Molecular Mechanism Underlying Inverse Agonist of Angiotensin II Type 1 Receptor

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 and the were with binding The ligand was determined from the on the Binding 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). was by the 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). of the AT1 structure of bovine was used as a for the modeling of the AT1 receptor K. T. T. H. I. T. M. M. 2000; PubMed Scopus Google Scholar). We the of the human AT1 receptor with that of bovine The mutations were performed with the followed by using at the modeling the structure was to that the AT1 structure was This model was used for olmesartan. was performed that interactions by experiments were interactions were between Tyr113 and the hydroxyl group of olmesartan, between Lys199 and the carboxyl group, and between Gln257 and the The conformation of olmesartan was from the structure of olmesartan and to the interactions described was performed with a olmesartan structure using a The interactions were in this results are as the of or independent in values were with an analysis of using and was at Binding of and to WT and AT1 we the Tyr113, Lys199, His256, and in the AT1 for specific binding sites of olmesartan on the molecular model of the AT1 receptor described in K. Y. A. Husain A. Karnik S.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. C. Y. K. Karnik S.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. To determine the specific that to olmesartan, we examined the binding affinity of olmesartan and its related compounds chemical structures of which are shown in Fig. to AT1 receptors mutated at the The results are shown in 1. The expression of the WT and mutated AT1 receptors were the same of The of II were the same in some and were in but not to the affinity to the WT AT1 binding and binding of II, olmesartan, and its related compounds to AT1 type and mutant receptors in of Kd 1 22 in a new The affinity of olmesartan was by in and with the WT AT1 receptor, suggesting that Tyr113, Lys199, His256, and in the AT1 receptor are in the binding to olmesartan. R-90929, which has a chemical structure to that of olmesartan but has hydroxyl group, a in binding affinity to the and with the WT AT1 receptor. R-239470, which has a group of the carboxyl group in olmesartan, also a in affinity in the and R-239471, which has and carboxyl groups of the and groups in olmesartan, a of affinity to the mutant and a of binding affinity to the and results that Tyr113, Lys199, and Gln257 are essential for binding between olmesartan and the AT1 receptor. and R-239470, which have a hydroxyl group in the imidazole group, in binding affinity in the R-90929, which not have a hydroxyl group, a in binding that the hydroxyl group to Tyr113 of the AT1 receptor. the binding affinity of in the mutant was to that in the wild-type receptor, of the of between the group of and the hydroxyl group of Tyr113 in the for Lys199, olmesartan, R-90929, and R-239471, which have a carboxyl group in the imidazole group, a decrease in binding affinity and in the the R-239470, which has a group of a carboxyl group, a of that Lys199 of the AT1 receptor has interaction with the carboxyl group in the imidazole a in binding affinity in the the related compounds a slight in binding affinity the presence of a carboxyl group 1). results not interaction between the carboxyl group and His256, the of binding by the of His256 have been for by interaction between the carboxyl group and Olmesartan, R-90929, and in binding affinity and in the Because the common group in all of the tested compounds is the group, the group of Gln257 with the R-239471, in which the group was with a carboxyl group, a of binding affinity in this has been that ARBs a group are potent a carboxyl group, the carboxyl and groups have and is available on the interaction between or in the AT1 receptor and Although olmesartan the binding affinity in by related compounds in affinity in is not important for binding to olmesartan. the binding affinity also in by but is for binding to olmesartan in molecular model, which is described We the interaction of and with olmesartan of the between these and olmesartan in have been reported to in the activation of the AT1 receptor T. C. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). between and in the inactive of the AT1 receptor be by Ang II to the new interaction between and T. C. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which is the in the activation of the AT1 receptor to a in the conformation of the receptor J. S. