activation in R167K. CONCLUSIONS: The interaction between 5-oxo-1,2,4-oxadiazole in AZL and Arg167 in the AT1R appears to be more important than the interaction between the tetrazole ring in CAN and Arg167.

Advanced glycation of high-density lipoprotein and the functionality of aldosterone release in type 2 diabetes.

Patients with type 2 diabetes mellitus (DM) exhibit modification of high-density lipoprotein (HDL), which is likely to have an important role in the development of atherosclerotic cardiovascular disease (ASCVD). Excess production of aldosterone (Ald) results in hypertension as well as ASCVD. However, the biological activity of modified HDL in steroidogenesis is not clear. We measured the accumulation of thiobarbituric acid-reactive substances (TBARSs) and Nɛ-(carboxymethyl)lysine (CML) levels (markers of oxidation and glycoxidation, respectively) in isolated HDL from 41 patients with type 2 diabetes mellitus (DM group) and 41 age- and gender-matched patients in a non-DM group. We quantified angiotensin II-sensitized and -nonsensitized Ald release using a validated living adrenocortical cell assay. TBARS levels in isolated HDL were similar between patients in the DM and non-DM groups, whereas the CML content of HDL in the DM group was lower than that in the non-DM group, irrespective of higher blood glucose and hemoglobin A1c levels. There was no difference in the HDL-induced ex vivo Ald release between the groups. Although sustained hyperglycemia was not a determinant of HDL-induced Ald release, the degree of HDL glycoxidation was inversely associated with HDL-induced Ald release (r=-0.40, P<0.001). In conclusion, in vivo advanced glycoxidation of HDL had an inverse effect on HDL-induced Ald release, independent of the prevalence of type 2 DM.

The angiotensin II type 1 receptor-neprilysin inhibitor LCZ696 blocked aldosterone synthesis in a human adrenocortical cell line.

A recent clinical study indicated that an angiotensin II (Ang II) type 1 (AT1) receptor-neprilysin inhibitor (ARNi) designated LCZ696 (sacubitril/valsartan, as combined sodium complex) was superior to enalapril at reducing the risks of death and hospitalization due to heart failure. Therefore, we investigated the possible mechanisms of the beneficial effect of LCZ696, in which the inhibition of neprilysin enhances atrial natriuretic peptide (NP) or brain NP (ANP or BNP)-evoked signals that can block Ang II/AT1 receptor-induced aldosterone (Ald) synthesis in human adrenocortical cells. The binding affinity of valsartan+LBQ657 (active moiety of sacubitril) to the AT1 receptor was greater than that of valsartan alone in an AT1 receptor-expressing human embryonic kidney cell-based assay. There was no difference in the dissociation from the AT1 receptor between valsartan+LBQ657 and valsartan alone. In Ang II-sensitized human adrenocortical cells, ANP or BNP alone, but not LBQ657 or valsartan alone, significantly decreased Ald synthesis. The level of suppression of Ald synthesis by ANP or BNP with LBQ657 was greater than that by ANP or BNP without LBQ657. The suppression of ANP was blocked by inhibitors of regulator of G-protein signaling proteins and cyclic GMP-dependent protein kinase. The inhibition of neprilysin did not change the mRNA levels of the AT1 receptor, ANP receptor A, regulator of G-protein signaling protein, renin or 3ß-hydroxysteroid dehydrogenases. In conclusion, the inhibition of neprilysin by LBQ657 enhances the NP-evoked signals that can block Ang II/AT1 receptor-induced Ald synthesis in human adrenocortical cells.

The ApoA-I mimetic peptide FAMP promotes recovery from hindlimb ischemia through a nitric oxide (NO)-related pathway.

