Next Article in Journal
Comparative Evaluation of the Cytotoxicity of Doxorubicin in BT-20 Triple-Negative Breast Carcinoma Monolayer and Spheroid Cultures
Previous Article in Journal
Histologic Analysis of Idiopathic Pulmonary Fibrosis by Morphometric and Fractal Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biomedicines
Volume 11
Issue 5
10.3390/biomedicines11051486
biomedicines-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Yoshino Matsuo
1,
Yasunori Suematsu
1,
Hidetaka Morita
1 and
Shin-ichiro Miura
1,2,*
1
Department of Cardiology, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan
2
Department of Internal Medicine, Fukuoka University Nishijin Hospital, Fukuoka 814-8522, Japan
*
Author to whom correspondence should be addressed.
Biomedicines 2023, 11(5), 1486; https://doi.org/10.3390/biomedicines11051486
Submission received: 10 April 2023 / Revised: 14 May 2023 / Accepted: 16 May 2023 / Published: 19 May 2023
(This article belongs to the Section Drug Discovery, Development and Delivery)
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. Introduction

Angiotensin II (Ang II) type 1 (AT1) receptor, which is a member of the G-protein-coupled receptor (GPCR) family, mediates most known pathological cardiovascular functions in addition to physiological functions [1,2]. There are many interesting aspects of the functions of GPCRs [3], such as homo- and hetero-oligomerization, constitutive or spontaneous activity in the absence of agonists, nuclear localization, trafficking, intracellular protein-evoked receptor activation, and the GPCR receptor-associated proteins. Studies on these topics may lead to new and improved therapeutics for cardiovascular diseases.
AT1 receptor blockers (ARBs) block the diverse effects of Ang II, such as vasoconstriction and cardiac hypertrophy. A biased ligand is a ligand that, when bound, activates the β-arrestin signal but not the Gaq signal, for example [4,5]. We previously reported that [Sar1, Ile4, Ile8]Ang II, which is an Ang II analogue, did not increase Gq-dependent inositol phosphate (IP) production. On the other hand, it still activated Gq-independent extracellular signal-regulated kinase (ERK) 1/2 in human coronary artery smooth muscle cells, as well as in a stably expressed AT1 receptor cell line [5]. Several ARBs show variable efficacies in addition to anti-hypertensive effects toward protection against organ damage in diabetic nephropathy, cardiac hypertrophy, arrhythmia, and renal failure [6]. Over the past several years, the efficacies of ARBs have been compared and differences have been observed, such as in reducing left ventricular mass, increasing nitric oxide production, lowering monocyte chemoattractant protein-1 and plasma plasminogen activator inhibitor type-1 antigen, and inhibiting platelet activation, except with regard to the lowering of blood pressure (as a pleiotropic effect) [7]. In addition, many studies have suggested that additional tissue-protective benefits of ARB therapy may be mediated by AT1 receptor β-arrestin signaling, although the structural basis for the differential activation of this mechanism by ARBs is not known [8,9].
Peptide-type AT1 receptor-selective agonists, which are biased ligands, have been developed, and one of them, TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-DAla-OH), competitively antagonizes Ang-II-stimulated G protein signaling. It stimulates β-arrestin recruitment and activates several kinase pathways including ERK 1/2, Src, and so on [10]. In addition, the AT1 receptor biased ligands TRV120023 and TRV120067 were reported to be useful in a mouse model of familial dilated cardiomyopathy [11] and in a mouse model of acute heart failure and dilated cardiomyopathy [12], respectively. On the other hand, a phase II trial with TRV027, which is similar to TRV120067, did not show an improved prognosis at day 30 compared with a placebo in patients with heart failure [13]. Peptide forms may be readily degradable, unstable, and inconsistent in their efficacy. In addition, although it activates the β-arrestin pathway, which is useful for treating heart failure, it is not clear whether it can activate other pathways that exacerbate heart failure because of the diversity of intracellular signals. If non-peptide AT1 receptor ligands could be developed, it would be possible to verify these points in detail. Most ARBs have common molecular structures (biphenyl-tetrazol and imidazole groups), and it is clear that ARBs have ‘class effects’ [14]. On the other hand, recent studies have demonstrated that not all ARBs have the same effects. Some benefits conferred by ARBs may not be class effects, and instead may be ‘molecular effects’ [14]. In addition, small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism, or inverse agonism toward a GPCR. A small difference in the molecular structure of angiotensin II receptor blockers induces agonism, neutral antagonism, or inverse agonism toward AT1 receptor [15]. Based on these reports, we thought that it may be possible to develop non-peptide AT1 receptor biased ligands by slightly changing the structure of ARBs.
Therefore, by using olmesartan as the basic structure of ARBs, we created compounds with various slight structural changes and used them as candidates of biased ligands. For example, the carboxyl group of olmesartan was converted to a carbamoyl group, which eliminated its potent inverse agonism. Instead, we designed and produced an olmesartan-related compound by adding a 4-hydroxybenzyl group to the biphenyl group so that it may have agonistic action. Here, we developed a non-peptide AT1 receptor compound as a biased ligand.

