6. Fujino,et al Hypertension Research 2010;331044-1052

ORIGINAL ARTICLE A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects Masahiro Fujino1, Shin-ichiro Miura1,2, Yoshihiro Kiya1, Yukio Tominaga3, Yoshino Matsuo1, Sadashiva S Karnik2 and Keijiro Saku1 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-c 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. Hypertension Research (2010) 33, 1044–1052; doi:10.1038/hr.2010.135; published online 29 July 2010 Keywords: angiotensin II type 1 receptor blocker; molecular structure; inverse agonism; insurmountability; monocyte chemoattractant protein-1 INTRODUCTION Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are highly selective for the (AT1 receptor, which is a member of the G protein- coupled receptor superfamily, and these agents block the diverse effects of Ang II. In addition to their blood pressure-lowering effects, ARBs provide cardiovascular and renal protection.1 Many ARBs are available for clinical use, but recent clinical studies have shown that not all ARBs have the same effects;2 therefore some of the benefits conferred by ARBs may not be class effects (common effect).3–5 This notion of drug-specific effects is referred to as a ‘molecular effect (off-target or drug effect)’. Most ARBs have a common chemical structure that includes a biphenyl- tetrazole group and an imidazole group. We previously reported that olmesartan has this common chemical structure, as well as a hydroxyl and a carboxyl group, and shows strong inverse agonism.6 The interactions between the AT1 receptor and the hydroxyl and carboxyl groups of olmesartan have an important role in inverse agonism. We hypothesized that small differences in the molecular structures among ARBs could lead to different degrees of inverse agonism. Small differences in the molecular structure of a ligand for a G protein- coupled receptor can lead to different pharmacological effects;7,8 however, the molecular mechanisms of such receptor-dependent and -independent beneficial effects are not well understood. Irbesartan inhibited basal production, as well as low-density lipo- protein- and platelet-activating factor-stimulated the monocyte chemoattractant protein-1 (MCP-1) production in isolated human monocytes, independent of Ang II stimulation.9 In addition, irbesar- tan has been identified as a ligand of peroxisome proliferator-activated receptor (PPAR)-g,10 and irbesartan-induced adiponectin upregulation Received 16 March 2010; revised 7 April 2010; accepted 19 May 2010; published online 29 July 2010 1Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan; 2Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, OH, USA and 3Genomic Science Laboratories, Dainippon Sumitomo Pharma Company Limited, Osaka, Japan Correspondence: Dr S Miura, Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail: miuras@cis.fukuoka-u.ac.jp Hypertension Research (2010) 33, 1044–1052 & 2010 The Japanese Society of Hypertension All rights reserved 0916-9636/10 $32.00 www.nature.com/hr was observed in the absence of Ang II.11 Thus, irbesartan may have beneficial effects independent of AT1 receptor-mediated signaling. Because irbesartan was derived from losartan, both ARBs have common chemical structures (biphenyl-tetrazole and imidazole groups). However, irbesartan has a cyclopentyl group instead of the chloride group found in losartan. We speculated that this small difference between the molecular structures of these ARBs could induce both AT1 receptor-dependent and -independent effects. To explore this hypothesis, we systematically examined the binding affinity to and dissociation from the AT1 receptor, as well as the inverse agonism and insurmountability toward inositol phosphate (IP) production as AT1 receptor dependent-effects and determined the unique binding behavior of irbesartan to the AT1 receptor. In addition, we analyzed whether irbesartan inhibited MCP-1 production and adiponectin secretion from cells independent of the AT1 receptor, and whether these effects were directly mediated by nuclear factor- kappa B (NF-kB) and PPAR-g. These experiments address the molecular mechanisms that may underlie the multiple pharmacologically beneficial effects induced by the small differences in the molecular structures of ARBs for the AT1 receptor. METHODS Materials The following antibodies and reagents were purchased: ARBs, irbesartan and losartan (Toronto Research Chemicals, Ontario, Canada); Ang