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• W2155777324 abstract "To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity. To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity. Angiotensin II (Ang II) 3The abbreviations used are: Ang II, angiotensin II; ARB, Ang II receptor antagonist; AT1, Ang II type 1; GPCR, G protein-coupled receptor; IP, inositol phosphate; TM, transmembrane; WT, wild-type. 3The abbreviations used are: Ang II, angiotensin II; ARB, Ang II receptor antagonist; AT1, Ang II type 1; GPCR, G protein-coupled receptor; IP, inositol phosphate; TM, transmembrane; WT, wild-type. receptor antagonists (ARBs) are highly selective for the Ang II type 1 (AT1) receptor, which is a member of the G protein-coupled receptor (GPCR) superfamily, and block the diverse effects of Ang II. In addition to their blood pressure-lowering effects in hypertensive patients, ARBs have been shown to promote regression of left ventricular hypertrophy and decrease cardiovascular morbidity and mortality in patients with heart failure or hypertensive diabetic nephropathy with proteinuria (1De Gasparo M. Catt J. Inagami T. Wright J.W. Unger T. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). Many ARBs are available for clinical use. Because not all ARBs have the same effects, some benefits conferred by ARBs may not be class effects (2Miura S. Fujino M. Saku K. Curr. Hypertens. Rev. 2005; 1: 115-121Crossref Google Scholar). This notion represents an exciting new area in ARB-based therapy, which holds the promise of reducing the incidence of cardiovascular disease. Inverse agonists, such as the opioid receptor ligand ICI174864 (3Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (475) Google Scholar), block agonist-independent signal transduction by GPCRs. Many clinically important medications have been shown to behave as inverse agonists when tested against either wild-type (WT) or mutated GPCRs; e.g. olanzapine in the 5-hydroxytryptamine 2C receptors (4Herrick-Davis K. Grinde E. Teitler M. J. Pharmacol. Exp. Ther. 2000; 295: 226-232PubMed Google Scholar) and metoprolol in the β-adrenoreceptor (5Engelhardt S. Grimmer Y. Fan G.H. Lohse M.J. Mol. Pharmacol. 2001; 60: 712-717PubMed Google Scholar). Spontaneous receptor mutations leading to constitutive activity have been implicated in some human diseases (6Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar, 7Parma J. Duprez L. Van Sande J. Cochaux P. Gervy C. Mockel J. Dumont J. Vassart G. Nature. 1993; 365: 649-651Crossref PubMed Scopus (842) Google Scholar). However, such spontaneous mutations have not been reported for the AT1 receptor, and the WT AT1 receptor shows slight constitutive activity (2Miura S. Fujino M. Saku K. Curr. Hypertens. Rev. 2005; 1: 115-121Crossref Google Scholar). A recent study demonstrates that the WT AT1receptor is activated by mechanical stretching of cultured rat myocytes without the involvement of Ang II, and this was suppressed by an inverse agonist (8Zou Y. Akazawa H. Qin Y. Sano M. Takano H. Minamino T. Makita N. Iwanaga K. Zhu W. Kudoh S. Toko H. Tamura K. Kihara M. Nagai T. Fukamizu A. Umemura S. Iiri T. Fujita T. Komuro I. Nat. Cell Biol. 2004; 6: 499-506Crossref PubMed Scopus (516) Google Scholar). The same study also demonstrates that cardiac hypertrophy induced by constricting the transverse aorta in angiotensinogen knock-out mice was attenuated by an inverse agonist, suggesting that the WT AT1 receptor is activated independent of Ang II by mechanical stimulation of the heart in vivo. In this way, an inverse agonist for the AT1 receptor may have pharmacotherapeutic relevance for the cardiovascular system. We have previously reported that EXP3174, an active metabolite of losartan, but not losartan itself, can act as an inverse agonist in the inositol phosphate (IP) production assay using a constitutively active AT1-N111G mutant (9Miura S. Saku K. Karnik S.S. Hypertens. Res. 2003; 26: 937-943Crossref PubMed Scopus (94) Google Scholar). Most ARBs, including EXP3174 and losartan, have biphenyltetrazole and imidazole groups as common chemical moieties (see Fig. 1). In addition, EXP3174, but not losartan, has a carboxyl group in the imidazole ring. This difference in the chemical structure may be important for inverse agonist activity. Olmesartan, a potent ARB, has a hydroxyl group in the imidazole ring in addition to the carboxyl group and exhibits potent inverse agonist activity. We speculated that the hydroxyl group, together with the carboxyl group, may