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A has been to for G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has a to and the of to a in the conformation of the AT1 receptor, which results in a in the binding affinity to Inverse of and in WT, and AT1 mutant AT1 receptor that constitutive activity has been a WT AT1 receptor at a constitutive activity. The WT AT1 receptor some constitutive activity and can be used for 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. olmesartan suppressed production the compounds tested (Fig. However, the activity of the WT AT1 receptor is to the inverse agonist activity of the related We have previously reported that the AT1-N111G mutant receptor had activity in the of Ang II Y.H. S. Husain A. Karnik S.S. PubMed Scopus Google Scholar, K. Feng Y.H. Y. Husain A. Karnik S.S. 1996; PubMed Scopus Google Scholar) and that the AT1 mutant receptor induced a in the conformation of transmembrane (TM) to activity with the mutant S. J. J. Karnik S.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the production in the mutant was with that induced by Ang II in the WT AT1 receptor (Fig. In the olmesartan and related compounds, for R-239470, which not have a carboxyl group, suppressed production (Fig. to production in the the potent binding affinity to the mutant the compounds tested 2), that binding affinity to the receptor not the inverse agonist activity. interaction of the carboxyl group with the receptor may be important for the inverse agonist as 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 active mutant (Fig. olmesartan and R-89059, which have hydroxyl and carboxyl groups in the imidazole group, suppressed but the compounds, such as and R-239471, to was of binding affinity to the mutant results that the of the carboxyl and hydroxyl groups in the imidazole group are required for the potent inverse agonist activity of binding and binding of II, olmesartan, and its related compounds to AT1 type and mutant receptors in of Kd 10 10 in a new Inverse of and in AT1 delineate the of the interaction between olmesartan and the AT1 receptor in the inverse agonist we examined the inverse agonist activity of olmesartan in receptors that had been mutated at Tyr113, Lys199, His256, and Gln257 of and The results are shown in Fig. at Tyr113, of or not decrease inverse agonist activity of olmesartan. However, olmesartan inverse agonist activity in the same binding affinity to and as shown in A was in results that cooperative interactions between the hydroxyl group and Tyr113 and between the carboxyl group and His256 are for the potent inverse agonist activity of olmesartan. suppressed constitutive production in but not in with these results, olmesartan a decrease in binding affinity to with binding to the WT AT1 but not a of affinity to of the affinity to the WT AT1 as shown in suggesting that interaction between the group and Gln257 is a in the inverse agonist activity of olmesartan. To the 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 that had been mutated at Tyr113, Lys199, His256, and and the results are shown in Fig. of and had the of olmesartan in the mutant (Fig. the compounds, which have a hydroxyl or carboxyl group, not inverse agonist activity. results also that of the hydroxyl and carboxyl groups is essential for inverse agonist activity. and of production with olmesartan in the mutant but not inverse agonist activity in the results that the interaction of His256 with the carboxyl group is important that of Lys199 for inverse agonist Lys199 and His256 to the carboxyl In the (Fig. and (Fig. the inverse agonist activity of the compounds, for R-239470, was not that interaction of the group with Gln257 is not important for the inverse agonist activity. of the and the AT1 molecular model was constructed the interactions between the AT1 receptor and olmesartan that were from the experiments, between Tyr113 and the hydroxyl group of olmesartan, between Lys199 and the carboxyl group, and between Gln257 and the and we constructed a molecular model the interactions (Fig. The between Tyr113 and the hydroxyl group, Lys199 and the carboxyl group, and Gln257 and the group are and which are for to Lys199 is also to the group, the the same as that between Lys199 and the suggesting the presence of interaction between Lys199 and the J. Chem. PubMed Scopus Google Scholar) report that an group, such as a carboxyl or group, in the is important for binding to the AT1 receptor; of the group by a group, such as a group, to a decrease in binding suggesting the presence of interaction between the group in the and the in the AT1 receptor. Lys199 was the for an with the group in The carboxyl group of olmesartan is at a of from Gln257 in model, which is with the of a in which the between of and Gln257 was determined to be T. C. Y. K. Karnik S.