BACKGROUND/OBJECTIVE: HDL has various atheroprotective functions and improves endothelial function. Apolipoprotein A-I (apoA-I) is a major protein of HDL and plays a crucial role in HDL functions. We developed a novel apoA-I mimetic peptide, FAMP (Fukuoka University ApoA-I Mimetic Peptide). It is unclear whether an apoA-I mimetic peptide can promote neovascularization in vivo. Here, we investigated the effect of FAMP on endothelial nitric oxide synthase (eNOS) activation and angiogenesis in a murine hindlimb ischemia model. METHODS AND RESULTS: Intramuscular administration of FAMP significantly enhanced blood flow recovery and increased capillary density in the ischemic limb of mice fed a high-cholesterol diet (HCD). In a gait analysis, FAMP ameliorated functional recovery compared with that in the control group. FAMP significantly activated Akt, ERK, and eNOS phosphorylation in endothelial cells, and improved the migratory functions of human aortic endothelial cells (HAECs). LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), significantly inhibited the activation of eNOS by FAMP. FAMP had no beneficial effects on blood flow recovery in eNOS(-/-) mice. CONCLUSIONS: FAMP promoted recovery from hindlimb ischemia through a nitric oxide (NO)-related pathway by activation of a PI3K/Akt pathway. FAMP may become a new therapeutic agent for the future clinical treatment of critical limb ischemia (CLI).

LCZ696, an angiotensin receptor-neprilysin inhibitor, improves cardiac function with the attenuation of fibrosis in heart failure with reduced ejection fraction in streptozotocin-induced diabetic mice.

AIMS: Angiotensin receptor-neprilysin inhibitors (ARNis) acts an ARB and neprilysin inhibitor. Diabetes mellitus significantly increases the risk of cardiovascular disease and heart failure (HF). Therefore, we evaluated the effects and mechanisms of ARNi in HF with reduced ejection fraction (HFrEF) in streptozotocin-induced diabetic mice. METHODS AND RESULTS: Male C57BL/6J mice were injected with streptozotocin to produce diabetic mice. After myocardial reperfusion injury, diabetic mice were randomized to treatment for 4 weeks with LCZ696 (60 mg/kg), valsartan (30 mg/kg), or no treatment (n = 26-28 in each group). Cardiac function was assessed by a pressure-volume Millar catheter. The ratios of heart weight to body weight in the valsartan (P = 0.02) and LCZ696 (P = 0.005) groups were significantly less than that in the control group. Treatment with LCZ696 improved LVEF (43 ± 3.4%) with a significantly reduction of atrial natriuretic peptide mRNA in the left ventricle compared with that in the control group (29 ± 3.2%) (P = 0.006). The fibrotic area in the LCZ696 group was significantly suppressed compared with those in the control (P = 0.003) and valsartan (P = 0.04) groups. Moreover, the mRNA level of transforming growth factor-ß (TGF-ß) in the left ventricle was suppressed in the LCZ696 group compared with that in the control (P = 0.002) group. CONCLUSION: The ARNi LCZ696 improved cardiac function with the reduction of fibrosis in an HF-rEF model in diabetic mice, by suppressing TGF-ß. This effect may be due to the specific inhibition of neprilysin, beyond the ARB effect of LCZ696.

A novel inducible cholesterol efflux peptide, FAMP, protects against myocardial ischemia reperfusion injury through a nitric oxide pathway.

OBJECTIVE: We evaluated whether a novel inducible cholesterol efflux (iCE) peptide [Fukuoka University Apolipoprotein A-I Mimetic Peptide (FAMP)] protects against myocardial ischemia reperfusion injury (IRI) through a nitric oxide (NO) pathway by an improvement of high-density lipoprotein (HDL) functionality. METHODS AND RESULTS: Male C57BL6/J mice were intraperitoneally injected with phosphate buffer as a control, low-dose (10 mg/kg) or high-dose (50 mg/kg) of FAMP before 18 h of IRI (n=6-12 in each group). After 30 min of ischemia followed by 6h of reperfusion, blood pressure, and infarct size were measured and cardiac function was evaluated by a Millar catheter. FAMP significantly improved stroke volume, cardiac output, left ventricular ejection fraction, and infarct size. FAMP significantly preserved cytochrome C in the mitochondrial fraction and inhibited its release into the cytosolic fraction in the heart, but did not significantly reduce mRNA levels of monocyte chemoattractant protein-1 or interluekin-6. In a TdT-mediated dUTP nick end labeling (TUNEL) assay, FAMP significantly suppressed the appearance of TUNEL-positive nuclear. We also performed same experiments with endothelial nitric oxide synthase-knockout (eNOS-KO) mice and FAMP-induced improvements of cardiac function were not observed in eNOS-KO mice. CONCLUSIONS: FAMP activated HDL-functionality and improved cardiac function in a model of myocardial IRI. It may have anti-apoptotic effects by protecting mitochondria through a NO pathway as a pleiotropic effect.