play an important role in inverse agonist activity. To explore this hypothesis, we systematically mutated the AT1 receptor and examined the interactions between the AT1 receptor and the hydroxyl and carboxyl groups of olmesartan. We also analyzed whether these interactions could be important for inducing inverse agonism. Based on the results of these experiments, molecular modeling was performed to determine the specific sites for the interaction between olmesartan and the AT1 receptor. This report will address the molecular mechanism underlying the inverse agonist activity of ARB. Materials—Olmesartan and its related compounds, R-89059, R-90929, R-239470, and R-239471, were synthesized in the Research Laboratories of Sankyo Co., Ltd. The chemical structures of these compounds are shown in Fig. 1. 125I-[Sar1,Ile8]Ang II was purchased from Amersham Biosciences. Cell Cultures and Transfections—COS1 cells were maintained in 10% fetal bovine serum and penicillin- and streptomycin-supplemented Dulbecco's modified Eagle's essential medium (Invitrogen) in 5% CO2 at 37 °C. Cell viability was >95% by trypan blue exclusion analysis in the control experiments. The WT and mutant AT1 receptors were transfected into COS1 cells using Lipofectamine 2000 liposomal reagent (Roche Applied Science) according to the manufacturer's instructions. Mutagenesis and Expression of the AT1 Receptor and Membrane Preparation—The synthetic rat AT1 receptor gene, cloned in the shuttle expression vector pMT-2, was used for expression and mutagenesis (Fig. 2), as described previously (10Miura S. Feng Y.H. Husain A. Karnik S.S. J. Biol. Chem. 1999; 274: 7103-7110Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To express the AT1 receptor protein, 10 μg of purified plasmid DNA/107 cells was used in the transfection. COS1 cells (American Type Culture Collection, Manassas, VA) were grown in 5% CO2 at 37 °C. Transfected cells that had been cultured for 72 h were harvested, and cell membranes were prepared by the nitrogen Parr bomb disruption method in the presence of protease inhibitors. Competition Binding Studies—The Kd and Bmax values of the receptor binding were determined by 125I-[Sar1,Ile8]Ang II-binding experiments under equilibrium conditions, as previously described (10Miura S. Feng Y.H. Husain A. Karnik S.S. J. Biol. Chem. 1999; 274: 7103-7110Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Membranes expressing the WT or mutant AT1 receptor were incubated with 100 pm 125I-[Sar1,Ile8]Ang II for 1 h at 22 °C in a volume of 125 μl. Binding was stopped by filtering the incubation mixture through Whatman GF/C glass fiber filters, and the residues were extensively washed further with binding buffer. The bound ligand fraction was determined from the counts/min remaining on the membrane. Binding kinetics values were determined as previously described (10Miura S. Feng Y.H. Husain A. Karnik S.S. J. Biol. Chem. 1999; 274: 7103-7110Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). IP Formation Studies—Total soluble IP was measured by the perchloric acid extraction method, as described previously (10Miura S. Feng Y.H. Husain A. Karnik S.S. J. Biol. Chem. 1999; 274: 7103-7110Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Molecular Modeling of the AT1 Receptor—The structure of bovine rhodopsin (Protein Data Bank code 1F88) was used as a template for the modeling of the AT1 receptor (11Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yomamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4971) Google Scholar). We aligned the primary sequence of the human AT1 receptor with that of bovine rhodopsin. The amino acid mutations were performed with the program Modeler (Accelrys) followed by energy minimization using CHARMm (Chemistry at Harvard Macromolecular Mechanics) (Accelrys). Throughout the entire modeling process, the three-dimensional structure was monitored to ensure that the AT1 secondary structure was conserved. This model was used for docking olmesartan. First, manual docking was performed so that three interactions deduced by mutation experiments were satisfied. These interactions were between Tyr113 and the hydroxyl group of olmesartan, between Lys199 and the carboxyl group, and between Gln257 and the tetrazole group. The conformation of olmesartan was derived from the x-ray-determined structure of olmesartan and adjusted manually to satisfy the interactions described above. Energy minimization was then performed with a flexible olmesartan structure using a CHARMm