2. Materials and Methods

2.1. Materials

We purchased the following reagents: Ang II and [Sar1, Ile8]Ang II (Peptide Institute Inc., Osaka, Japan); olmesartan (Toronto Research Inc., Toronto, ON, Canada); and 125I-[Sar1, Ile8]Ang II (PerkinElmer Japan Co. Ltd., Tokyo, Japan). Three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) as candidates of biased ligands were provided by Daiichi Sankyo Co., Ltd., Tokyo, Japan (Figure 1A).

2.2. AT1 Mutant Receptors

The secondary structure of the AT1 receptor is shown in Figure 1B. We mutated four amino acids in transmembrane (TM) III, VI, and VII in the AT1 receptor (L112A, Q257A, Y292A, and N295A receptors). We determined the mutation sites of the AT1 receptor according to ARB, which produced olmesartan derivatives with different interactions and functions toward the AT1 receptor, as previously reported by Zhang et al. [16], since the additional 4-hydroxybenzyl groups of R781253 and R794847 were predicted to extend to a sub-pocket on the bottom of the AT1 receptor ligand-binding pocket consisting of Leu112 and Gln257. In addition, since the biased ligand TRV027 induced changes near the NPxxY motif in TM domain 7 that are related to receptor activation [17], Tyr292 and Asn295 in the AT1 receptor were mutated.

2.3. Cell Cultures, Transfection, and Membrane Preparation

The synthetic rat AT1 receptor gene, which was cloned in the shuttle expression vector pMT-2, was used for expression and mutagenesis, as described previously [9,15,18]. COS7 cells (No. CRL-1651, ATCC, Manassas, VA, USA) were cultured in 10% fetal bovine serum (FBS) and penicillin- and streptomycin-supplemented Dulbecco’s modified Eagle’s essential medium (PS-DMEM) in 5% CO2 at 37 °C. In the experiments, cells were grown without a cell-growth supplement. To express the AT1 receptor protein, 10 µg of purified plasmid DNA per 107 cells was used in the transfection. Wild-type and mutated AT1 receptors were transiently transfected into cells using Lipofectamine 2000 liposomal reagent (Roche Applied Science, Penzberg, Germany). Cells were plated and grown and were 70–90% confluent at the time of transfection. We incubated plasmid DNA (10 µg)–lipid (10 µL) complexes for 5 min at room temperature and added and incubated the complexes with cells for 24 h. We then analyzed the parameters in transfected cells. Cell viability was >95% according to trypan blue exclusion analysis in the control experiments. Transfected cells that had been cultured for 48–72 h were harvested. Cell membranes were prepared using the nitrogen Parr bomb disruption or freeze–thaw methods.

2.4. Competition Binding Study

The Kd (nM) values of receptor binding were determined with 125I-[Sar1, Ile8]AngII-binding experiments under equilibrium conditions. Binding kinetics were determined following the methods described in the literature [9,15,18]. We carried out binding by using 125I-[Sar1, Ile8] AngII (2200 Ci/mmol) and an increasing concentration (10−11–10−5 M) of each unlabeled compound. Nonspecific binding of 125I-[Sar1, Ile8] Ang II was determined in the presence of a high concentration (10−6 M) of unlabeled [Sar1, Ile8]. Next, the incubation mixture was separated by vacuum filtration through Whatman GF/C glass filters (Disposable Filter Funnels Grade GF/C, GE Healthcare), and presoaked with 0.1% polyethylenimine. The filter was washed with 3 mL of ice-cold stop buffer (25 mM Tris–HCl, pH 7.4, 1 mM MgCl2). The radioactivity trapped on the filters was quantified using an automatic γ-counter. The percentage (%) of specific binding of 125I-[Sar1, Ile8]Ang II was calculated using the following formula: 100 × (% total binding of 125I-[Sar1, Ile8]Ang II—nonspecific binding of 125I-[Sar1, Ile8]Ang II)/(% total binding of 125I-[Sar1, Ile8]Ang II).