II (Sigma- Aldrich, St Louis, MO, USA); 125I-[Sar1, Ile8]Ang II (Amersham Biosciences, Buckinghamshire, UK); hygromycin and doxycycline (Clontech Laboratories, Mountain View, CA, USA) and geneticin (G418, MP Biomedics, Solon, OH, USA). The molecular structures of the ARBs are shown in Figure 1a. Mutagenesis and expression of the AT1 receptor and membrane preparation The synthetic wild-type (WT) AT1 receptor gene, cloned in the shuttle expression vector pMT-3, was used for expression and mutagenesis (Table 1), as described previously.12 Cell cultures, transfections and membrane preparation COS1 cells, human coronary endothelial cells (HCECs) and mouse 3T3-L1 proadipocytes were cultured. COS1 cells were maintained in 10% fetal bovine serum and penicillin- and streptomycin-supplemented Dulbecco’s modified Eagle’s essential medium (Invitrogen, Carlsbad, CA, USA) in 5% CO2 at 37 1C. The HCECs were grown in media. In these experiments, cells supplemented without cell-growth supplement were used. Cell viability was495% by trypan blue exclusion analysisin control experiments. WT and mutant AT1 receptors were transiently transfected into COS1 cells using Lipofectamine 2000 liposo- mal reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. Cell membranes were prepared by the nitrogen Parr bomb disruption method in the presence of protease inhibitors. In addition, mouse 3T3-L1 proadipocytes were cultured and differentiated as previously described13 using a standard differentiation mixture (dexametha- sone, 3-isobutyl-methylxanthine, insulin and 10% fetal bovine serum). Tetracycline-inducible system using HEK293 cells expressing the WT AT1 receptor A tetracycline-inducible (Tet-ON) gene expression system was used in HEK293 cells stably transfected with the WT AT1 receptor (Clontech Laboratories). Briefly, stably transformed HEK293 cells were transfected with the neomycin- resistant pTet-ON regulator plasmid encoding the reverse tetracycline-con- trolled transactivator (rtTA) protein. These stably transformed cells were grown in a medium containing 100mgml�1 G418. The Tet-ON inducible HEK293 cells were used for the transfection of WT AT1 receptor-TRE-2-hyg plasmids with Lipofectamine 2000, and selected with 150mgml�1 hygromycin. The transfected cells with TRE-2-hyg and WT AT1 receptor-TRE-2-hyg plasmids were maintained in aa medium with 100mgml�1 G418 and 100mgml�1 hygromycin. Dose- and time-dependent experiments on stably transfected Tet-ON cells showed a maximal induction of the WT AT1 receptor at 400mgml�1 doxycycline after 4 days in culture. Experiments used a pooled population of cells with the WT AT1 receptor induced by 0, 100 and 400mgml�1 doxycycline for 4 days. Competition binding study The bindinf affinity (Kd) and maximal binding capacity (Bmax) values for receptor binding were determined by 125I-[Sar1, Ile8]Ang II-binding experi- ments under equilibrium conditions as previously described.12 0.01 μM 0.01 μM 0.1 μM 0.1 μM 1 μM 1 μM 1 μM ARB(-) losartan WT(%) Cyclopentyl group N OH3C Irbesartan Imidazol group Y113A N N Tetrazol group Biphenyl group Irbesartan WT N N NH Losartan Chloride group IP p ro du ct io n (% of m ax im al) N N Cl OH H3C 100 60 80 40 20 0 N N N NH Ang II [log (M)] -10-11 -5-6-7-8-9 Figure 1 (a) Molecular structures of irbesartan and losartan. (b) The effect of preincubation with either irbesartan or with losartan on Ang II-mediated inositol phosphate (IP) production in COS cells with transiently transfected wild-type (WT) and Y113A AT1 receptors. Cells were preincubated with or without the indicated concentrations of irbesartan or losartan for 30min at 37 1C, and then further incubated for 5 min with increasing concentrations of Ang II. The percentage of maximal IP production in control cells without angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) (ARB(�)) with WT and Y113A AT1 receptors was adjusted to 100% (5934±411 c.p.m. and 3502±263 c.p.m., respectively). Differences in beneficial effects of ARBs M Fujino et al 1045 Hypertension Research IP production study Total soluble IP was measured by the perchloric acid extraction method, which was described previously.12 Dissociation study by washing-out Prepared cell membranes expressing the WT and mutant AT1 receptors were incubated for 30min at 22 1C with or without the indicated concentrations of ARBs. After the membranes were washed-out 1–3 times with excess cold phosphate-buffered saline, they were