force field. The three interactions mentioned above were constrained in this process. Statistical Analysis—The results are expressed as the mean ± S.D. of four or more independent determinations. Significant differences in measured values were evaluated with an analysis of variance using Fisher's t test and unpaired Student's t test. Statistical significance was set at <0.05. Binding of Olmesartan and Its Related Compounds to WT and Mutant AT1 Receptors—First, we selected the candidate residues (Ser105, Val108, Ser109, Tyr113, His166, His183, Lys199, His256, Gln257, Thr260, Met284, Thr287, Tyr292, and Asn295 in the AT1 receptor) for specific binding sites of olmesartan based on the molecular model of the AT1 receptor complex described in previous reports (12Noda K. Saad Y. Kinoshita A. Boyle T.P. Graham R.M. Husain A. Karnik S.S. J. Biol. Chem. 1995; 270: 2284-2289Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 13Takezako T. Gogonea C. Saad Y. Noda K. Karnik S.S. J. Biol. Chem. 2004; 279: 15248-15257Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (Fig. 2). To determine the specific amino acids that bind to olmesartan, we examined the binding affinity of olmesartan and its related compounds (the chemical structures of which are shown in Fig. 1) to AT1 receptors mutated at the candidate amino acids mentioned above. The results are shown in Table 1. 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 mutants and were decreased in other mutants but not to <0.1 the affinity to the WT AT1 receptor.TABLE 1Maximal binding capacities (Bmax) and binding affinities (Kd) of [Sar1,Ile8]Ang II, olmesartan, and its related compounds to AT1 wild type and mutant receptors Numbers in parentheses show ratio of Kd (mutant)/Kd (wild type).ReceptorBmaxKd[Sar1, Ile8]Ang IIOlmesartanR-90929R-239470R-239471pmol/mg proteinnmWild type0.52 ± 0.040.7 ± 0.2 (1.0)2.2 ± 0.7 (1.0)24 ± 2 (1.0)0.8 ± 0.3 (1.0)61 ± 21 (1.0)S105A0.46 ± 0.061.7 ± 0.6 (2.4)8.4 ± 3.3 (3.8)66 ± 15 (2.8)4.5 ± 1.0 (5.6)177 ± 13 (2.9)V108G0.64 ± 0.050.5 ± 0.2 (0.7)15 ± 3 (6.8)52 ± 9 (2.2)7.7 ± 1.4 (9.6)817 ± 87 (13)V108A0.68 ± 0.150.7 ± 0.4 (1.0)4.7 ± 1.3 (2.1)25 ± 4 (1.0)1.1 ± 0.1 (1.4)56 ± 6 (0.9)S109A0.57 ± 0.081.4 ± 0.1 (2.0)7.6 ± 1.6 (3.5)25 ± 5 (1.0)1.7 ± 0.4 (2.1)90 ± 6 (1.5)Y113A0.39 ± 0.010.7 ± 0.2 (1.0)9.3 ± 2.7 (4.2)Y113F0.68 ± 0.132.6 ± 0.6 (3.7)52 ± 7 (24)103 ± 17 (4.3)17 ± 3 (21)3110 ± 311 (51)H166E0.28 ± 0.073.2 ± 0.3 (4.6)19 ± 4 (8.6)160 ± 12 (6.7)7.1 ± 1.4 (8.9)1813 ± 206 (30)H183E0.82 ± 0.091.2 ± 0.3 (1.7)11 ± 1 (5.0)52 ± 5 (2.2)2.5 ± 0.6 (3.1)172 ± 15 (2.8)K199Q0.23 ± 0.032.5 ± 1.3 (3.6)33 ± 6 (15)362 ± 43 (15)1.8 ± 0.3 (2.3)2655 ± 295 (44)H256A0.24 ± 0.060.6 ± 0.2 (0.9)36 ± 4 (16)34 ± 6 (1.4)1.1 ± 0.2 (1.4)242 ± 46 (4.0)Q257A0.08 ± 0.165.2 ± 2.0 (7.4)226 ± 4 (103)468 ± 39 (20)20 ± 2 (25)>105 (>1639)T260A0.72 ± 0.083.9 ± 1.9 (5.6)26 ± 3 (12)60 ± 13 (2.5)5.7 ± 1.3 (7.1)161 ± 12 (2.6)M284A0.47 ± 0.021.0 ± 0.3 (1.4)2.8 ± 0.6 (1.3)T287A0.43 ± 0.061.3 ± 0.2 (1.9)25 ± 4 (11)131 ± 25 (5.4)16 ± 6 (20)2400 ± 364 (39)Y292A0.73 ± 0.032.9 ± 1.8 (4.1)47 ± 15 (21)87 ± 18 (3.6)6.3 ± 3.8 (7.9)1585 ± 581 (26)Y292F0.79 ± 0.021.1 ± 0.3 (1.6)25 ± 4 (11)93 ± 22 (3.9)4.0 ± 0.8 (5.0)890 ± 345 (15)N295I0.46 ± 0.012.0 ± 0.5 (2.9)25 ± 3 (11)95 ± 5 (4.0)3.8 ± 0.5 (4.8)919 ± 140 (15) Open table in a new tab The affinity of olmesartan was reduced by >10-fold in Y113F, K199Q, H256A, Q257A, T260A, T287A, Y292A, Y292F, and N295I mutants compared with the WT AT1 receptor, suggesting that Tyr113, Lys199, His256, Gln257, Thr260, Thr287, Tyr292, and Asn295 in the AT1 receptor are involved in the binding to olmesartan. R-90929, which has a chemical structure similar to that of olmesartan but has no hydroxyl group, exhibited a >10-fold reduction in binding affinity to the K199Q (15-fold reduction) and Q257A (20-fold reduction) mutants compared with the WT AT1 receptor. R-239470, which has a non-acidic carbamoyl group instead of the carboxyl group in olmesartan, also exhibited a >10-fold reduction in affinity in the Y113F (21-fold reduction), Q257A (25-fold reduction), and T287A (20-fold reduction) mutants. R-239471, which has methoxymethyl and carboxyl groups instead of the hydroxyisopropyl and tetrazole groups in olmesartan, respectively, exhibited a total loss of affinity to the Q257A mutant and a >10-fold reduction of binding affinity to the Y113F, H166E, K199Q, T287A, Y292A, Y292F, and N295I mutants. These results suggest that Tyr113, Lys199, and Gln257 are essential for binding between olmesartan and the AT1 