2.5. IP Production Assay

Ligand (such as agonist or antagonist)-induced IP production through the AT1 receptor in transfected COS7 cells transiently expressing the AT1 receptor was measured to evaluate cell signaling as a second messenger for controlling blood pressure, since the increases in the intracellular Ca2+ concentration were the result of IP accumulation, which indicated vasoconstriction. As a surrogate model with higher levels of AT1 receptor expression, COS7 cells are suitable for IP measurement because it is easy to detect differences in IP production. Cells were labeled for 24 h with [3H]-myoinositol at 37 °C in DMEM containing 10% FBS. Next, the cells were washed with buffer 3 times and incubated with a medium containing 10 mM LiCl for 20 min; 0.1 µM Ang II or 1 µM ligands were added and incubation was continued for another 30 min at 37 °C. After incubation, the medium was removed, and total soluble IP was extracted from the cells using the perchloric acid extraction method, as described previously [9,15,18]. Cells were plated at a field density of 5 × 103 cells/cm2 in 60 mm dishes and cultured in a fresh growth medium at 37 °C in a DMEM 5% CO2 incubator. After 24 h of plating, the cells were transfected with empty vector or expression vector for wild-type or mutant AT1 receptors. The culture medium was changed 48 h after transfection and 1.5 mL of 1 μCi/mL of [3H]-myoinositol was added to each 60-mm dish at 37 °C in DMEM containing 10% FBS. After labeling, the cells were washed with buffer 3 times and incubated with a medium containing 10 mM LiCl for 20 min. The indicated concentration of ligands was added and incubation was continued for an additional 30 min at 37 °C. After incubation, the medium was removed, and total soluble IP was extracted from the cells using the perchloric acid extraction method. Total IP production was expressed as the sum of radioactivity, as described previously [9,15,18].

2.6. Immunoblotting of ERK 1/2 Activation

In in vitro studies, ERK 1/2 activation by 1 µM ligands or 0.1 µM Ang II incubated for 10 min was measured using COS7 cells that overexpressed AT1 wild-type and mutant receptors. ERK activity was measured in cells maintained for 18 h in DMEM containing 0.1% serum. At the end of the stimulation, the culture medium was removed by aspiration, the cells were washed with ice-cold phosphate-buffered saline, and the dishes were frozen in liquid nitrogen. The cells were scraped using a scraper after melting, and the scraped cells were transferred to a tube. We homogenized the cells in lysis buffer plus protease inhibitors and determined the protein concentration using the bicinchoninic acid method. Immunoblotting was performed with primary antibodies as the specified total ERK (#9102, Cell Signaling, Danvers, MA, USA) or phospho-ERK (#9106, Cell Signaling, Danvers, MA, USA) antibody in each sample [5]. We fractionated total proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis; we then transferred the proteins to a Hybond nitrocellulose membrane and blocked the blotted membranes with 10% bovine serum albumin in tris-buffered saline buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl) for 1 h at room temperature. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (BIO-RAD170-6515 or 170-6516, Bio-Rad Laboratories Inc., Hercules, CA, USA) against total or phosphorylated ERK overnight at 4 °C. An enhanced chemiluminescent substrate system (Amersham, Buckinghamshire, UK) was used. The signal was independently quantified by digital image analysis (ImageJ, National Institutes of Health, Bethesda, MD, USA) [19].

2.7. Statistical Analysis

All data are expressed as the mean ± standard error. Each experiment was performed by three or more independent determinations. The significance of differences among the measured values was evaluated with an analysis of variance using an unpaired or paired Student’s t-test and Fisher’s t-test, as appropriate. Statistical significance was considered to be present at <0.05.

3. Results

3.1. Binding Affinities of [Sar1,Ile8]Ang II, Olmesartan, R239470, R781253, and R794847 to AT1 Wild-Type and Mutant Receptors

The Kd values of [Sar1,Ile8]Ang II, olmesartan, R239470, R781253, and R794847 for the AT1 wild-type receptor, as determined by a competition binding study, were 0.8, 2.3, 0.8, 21, and 48 nM, respectively (Table 1). Those of R239470, R781253, and R794847 for the AT1 L112A receptor were 37, 23, and 31 nM, respectively. The Kd values of R239470, R781253, and R794847 were comparable to the Kd value of olmesartan for the receptor. The Kd values of R239470, R781253, and R794847 for the AT1 Q257 receptor were better than that of olmesartan. Although R239470 could bind to the AT1 N295A receptor, R781253 and R794847 could not (Kd > 10,000 nM).

3.2. Levels of IP Production Using Various Ligands in AT1-WT, -L112A and Q257A Receptors

The levels of IP production using various ligands in AT1 wild-type, -L112A, and Q257A receptors are shown in Figure 2. Ang II significantly increased IP production in AT1 wild-type, L112A, and Q257A receptors in this cell system. Olmesartan induced a significant reduction in basal IP production levels in the AT1 wild-type, L112A, and Q257A receptors and showed inverse agonism. In the AT1 wild-type receptor, R781253, and R794847 significantly decreased and increased IP production, respectively, whereas R239470 did not change IP production (Figure 2). In the AT1-L112A and -Q257A receptors, none of the compounds (R239470, R781253, and R794847) affected IP production, although olmesartan induced a significant reduction in basal IP production levels in the receptors.