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The in binding affinity of olmesartan and its related compounds in the mutant be by the of the interaction between the carboxyl or group of the tested compounds and the group of The His256, the mutant of which a in binding affinity to olmesartan, is from the carboxyl group, suggesting the of interaction between The between the group and His256 is which is that reported for T. C. Y. K. Karnik S.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the for binding to olmesartan, with olmesartan but have which could the decrease in affinity in the The and to be to to olmesartan. In addition, the mutant a of binding affinity for olmesartan with the WT AT1 receptor. is to to olmesartan, but this is of a with His256 in The group is in the area of and in the model which is in the class A of K. M. H. J. Chem. 2004; PubMed Scopus Google Scholar). A recent study on the binding model between losartan and the AT1 receptor constructed by analysis that Tyr113 with the group and the group of losartan C. Karnik S. J. Mol. PubMed Scopus Google Scholar). In the R-90929, which has and groups but not a hydroxyl group, a slight in binding affinity in the mutant as The group to Tyr113 in the receptor binding model was the hydroxyl group and not the or This that the hydroxyl group is for interaction with Tyr113 the or The Lys199, His256, and which are in olmesartan binding and inverse agonist were also to with However, olmesartan is in the TM losartan, and the hydroxyl and carboxyl groups of olmesartan, which are not in losartan, with the AT1 receptor. This of olmesartan which is from that of losartan, the potent inverse agonist activity. and an Inverse to the TMs but in the AT1 binding and molecular model studies that the interactions of the and groups of olmesartan with Tyr113, Lys199, His256, and Gln257 in the AT1 receptor play important in the binding between olmesartan and the receptor. these interactions, that between the hydroxyl group and Tyr113 and that between the carboxyl group and His256 are important for the inverse agonist activity of olmesartan. C. K. 1996; 274: PubMed Scopus Google Scholar) and U. S. P. H. J. PubMed Google Scholar) that the activation of by is initiated by anti-clockwise rotation of TM III and TM VI followed by changes in the conformation of the changes in conformation are to in constitutively active mutant receptors G. P. Lohse M. M. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the of the AT1 receptor, Ang II binding to in TM III and to His256 in TM VI is for receptor activation (9Miura S. Saku K. Karnik S.S. Hypertens. Res. 2003; 26: 937-943Crossref PubMed Scopus (94) Google Scholar). changes in conformation in the constitutively active and mutant AT1 receptors Y.H. S. Husain A. Karnik S.S. PubMed Scopus Google Scholar, G. P. Lohse M. M. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive which not G The agonist Ang II the AT1 receptor in the active to the active the inverse agonist olmesartan may the receptor in the to the inactive through changes of TM III and TM of Inverse AT1 to which inverse agonism could a on the role of constitutive activity in the A area this be is has been shown that the of in cells by constitutive activity can to cell J. G. L. K. H. E. PubMed Scopus Google Scholar). receptors such as the have been shown to be agonist-independent S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). active may also be important in J. Pharmacol. 2000; PubMed Scopus 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 is the pharmacotherapeutic relevance of ARBs as inverse agonists, we the results of two which not carboxyl or hydroxyl Ang but not mechanical in human cells G. S. W. G. L. G. J. 1999; PubMed Google Scholar). Ang II and stretching can suggesting that Ang and AT1 receptor may losartan is an inverse agonist, the signal be To this (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 the AT1 receptor independent of Ang II, and this activation can be by the inverse agonist examined on the heart by constricting the transverse of angiotensinogen knock-out Although with not blood attenuated the development of cardiac results that mechanical can cardiac hypertrophy in by the AT1 receptor independent of Ang II. a mechanism by which mechanical the AT1 receptor without Ang II. of the cell may the conformation of the AT1 receptor, and which the carboxyl group, may such changes as an inverse The study to the molecular mechanisms underlying inverse agonist activity of an and a new in the of angiotensin A better understanding of the mechanism of inverse agonism be useful for the development of new