centrifuged for 10min at 16 000 g at 4 1C. The membrane fractions were used in the assay for the specific binding of 125I-[Sar1, Ile8]Ang II. The percentage of ARB dissociated from the AT1 receptor was calculated by the following formula: 100�((specific binding using cell membrane without ARB treatment with no wash-out)�(specific binding using cell membrane with ARB treatment at the indicated wash-out times)/(specific binding using cell membrane without ARB treatment with no wash-out)�(specific binding using cell membrane with ARB treatment with no wash-out))�100 (%). Molecular modeling of AT1 receptor-ARBs A binding model of irbesartan or losartan with the AT1 receptor was constructed. InsightII software (Accelrys, San Diego, CA, USA) was used to construct a homology model of the human AT1 receptor. The structure of bovine rhodopsin (Protein Data Bank code 1U19)14 was used as a template for modeling the AT1 receptor. The primary sequences of the AT1 receptor and bovine rhodopsin were aligned in a manner consistent with a previous report.15 Based on this alignment, the AT1 receptor model was constructed and then subjected to a simulated annealing protocol by means of the Modeller program.16 We selected important amino-acid residues of the AT1 receptor to bind to irbesartan by site-directed mutagenesis studies. Although keeping the results of the mutagenesis study in mind, we manually docked irbesartan in the AT1-receptor model, and the ligand-receptor model was then energy-mini- mized using an OPLS_2005 force field. The model was further refined according to the Induced Fit Docking Procedure based on Glide 4.5 and Prime 1.6, as implemented in the Schro¨dinger software package (Schro¨dinger, LLC, Portland, OR, USA). A binding model of losartan with the AT1 receptor was also constructed by the Induced Fit Docking procedure, but in this case, the structure of the AT1 receptor was obtained from the refined irbesartan-bound AT1 receptor model. Measurement of MCP-1 production and NF-jB activation The HCECs were grown under serum-free conditions for 24 h with or without the indicated concentrations of ARBs. MCP-1 secretion in the medium from HCECs was measured by an ELISA kit (R&D Systems, Minneapolis, MN, USA). In addition, nuclear extracts from HCECs were prepared and NF-kB activation was measured by EZ-DetectTM Transcription Factor Kits for NF-kB p50 or p65 (Pierce, Rockford, IL, USA). Receptor cofactor assay system for PPAR-c A receptor cofactor assay using the indicated concentrations of ARBs was carried out using EnBio receptor cofactor assay system for PPAR-g (EnBioTec Laboratories, Tokyo, Japan). PPAR-c DNA-binding activity PPAR-g DNA-binding activities were examined with the PPAR-g transcription factor assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) using nuclear extracts from 3T3L1 adipocytes after 11 days of differentiation with and without the indicated concentrations of ARBs. Statistical analysis Results are expressed as the mean±s.d. of three or more independent trials. Significant differences in measured values were evaluated with an analysis of variance using Fisher’s t-test and paired or unpaired Student’s t-test, as appropriate. Statistical significance was set at o0.05. RESULTS Binding of irbesartan and losartan to WTand mutant AT1 receptors The Kd of irbesartan was significantly lower than that of losartan for WT AT1 receptors (Table 1). Next, we selected candidate residues (Val108, Ser109, Leu112, Tyr113, Tyr184, Lys199, Asn200, Phe204, His256, Gln257 and Met284 in the AT1 receptor) for specific binding sites of irbesartan and losartan, based on the molecular model of the AT1 receptor complex described by previous reports.6,17–19 To determine the specific amino acids that bind to these two ARBs, we examined the binding affinities of ARBs to AT1 receptors mutated at the candidate amino acids mentioned above. The expression levels of the WT and mutated AT1 receptors were within the same order of magnitude. The affinities of [Sar1, Ile8]Ang II were almost the same in some of the mutants and decreased in other mutants, but they were not less than 1/10 the affinity for the WT AT1 receptor, except for F204A. F204A was not used in further analyses because the mutation itself affected the conformation of the AT1 receptor. The affinity of irbesartan was reduced by more than 10-fold in V108A and Y113A Table 1 Maximal binding capacities (Bmax) and binding affinities (Kd) of [Sar1,Ile8]Ang II, irbesartan and losartan to AT1 wild-type (WT) and mutants receptors Kd (nM) Receptor Bmax (pmolmg �1 protein) [Sar1,Ile8]Ang II Irbesartan Losartan WT 0.62±0.07 0.8±0.3 (1.0) 1.9±1.1 (1.0) 11±3 (1.0) V108A 0.60±0.11 0.7±0.4 (0.9) 19±5 (10) 416±149 (38) S109A 0.51±0.03 0.9±0.6 (1.1) 2.1±0.6 (1.1) 33±13 (3.0) L112A 0.57±0.04 0.8±0.3 (1.0) 10±1 (5.3) 593±88 (54) Y113F 0.71±0.12 1.8±1.0 (2.3) 4.6±0.7 (2.4) 179±18 (10) Y113A 0.30±0.02 0.8±0.2 (1.0) 49±5 (26) 1455±431 (132) Y184F 0.65±0.04 0.9±0.4 (1.1) 3.0±1.3 (1.6) 40±17 (3.6) Y184A 0.56±0.03 0.9±0.3 (1.1) 2.1±1.1 (1.1) 21±3 (1.9) K199Q 0.25±0.04 2.0±0.9 (2.5) 2.0±1.0 (1.1) 21±9 (1.9) N200A 0.29±0.02 1.8±1.2 (2.3) 1.8±0.2 (0.9) 26±13 (2.4) F204A 0.15±0.02 24±9 (30) — — H256A 0.29±0.09 0.8±0.4 (1.0) 2.4±1.4 (1.3) 41±17 (3.7) Q257A 0.15±0.07 5.0±2.1 (6.3) 3.2±0.7 (1.7) 148±29 (13) M284G 0.43±0.01 0.6±0.1 (0.8) 0.7±0.1 (0.4) 7.8±2.3 (0.7) M284A 0.41±0.03 1.3±0.7 (1.6) 0.5±0.1 (0.3) 9.0±0.9 (0.8) Differences in beneficial effects of ARBs M Fujino et al 1046 Hypertension Research receptors and fivefold in L112A receptor compared with the WT AT1 receptor. These results suggest that Val108, Leu112 and Tyr113 in the AT1 receptor are involved in binding to irbesartan. However, losartan may bind to Val108 Lue112, Tyr113 and Gln257 because the affinity of losartan was reduced by more than 10-fold in V108A, L112A, Y113A and Q257A receptors compared with the WT AT1 receptor. Irbesartan, which has a chemical structure similar to that of losartan and a cyclopentyl group, did not show a reduction in binding affinity to the Y113F (only a 2.4-fold reduction) mutant compared with the WTAT1 receptor. Losartan, which has a chloride group instead of the cyclo- pentyl group found in irbesartan, showed a greater than 10-fold reduction in affinity for the Y113F mutant receptor. Although irbesartan showed a significant loss (26-fold reduction) in binding affinity for the Y113A receptor, losartan showed an even greater loss in binding affinity for the Y113A receptor (132-fold reduction). These results indicate that Tyr113 in the AT1 receptor is a key residue mediating the differences in the binding behavior between irbesartan and losartan. Insurmountabilily of irbesartan and losartan in WTand Y113A AT1 receptors The insurmountability of irbesartan and losartan in WT and Y113A AT1 receptors were tested, and these results are shown in Figure 1b. Preincubation of cells expressing WT AT1 receptor for 30min with irbesartan (0.01, 0.1 and 1mM) decreased the maximal response to subsequently added Ang II. The maximal response with 1mM losartan was significantly higher than that with the lowest concentration of irbesartan tested (0.01mM). In addition, a marked rightward shift of the Ang II concentration–response curve was observed with an increasing irbesartan concentration (0.01, 0.1 and 1mM), whereas a rightward shift was observed with 1mM losartan. Interestingly, the marked rightward shift and significant decrease in the maximal response with 1mM irbesartan in the WT AT1 receptor were not observed with 1mM irbesartan in the Y113A AT1 receptor. Thus, irbesartan had a higher degree of insurmountability for the AT1 receptor than losartan. The strong insurmountability with irbe- sartan was not observed in the Y113A AT1 receptor, indicating that Tyr113 is important for the strong irbesartan-induced insur- mountability. Inverse agonism of irbesartan and losartan in WT and mutant AT1 receptors The inverse agonist activities of irbesartan and losartan in the WTand mutant AT1 receptor were tested, and the results are shown in Figure 2. We previously reported that the mutant AT1 receptor (N111G) had high basal activity in the absence of Ang II and may have mimicked the pre-activated state of the WT AT1 receptor. 20,21 Only irbesartan significantly suppressed the basal IP production in WT and N111G AT1 receptors, in a dose-dependent manner. Interestingly, the inverse agonism observed with 1mM irbesartan was lost with the Y113A and N111G/Y113A AT1 receptors, thereby indicating that Tyr 113 was also important for the inverse agonism of irbesartan. Dissociation of irbesartan and losartan from WT and mutant AT1 receptors The degree of dissociation of irbesartan and losartan from the WT and mutant AT1 receptors was tested, and the results are shown in Figure 3. Irbesartan (0.1–1mM) showed a lower dissociation than losartan after the first wash-out, whereas a high concentrat