receptor. Olmesartan and R-239470, which have a hydroxyl group in the imidazole group, showed >10-fold decreases in binding affinity in the Y113F mutant, whereas R-90929, which does not have a hydroxyl group, showed only a 4.3-fold reduction in binding affinity, indicating that the hydroxyl group probably binds to Tyr113 of the AT1 receptor. Also, the binding affinity of R-239471 in the Y113F mutant was reduced to 1/51 that in the wild-type receptor, probably because of the lack of hydrogen bonding between the methoxymethyl group of R-239471 and the hydroxyl group of Tyr113 in the Y113F mutant. As for Lys199, olmesartan, R-90929, and R-239471, which have a carboxyl group in the imidazole group, showed a significant decrease in binding affinity (1/15, 1/15, and 1/40, respectively) in the K199Q mutant. On the other hand, R-239470, which has a carbamoyl group instead of a carboxyl group, showed only a 2.3-fold loss of affinity. These data suggest that Lys199 of the AT1 receptor has electrostatic interaction with the carboxyl group in the imidazole group. Olmesartan showed a 16-fold reduction in binding affinity in the H256A mutant, whereas the other related compounds showed only a slight reduction in binding affinity (1.4-4.0-fold reduction) despite the presence of a carboxyl group (Table 1). These results do not necessarily suggest interaction between the carboxyl group and His256, because the loss of binding caused by the mutation of His256 would have been compensated for by interaction between the carboxyl group and Lys199. Olmesartan, R-90929, and R-239470 showed large decreases in binding affinity (103-, 20-, and 25-fold decreases, respectively) in the Q257A mutant. Because the common functional group in all of the tested compounds is the tetrazole group, the carbamoyl group of Gln257 would interact with the tetrazole group. R-239471, in which the tetrazole group was replaced with a carboxyl group, showed a complete loss of binding affinity in this mutant. It has been known that ARBs having a tetrazole group are more potent than those having a carboxyl group, although the carboxyl and tetrazole groups have similar physicochemical and biological properties. There is very little information available on the interaction between Thr260 or Thr287 in the AT1 receptor and ARBs. Although olmesartan reduced the binding affinity in T260A by 12-fold, other related compounds showed <10-fold (only 2.5-7.1-fold) decreases in affinity in T260A. Thus, Thr260 is not very important for binding to olmesartan. Olmesartan reduced the binding affinity also in T287A by 11-fold, but Thr287 is located too far for direct binding to olmesartan in our molecular model, which is described below. We excluded the interaction of Tyr292 and Asn295 with olmesartan because of the large distances between these amino acids and olmesartan in our model. These amino acids have been reported to participate in the activation of the AT1 receptor (14Groblewski T. Maigret B. Larguier R. Lombard C. Bonnafous J.C. Marie J. J. Biol. Chem. 1997; 272: 1822-1826Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 15Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar, 16Schambye H.T. Hjorth S.A. Bergsma D.J. Sathe G. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7046-7050Crossref PubMed Scopus (116) Google Scholar, 17Balmforth A.J. Lee A.J. Warburton P. Donnelly D. Ball S.G. J. Biol. Chem. 1997; 272: 4245-4251Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Interaction between Tyr292 and Asn111 in the inactive state of the AT1 receptor would be cleaved by Ang II to allow the new interaction between Asp74 and Tyr292 (14Groblewski T. Maigret B. Larguier R. Lombard C. Bonnafous J.C. Marie J. J. Biol. Chem. 1997; 272: 1822-1826Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), which is the key event in the activation of the AT1 receptor to induce a change in the conformation of the receptor (15Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar). A similar event has been proposed to take place for Asn295 (16Schambye H.T. Hjorth S.A. Bergsma D.J. Sathe G. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7046-7050Crossref PubMed Scopus (116) Google Scholar, 17Balmforth A.J. Lee A.J. Warburton P. Donnelly D. Ball S.G. J. Biol. Chem. 1997; 272: 4245-4251Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Asn295 has a close spatial proximity to Asn111, and the mutation of Asn295 leads to a large change in the conformation of the AT1 receptor, which results in a serious reduction in the binding affinity to non-peptide ARBs. Inverse Agonist Activity of Olmesartan and Its Related Compounds in WT, N111G, and F77A/N111G AT1 Receptors—Although no naturally occurring mutant AT1 receptor that induces constitutive activity has been found, a recombinant WT AT1 receptor expressed at a high density showed constitutive activity. The native WT AT1 receptor has, therefore, some constitutive activity and can be used for analyzing the molecular mechanism underlying the inverse agonist activity. The inverse agonist activity of olmesartan and its related compounds in the WT AT1 receptor was tested and the results are shown in Fig. 3. Only olmesartan significantly suppressed basal IP production among the compounds tested (Fig. 3a). However, the basal activity of the WT AT1 receptor is too low to evaluate the inverse agonist activity of the related compounds. We have previously reported that the AT1-N111G mutant receptor had high basal activity in the absence of Ang II (18Feng Y.H. Miura S. Husain A. Karnik S.S. Biochemistry. 1998; 37: 15791-15798Crossref PubMed Scopus (85) Google Scholar, 19Noda K. Feng Y.H. Liu X.P. Saad Y. Husain A. Karnik S.S. Biochemistry. 1996; 35: 16435-16442Crossref PubMed Scopus (141) Google Scholar) and that the AT1 double mutant F77A/N111G receptor induced a change in the conformation of transmembrane (TM) VII to give higher basal activity than seen with the N111G mutant (20Miura S. Zhang J. Boros J. Karnik S.S. J. Biol. Chem. 2003; 278: 3720-3725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In fact, the basal IP production in the double mutant was comparable with that induced by Ang II in the WT AT1 receptor (Fig. 3c). In the N111G mutant, olmesartan and other related compounds, except for R-239470, which does not have a carboxyl group, significantly suppressed basal IP production (Fig. 3b). R-239470 failed to suppress IP production in the N111G mutant, although it showed the most potent binding affinity to the N111G mutant among the compounds tested (Table 2), indicating that binding affinity to the receptor does not necessarily parallel the inverse agonist activity. Electrostatic interaction of the carboxyl group with the receptor may be important for the inverse agonist activity, as observed in the difference between losartan and EXP3174 (9Miura S. Saku K. Karnik S.S. Hypertens. Res. 2003; 26: 937-943Crossref PubMed Scopus (94) Google Scholar). In the more active F77A/N111G mutant (Fig. 3c), olmesartan and R-89059, which have hydroxyl and carboxyl groups in the imidazole group, significantly suppressed high basal IP production, but the other compounds, such as R-90929 and R-239471, failed to do so, although there was no large loss of binding affinity to the F77A/N111G mutant (Table 2). These results suggest that the coexistence of both the carboxyl and hydroxyl groups in the imidazole group are required for the potent inverse agonist activity of olmesartan.TABLE 2Maximal binding capacities (Bmax) and binding affinities (Kd) of [Sar1,Ile8]Ang II, olmesartan, and its related compounds to AT1 wild type and mutant receptors Numbers in parentheses show ratio of Kd (mutant)/Kd (wild type).ReceptorBmaxKd[Sar1, Ile8]Ang IIOlmesartanR-89059R-90929R-239470R-239471pmol/mg proteinnmWild type0.52 ± 0.040.7 ± 0.2 (1.0)2.2 ± 0.7 (1.0)2.4 ± 1.0 (1.0)24 ± 2 (1.0)0.8 ± 0.3 (1.0)61 ± 21 (1.0)N111G0.38 ± 0.020.8 ± 0.6 (1.1)40 ± 18 (18)128 ± 24 (53)178 ± 11 (7.4)15 ± 10 (19)40 ± 10 (0.7)N111G/Y113F0.26 ± 0.111.0 ± 0.2 (1.4)25 ± 5 (11)54 ± 21 (23)1025 ± 230 (43)29 ± 8 (36)1350 ± 216 (22)N111G/K199Q0.31 ± 0.111.2 ± 0.9 (1.7)63 ± 27 (29)263 ± 124 (110)10 ± 5 (0.4)2.5 ± 1.4 (3.1)>105 (>1639)N111G/H256A0.24 ± 0.041.0 ± 0.5 (1.4)5.3 ± 1.6 (2.4)18 ± 4 (7.5)53 ± 7 (2.2)3.2 ± 0.7 (4.0)>105 (>1639)N111G/Q257A0.35 ± 0.031.0 ± 0.2 (1.4)26 ± 9 (12)41 ± 7 (17)272 ± 43 (11)6.1 ± 1.2 (7.6)579 ± 33 (9.5)N111G/Y113F/H256A0.15 ± 0.031.4 ± 0.2 (2.0)22 ± 7 (10)F77A/N111G0.54 ± 0.103.9 ± 0.5 (5.6)36 ± 6 (16)33 ± 6 (14)42 ± 9 (1.8)3.9 ± 1.1 (4.9)152 ± 18 (2.5)F77A/N111G/Y113F0.37 ± 0.092.3 ± 0.5 (3.3)228 ± 76 (104)F77A/N111G/K199Q0.36 ± 0.033.3 ± 0.9 (4.7)164 ± 25 (75)F77A/N111G/H256A0.39 ± 0.109.7 ± 2.3 (14)76 ± 11 (35)F77A/N111G/Q257A0.19 ± 0.0219 ± 5 (27)152 ± 32 (69)F77A/N111G/Y113F/H256A0.34 ± 0.081.6 ± 0.5 (2.3)29 ± 8 (13) Open table in a new tab Inverse Agonist Activity of Olmesartan and Its Related Compounds in Various Mutated AT1 Receptors—To delineate the participation of the interaction between olmesartan and the AT1 receptor in the inverse agonist