Addition of a Nitric Oxide Donor to an Angiotensin II Type 1 Receptor Blocker May Cancel Its Blood Pressure-Lowering Effects.

While physiological levels of nitric oxide (NO) protect the endothelium and have vasodilatory effects, excessive NO has adverse effects on the cardiovascular system. Recently, new NO-releasing pharmacodynamic hybrids of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) have been developed.We analyzed whether olmesartan with NO-donor side chains (Olm-NO) was superior to olmesartan (Olm) for the control of blood pressure (BP). Although there was no significant difference in binding affinity to AT1 wild-type (WT) receptor between Olm and Olm-NO in a cell-based binding assay, the suppressive effect of Olm-NO on Ang II-induced inositol phosphate (IP) production was significantly weaker than that of Olm in AT1 WT receptor-expressing cells. While Olm had a strong inverse agonistic effect on IP production, Olm-NO did not. Next, we divided 18 C57BL mice into 3 groups: Ang II (infusion using an osmotic mini-pump) as a control group, Ang II (n = 6) + Olm, and Ang II (n = 6) + Olm-NO groups (n = 6). Olm-NO did not block Ang II-induced high BP after 10 days, whereas Olm significantly decreased BP. In addition, Olm, but not Olm-NO, significantly reduced the ratio of heart weight to body weight (HW/BW) with downregulation of the mRNA levels of atrial natriuretic peptide.An ARB with a NO-donor may cancel BP-lowering effects probably due to excessive NO and a weak blocking effect by Olm-NO toward AT1 receptor activation.

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide.

Bifunctional angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) that can block the activation of not only AT1 receptor, but also neprilysin, which metabolizes vasoactive peptides including atrial natriuretic peptide (ANP), are currently being developed. However, the usefulness of the inactivation of ANP in addition to the AT1 receptor with regard to aldosterone (Ald) synthesis is not yet clear. We evaluated the inhibitory effects of various ARBs combined with or without ANP on Ang II-induced adrenal Ald synthesis using a human adrenocortical cell line (NCI-H295R). Ang II increased Ald synthesis in a dose- and time-dependent manner. Ald synthesis induced by Ang II was completely blocked by azilsartan, but not PD123319 (AT2 receptor antagonist). CGP42112 AT2 receptor agonist did not affect Ald synthesis. While most ARBs block Ang II-induced Ald synthesis to different extents, azilsartan and olmesartan have similar blocking effects on Ald synthesis. The different effects of ARBs were particularly observed at 10(-7) and 10(-8 )M. ANP attenuated Ang II-induced Ald synthesis, and ANP-mediated attenuation of Ang II-induced Ald synthesis were blocked by inhibitors of G-protein signaling subtype 4 and protein kinase G. ANP (10(-8) and 10(-7 )M) without ARBs inhibited Ald synthesis, and the combination of ANP (10(-7 )M) and ARB (10(-8 )M) had an additive effect with respect to the inhibition of Ald synthesis. In conclusions, ARBs had differential effects on Ang II-induced Ald synthesis, and ANP may help to block Ald synthesis when the dose of ARB is not sufficient to block its secretion.

Impact of cigarette smoking cessation on high-density lipoprotein functionality.

BACKGROUND: Smoking cessation reduces the risk of cardiovascular disease (CVD) and improves clinical outcomes in public health. We studied the effect of smoking cessation on high-density lipoprotein (HDL) functionality. METHODS AND RESULTS: We randomly treated 32 smokers with varenicline or a transdermal nicotine patch as part of a 12-week smoking cessation program (The VN-SEESAW Study). The plasma lipid profiles, plasma and HDL malondialdehyde (MDA) levels, HDL subfractions as analyzed by capillary isotachophoresis, cholesterol efflux capacity, and antiinflammatory activity of HDL were measured before and after the anti-smoking intervention. After smoking cessation, HDL-C, apoA-I levels and HDL subfractions were not significantly different from the respective baseline values. However, cholesterol efflux capacity and the HDL inflammatory index (HII) were significantly improved after smoking cessation. The changes in both parameters (%∆ cholesterol efflux capacity and ∆HII) were also significantly improved in the successful smoking cessation group compared with the unsuccessful group. The changes in cholesterol efflux capacity and HII also correlated with those in end-expiratory CO concentration and MDA in HDL, respectively. CONCLUSIONS: Our findings indicate that smoking cessation leads to improved HDL functionality, increased cholesterol efflux capacity and decreased HII, without changing HDL-C or apoA-I levels or HDL subfractions. This may be one of the mechanisms by which smoking cessation improves the risk of CVD.