activity, we examined the inverse agonist activity of olmesartan in receptors that had been further mutated at Tyr113, Lys199, His256, and Gln257 of N111G and F77A/N111G. The results are shown in Fig. 4. Interestingly, mutation at Tyr113, Lys199,orHis256 of N111G or F77A/N111G did not decrease inverse agonist activity of olmesartan. However, olmesartan lost inverse agonist activity in N111G/Y113F/H256A despite the same binding affinity to N111G and N111G/Y113F/H256A, as shown in Table 2. A similar result was observed in F77A/N111G/Y113F/H256A. These results again suggest that cooperative interactions between the hydroxyl group and Tyr113 (TM III) and between the carboxyl group and His256 (TM VI) are crucial for the potent inverse agonist activity of olmesartan. Olmesartan suppressed constitutive IP production in N111G/Q257A but not in F77A/N111G/Q257A. Consistent with these results, olmesartan showed a large decrease in binding affinity to F77A/N111G/Q257A (1/69 compared with binding to the WT AT1 receptor) but did not show a large loss of affinity to N111G/Q257A (1/12 of the affinity to the WT AT1 receptor), as shown in Table 2, suggesting that interaction between the tetrazole group and Gln257 is only a little involved in the inverse agonist activity of olmesartan. To elucidate the importance of the interaction of the hydroxyl and carboxyl groups with the AT1 receptor, we examined the inverse agonist activity of olmesartan and its related compounds in N111G mutants that had been further mutated at Tyr113, Lys199, His256, and Gln257, and the results are shown in Fig. 5. Olmesartan showed 60% inhibition of IP production, and R-89059 had ⅓ the ability of olmesartan in the N111G/Y113 mutant (Fig. 5a), whereas the other compounds, which have only a hydroxyl or carboxyl group, did not show inverse agonist activity. These results also suggest that coexistence of the hydroxyl and carboxyl groups is essential for inverse agonist activity. R-89059 and R-90929 each showed ∼60% inhibition of IP production compared with olmesartan in the N111G/K199Q mutant but did not show inverse agonist activity in the N111G/H256A mutant. These results suggest that the interaction of His256 with the carboxyl group is more important than that of Lys199 for inverse agonist activity, although both Lys199 and His256 bind to the carboxyl group. In the N111G (Fig. 3b) and N111G/Q257A (Fig. 5d) mutants, the inverse agonist activity of the test compounds, except for R-239470, was not reduced, indicating that interaction of the tetrazole group with Gln257 is not important for the inverse agonist activity. Molecular Model of the Interaction Between Olmesartan and the AT1 Receptor—A molecular model was constructed considering the three interactions between the AT1 receptor and olmesartan that were suggested from the mutation experiments, between Tyr113 and the hydroxyl group of olmesartan, between Lys199 and the carboxyl group, and between Gln257 and the tetrazole group. After manual docking and energy minimization, we constructed a molecular model containing the three interactions mentioned above (Fig. 6). The distances between Tyr113 and the hydroxyl group, Lys199 and the carboxyl group, and Gln257 and the tetrazole group are 2.7, 2.8, and 3.1 Å, respectively, which are reasonable distances for contributing to electrostatic and/or hydrogen bond interactions. Lys199 is also close to the tetrazole group, the distance (2.8 Å) being the same as that between Lys199 and the carboxylic acid, suggesting the presence of electrostatic interaction between Lys199 and the tetrazole group. Carini et al. (21Carini D.J. Duncia J.V. Aldrich P.E. Chiu A.T. Johnson A.L. Pierce M.E. Price W.A. Santella III, J.B. Wells G.J. Wexler R.R. Wong P.C. Yoo S.E. Timmerman P.B.M.W.M. J. Med. Chem. 1991; 34: 2525-2547Crossref PubMed Scopus (514) Google Scholar) report that an acidic group, such as a carboxyl or tetrazole group, in the biphenyl moiety is important for binding to the AT1 receptor; i.e. replacement of the acidic group by a non-acidic group, such as a carbamoyl group, leads to a remarkable decrease in binding affinity, suggesting the presence of ionic interaction between the acidic group in the biphenyl moiety and the basic amino acid in the AT1 receptor. Lys199 was the only candidate amino acid for forming an ionic bond with the tetrazole group in our model. The carboxyl group of olmesartan is located at a distance of 5.8 Å from Gln257 in our model, which is consistent with the finding of a previous paper in which the distance between α-COO- of candesartan and Gln257 was determined to be 4.98 Å (13Takezako T. Gogonea C. Saad Y. Noda K. Karnik S.S. J. Biol. Chem. 2004; 279: 15248-15257Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The large loss in binding affinity of olmesartan and its related compounds in the Q257A mutant would be explained by the loss of the hydrogen bond interaction between the carboxyl or carbamoyl group of the tested compounds and the carbamoyl group of Gln257. The other amino acid His256, the mutant of which (H256A) showed a 16-fold reduction in binding affinity to olmesartan, is located 4.4 Å from the carboxyl group, suggesting the existence of interaction between them. The distance between the tetrazole group and His256 is 6.3 Å, which is shorter than that reported for candesartan (7.31 Å) (13Takezako T. Gogonea C. Saad Y. Noda K. Karnik S.S. J. Biol. Chem. 2004; 279: 15248-15257Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Thr260, the other candidate amino acid for binding to olmesartan, cannot form hydrogen bonding with olmesartan but does have significant van der Waals contact, which could explain the decrease in affinity in the T260A mutant. The remaining candidates Tyr292 and Asn295 seemed to be too far away to directly bind to olmesartan. In addition, the T287A mutant showed a 10-fold loss of binding affinity for olmesartan compared with the WT AT1 receptor. Thr287 is located too far away to directly bind to olmesartan, but this amino acid is capable of forming a hydrogen bond with His256 in our model. The biphenyl group is positioned in the aromatic area consisting of Phe208, Phe249, and Trp253 in the model (3.7-4.5 Å), which is conserved in the class A family of GPCRs (22Bondensgaad K. Ankersen M. Thogersen H. Hansen B.S. Wulff B.S. Bywater R.P. J. Med. Chem. 2004; 47: 888-899Crossref PubMed Scopus (195) Google Scholar). A recent study on the binding model between losartan and the AT1 receptor constructed by computational analysis suggested that Tyr113 strongly interacted with the tetrazole group (2.70 Å) and the neighboring phenyl group (2.70 Å) of losartan (23Baleanu-Gogonea C. Karnik S. J. Mol. Model. 2006; 12: 325-337Crossref PubMed Scopus (28) Google Scholar). In the present study, however, R-90929, which has tetrazole and phenyl groups but not a hydroxyl group, showed only a slight reduction in binding affinity in the Y113F mutant (4.3-fold reduction) as mentioned above. The functional group closest to Tyr113 in the olmesartan-AT1 receptor binding model was the hydroxyl group (2.7 Å) and not the tetrazole (4.9 Å) or phenyl (4.0 Å) group. This suggests that the hydroxyl group is more suitable for interaction with Tyr113 than the tetrazole or phenyl group. The other amino acids Lys199, His256, and Gln257, which are involved in olmesartan binding and inverse agonist activity, were also suggested to interact with losartan. However, olmesartan is located deeper in the TM domain than losartan, and the hydroxyl and carboxyl groups of olmesartan, which are not present in losartan, interact with the AT1 receptor. This mode of olmesartan binding, which is distinct from that of losartan, affords the potent inverse agonist activity. An Agonist and an Inverse Agonist Bind to the Same TMs but Induce Different Conformational Changes in the AT1 Receptor—The binding affinities and molecular model studies suggest that the interactions of the hydroxyl, carboxyl, and tetrazole groups of olmesartan with Tyr113, Lys199, His256, and Gln257 in the AT1 receptor play important roles in the tight binding between olmesartan and the receptor. Among these interactions, that between the hydroxyl group and Tyr113 (TM III) and that between the carboxyl group and His256 (TM VI), are important for the inverse agonist activity of olmesartan. Farrens et al. (24Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1102) Google Scholar) and Gether et al. (25Gether U. Lin S. Ghanouni P. Ballesteros J.A. Weinstein H. Kobilka B.K. EMBO J. 1997; 16: 6737-6747Crossref PubMed Google Scholar) suggest that the activation of rhodopsin by ligands is initiated by anti-clockwise rotation of TM III and TM VI followed by changes in the conformation of the receptors. Similar changes in conformation are postulated to take place in constitutively active mutant receptors (26Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2699-2702Crossref PubMed Scopus (138) Google Scholar). In the case of the AT1 receptor, Ang II binding to Asn111 in TM III and to His256 in TM VI is critical for receptor activation (9Miura S. Saku K. Karnik S.S. Hypertens. Res. 2003; 26: 937-943Crossref PubMed Scopus (94) Google Scholar). Similar changes in conformation take place in the constitutively active β2 adrenergic and mutant AT1 receptors (18Feng Y.H. Miura S. Husain A. Karnik S.S. Biochemistry. 