Ability of the new AT1 receptor blocker azilsartan to block angiotensin II-induced AT1 receptor activation after wash-out.

INTRODUCTION: The recently approved angiotensin II (Ang II) type 1 (AT1) receptor blocker (ARB) azilsartan strongly reduces blood pressure (BP) in patients with hypertension. We previously reported that azilsartan showed unique binding behavior to the AT1 receptor because of its 5-oxo-1,2,4-oxadiazole moiety. However, the ability of azilsartan to block Ang II-dependent AT1 receptor activation is not yet clear. MATERIALS AND METHODS: Azilsartan and a derivative of azilsartan (azilsartan-7H) that lacks a carboxyl group at the benzimidazole ring were used. Ang II-induced inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) activation were analyzed in a cell-based wash-out assay. RESULTS: Azilsartan, but not azilsartan-7H, completely blocked Ang II-induced IP production and ERK activation. Our previous report demonstrated that azilsartan mainly interacts with Tyr(113), Lys(199), and Gln(257) in the AT1 receptor. The interactions between azilsartan and Tyr(113) and Gln(257), but not Lys(199), were critical for blocking Ang II-induced IP production and ERK activation after wash-out. CONCLUSIONS: Although our findings regarding the molecule-specific effects of azilsartan are based on basic research, they may lead to an exciting insight into the mechanism of azilsartan.

Azelnidipine, Not Amlodipine, Induces Secretion of Vascular Endothelial Growth Factor From Smooth Muscle Cells and Promotes Endothelial Tube Formation.

BACKGROUND: We previously reported that the calcium channel blocker (CCB) nifedipine-induced secretion of vascular endothelial growth factor (VEGF) from human coronary smooth muscle cells (HCSMCs) promoted human coronary endothelial cell (HCEC) tube formation. Therefore, we analyzed whether other CCBs, azelnidipine and amlodipine, also induced the secretion of VEGF and promoted HCEC tube formation, and the underlying molecular mechanisms. METHODS: To evaluate the tube formation, HCECs were grown on Matrigel for 18 hours in the supernatants from HCSMCs that had been treated with different kinds of reagents. Concentrations of VEGF in cultured HCSMCs were determined by specific enzyme immunoassays. Nuclear extracts from HCSMCs were prepared, and nuclear factor-kappa B (NF-κB) activation was measured by EZ-DetectTM Transcription Factor Kits for NF-κB p50 or p65. RESULTS: Although azelnidipine dose-dependently stimulated the significant secretion of VEGF from HCSMCs and this stimulation was abolished by a protein kinase C inhibitor, amlodipine-induced secretion of VEGF was significantly lower than that induced by azelnidipine. The medium derived from azelnidipine (at up to 2 µM)-treated HCSMCs led to HCEC tube formation, whereas that obtained with amlodipine did not. Azelnidipine-induced tube formation was blocked by an inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 tyrosine kinase. Azelnidipine at up to 2 µM induced NF-κB activation. CONCLUSIONS: Azelnidipine, but not amlodipine, stimulated the secretion of VEGF from HCSMCs and induced HCEC tube formation. This secretion is mediated at least in part via the activation of NF-κB. Azelnidipine may have a novel beneficial effect in improving coronary microvascular blood flow in addition to its main effect of lowering blood pressure.

Reassessment of the unique mode of binding between angiotensin II type 1 receptor and their blockers.

While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr(113), Tyr(184), Lys(199), His(256) and Gln(257) using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr(113) in the AT1 receptor and the hydroxyl group of olmesartan, between Lys(199) and carboxyl or tetrazole groups, and between His(256) or Gln(257) and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys(199), His(256) and Gln(257) of AT1 receptor. Lys(199) in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys(199). The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr(113). On the other hand, the n-butyl group of irbesartan may bind to Tyr(113). In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding.

Unique binding behavior of the recently approved angiotensin II receptor blocker azilsartan compared with that of candesartan.