1998; 37: 15791-15798Crossref PubMed Scopus (85) Google Scholar, 26Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2699-2702Crossref PubMed Scopus (138) Google Scholar). We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation, which does not allow G protein coupling. The agonist Ang II shifts the AT1 receptor in the [R′] state (partially active state) to the [R*] state (fully active state), whereas the inverse agonist olmesartan may shift the receptor in the [R′] state to the inactive state through conformational changes of TM III and TM VI. Therapeutic Relevance of Inverse Agonism toward AT1 Receptor—The extent to which inverse agonism could offer a therapeutic advantage depends on the role of constitutive GPCR activity in the pathology. A potential therapeutic area where this might be relevant is cancer. It has been shown that the chronic elevation of second messengers in cells produced by constitutive GPCR activity can lead to cell transformation (27Lyons J. Landis C.A. Harsh G. Vallar L. Grunewald K. Feichtinger H. Duh Q.Y. Clark O.H. Kawasaki E. Bourne H.R. Science. 1990; 249: 655-659Crossref PubMed Scopus (922) Google Scholar). For example, receptors such as the α1-adrenoreceptor have been shown to be agonist-independent proto-oncogenes (28Allen L.F. Lefkowitz R.J. Caron M.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11354-11358Crossref PubMed Scopus (292) Google Scholar). Constitutively active GPCRs may also be important in autoimmune disease (29de Ligt R.A. Kourounakis A.P. Ijzerman A.P. Br. J. Pharmacol. 2000; 130: 1-12Crossref PubMed Scopus (176) Google Scholar). Although the AT1 receptor also has slight constitutive activity (2Miura S. Fujino M. Saku K. Curr. Hypertens. Rev. 2005; 1: 115-121Crossref Google Scholar) and there is no evidence regarding the pharmacotherapeutic relevance of ARBs as inverse agonists, we should consider the results of two very interesting studies. Losartan, which does not contain carboxyl or hydroxyl groups, prevented Ang II-induced, but not mechanical stretch-induced, vascular endothelial growth factor protein secretion in human mesangial cells (30Gruden G. Thomas S. Burt D. Zhou W. Chusney G. Gnudi L. Viberti G. J. Am. Soc. Nephrol. 1999; 10: 730-737Crossref PubMed Google Scholar). Ang II and stretching can each induce vascular endothelial growth factor production, suggesting that both Ang II- and stretch-induced AT1 receptor signals may exist. If losartan is an inverse agonist, the stretch-induced signal might be blocked. To resolve this question, Zou et al. (8Zou Y. Akazawa H. Qin Y. Sano M. Takano H. Minamino T. Makita N. Iwanaga K. Zhu W. Kudoh S. Toko H. Tamura K. Kihara M. Nagai T. Fukamizu A. Umemura S. Iiri T. Fujita T. Komuro I. Nat. Cell Biol. 2004; 6: 499-506Crossref PubMed Scopus (516) Google Scholar) reported that mechanical stress activates the AT1 receptor independent of Ang II, and this activation can be inhibited by the inverse agonist candesartan. They examined pressure overload on the heart by constricting the transverse aortae of adult male angiotensinogen knock-out mice. Although treatment with candesartan did not reduce blood pressure, candesartan significantly attenuated the development of cardiac hypertrophy. These results suggest that mechanical stress can induce cardiac hypertrophy in vivo by activating the AT1 receptor independent of Ang II. They suggested a mechanism by which mechanical stress might activate the AT1 receptor without Ang II. Stretching of the cell membrane may directly change the conformation of the AT1 receptor, and candesartan, which contains the carboxyl group, may prevent such changes as an inverse agonist. The present study attempted to elucidate the molecular mechanisms underlying inverse agonist activity of an ARB and provided a new perspective in the research of angiotensin receptors. A better understanding of the mechanism of inverse agonism would be useful for the development of new GPCR antagonists." @default.
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• W2155777324 title "Molecular Mechanism Underlying Inverse Agonist of Angiotensin II Type 1 Receptor" @default.
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