The angiotensin II type 1 (AT(1)) receptor blocker (ARB) candesartan strongly reduces blood pressure (BP) in patients with hypertension and has been shown to have cardioprotective effects. A new ARB, azilsartan, was recently approved and has been shown to provide a more potent 24-h sustained antihypertensive effect than candesartan. However, the molecular interactions of azilsartan with the AT(1) receptor that could explain its strong BP-lowering activity are not yet clear. To address this issue, we examined the binding affinities of ARBs for the AT(1) receptor and their inverse agonist activity toward the production of inositol phosphate (IP), and we constructed docking models for the interactions between ARBs and the receptor. Azilsartan, unlike candesartan, has a unique moiety, a 5-oxo-1,2,4-oxadiazole, in place of a tetrazole ring. Although the results regarding the binding affinities of azilsartan and candesartan demonstrated that these ARBs interact with the same sites in the AT(1) receptor (Tyr(113), Lys(199) and Gln(257)), the hydrogen bonding between the oxadiazole of azilsartan-Gln(257) is stronger than that between the tetrazole of candesartan-Gln(257), according to molecular docking models. An examination of the inhibition of IP production by ARBs using constitutively active mutant receptors indicated that inverse agonist activity required azilsartan-Gln(257) interaction and that azilsartan had a stronger interaction with Gln(257) than candesartan. Thus, we speculate that azilsartan has a unique binding behavior to the AT(1) receptor due to its 5-oxo-1,2,4-oxadiazole moiety and induces stronger inverse agonism. This property of azilsartan may underlie its previously demonstrated superior BP-lowering efficacy compared with candesartan and other ARBs.

Small molecules with similar structures exhibit agonist, neutral antagonist or inverse agonist activity toward angiotensin II type 1 receptor.

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT(1) receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).

Abilities of candesartan and other AT(1) receptor blockers to impair angiotensin II-induced AT(1) receptor activation after wash-out.

Angiotensin II (Ang II) binds to Ang II type 1 (AT(1)) receptor and evokes cell signaling, and subsequently stimulates vasoconstriction and cell proliferation, which eventually lead to cardiovascular disease. Since most AT(1) receptor blockers (ARBs) have molecular (differential) effects, we evaluated the specific features of candesartan and compared the abilities of candesartan and other ARBs (olmesartan, telmisartan, valsartan, irbesartan and losartan) to bind to and activate AT(1) receptors using a cell-based wash-out assay. Each ARB blocked Ang II-induced extracellular signal-regulated kinase (ERK) activation and inositol phosphate production to different degrees after wash-out. In addition, a small difference in the molecular structure, i.e. a carboxyl group, between candesartan and candesartan-7H was associated with a difference in the degree of this blocking effect. In addition, interaction between Gln(257) in the AT(1) receptor and the carboxyl group of candesartan may be partially associated with the effect of candesartan after wash-out. Although our findings regarding the molecular effects of ARB are based on basic research, these findings may lead to an exciting new area in the clinical application of ARBs.

A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects.

Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) induce multiple pharmacological beneficial effects, but not all ARBs have the same effects and the molecular mechanisms underlying their actions are not certain. In this study, irbesartan and losartan were examined because of their different molecular structures (irbesartan has a cyclopentyl group whereas losartan has a chloride group). We analyzed the binding affinity and production of inositol phosphate (IP), monocyte chemoattractant protein-1 (MCP-1) and adiponectin. Compared with losartan, irbesartan showed a significantly higher binding affinity and slower dissociation rate from the AT1 receptor and a significantly higher degree of inverse agonism and insurmountability toward IP production. These effects of irbesartan were not seen with the AT1-Y113A mutant receptor. On the basis of the molecular modeling of the ARBs-AT1 receptor complex and a mutagenesis study, the phenyl group at Tyr(113) in the AT1 receptor and the cyclopentyl group of irbesartan may form a hydrophobic interaction that is stronger than the losartan-AT1 receptor interaction. Interestingly, irbesartan inhibited MCP-1 production more strongly than losartan. This effect was mediated by the inhibition of nuclear factor-kappa B activation that was independent of the AT1 receptor in the human coronary endothelial cells. In addition, irbesartan, but not losartan, induced significant adiponectin production that was mediated by peroxisome proliferator-activated receptor-γ activation in 3T3-L1 adipocytes, and this effect was not mediated by the AT1 receptor. In conclusion, irbesartan induced greater beneficial effects than losartan due to small differences between their molecular structures, and these differential effects were both dependent on and independent of the AT1 receptor.