3.3. Levels of ERK Activities Using Various Ligands in AT1-WT, -L112A, and Q257A Receptors

Ang II significantly activated ERK1/2 in AT1 wild-type, L112A, and Q257A receptors in this cell system (Figure 3). Olmesartan did not change ERK1/2 activation in AT1 wild-type, L112A, or Q257A receptors. In the AT1 wild-type receptor, R781253 and R794847, but not R239470, activated ERK1/2. R781253 and R794847, but not R239470, activated ERK1/2 in the AT1-L112A receptor. None of the compounds activated ERK1/2 in the AT1-Q257A receptor.

4. Discussion

In this study, we developed a non-peptide AT1 receptor compound, R781253, as a biased ligand (Figure 4). The positions of Lys112 and Gln257 in the AT1 receptor may play an important role in IP production with regard to R781253 as a biased ligand, whereas the position of Gln257, but not Lys112, plays an important role in the ERK1/2 activation of R781253. Thus, a small difference in the molecular structure produced a biased ligand. In addition, the differences in the binding position of the AT1 receptor relative to R781253 influenced the function of the biased ligand.
We confirmed that the non-peptide AT1 receptor compound R781253 acted as a biased ligand (Figure 4). R239470, R781253, and R794847 were a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. We previously reported that small differences in the chemical structures of ARBs can be responsible for agonism, neutral antagonism, or inverse agonism regarding IP production toward the AT1 receptor [15]. We analyzed the ligand-specific changes in the receptor conformation with respect to stabilization around TM3. Although the agonist-, neutral antagonist and inverse-agonist-binding sites in the AT1 receptor are similar, each ligand induced specific conformational changes in TM3. The hydroxyphenyl moiety, which represents the difference between the chemical structures of olmesartan and R794847, interacts with the Leu112 of the AT1 receptor. Coupled movements of Leu112 and Asn111 occur in molecular dynamics simulations of the AT1 receptor [20]. The flipping of Asn111 is essential for Gq-dependent signaling. In this study, we also analyzed the positions of Lys112, which is adjacent to Asn111, as well as Gln257, in the AT1 receptor. Leu112, in addition to Gln257, may play an important role in Gq-dependent IP production with regard to R781253 as a biased ligand. On the other hand, R781253 and R794847, but not R239470, were found to be agonists in terms of ERK1/2 activation. The additional 4-hydroxybenzyl groups of both R781253 and R794847 were predicted to extend to a sub-pocket on the bottom of the AT1 receptor ligand-binding pocket, which includes Leu112, Lys199, Asn200, Trp253, His256, Gln257, and Thr260 in the AT1 receptor [16]. Since the position of Gln257, but not of Lys112, is important for the ERK1/2 activation by R781253, the hydroxy group in the 4-hydroxybenzyl group of R781253 may be crucial for the binding to Gln257 in the AT1 receptor for ERK1/2 activation. In addition, the carbamoyl moieties of R239470 and R794847 cannot form a salt bridge to Arg167 in the AT1 receptor. Therefore, R781253 retains inverse agonism, while R239470 and R794847 do not. In addition, Arg167 forms extensive networks of hydrogen bonds and salt bridges with the acidic tetrazole ring and the carboxyl group on the imidazole moiety of olmesartan in the AT1 receptor–olmesartan complex. We might have been able to analyze IP production and ERK1/2 activation more effectively using the position of the Arg167 mutated AT1 receptor. Lys199 also played an important role in Ang II binding, as the K199A mutant completely abolishes Ang II binding, and we did not perform any further analysis.
The β-arrestin-biased ligand stabilizes an alternative TM7 conformation in the AT1 receptor, whereas G-protein-biased ligands do not favor this conformation [21]. The binding of partial or β-arrestin-biased peptide agonists triggers a shift of TM6, which is a hallmark of GPCR activation, and a change in the conformation of Asn295 [20]. In addition, Tyr292 and Leu112 in the AT1 receptor play important roles in coupling Ang II binding to Gq activation. In the active state of AT1 receptor structures, the outward rotation of TM6 and its associated inward rotation of TM7 disrupts an inactive state for stabilizing interactions between Asn111 and Asn295 underneath the ligand-binding pocket. Tyr292 is also coupled to the ligand through two adjacent residues, Asn111 and Leu112, on TM3 [20]. To analyze the importance of the change in TM7 conformation by a non-peptide biased ligand R781253 in this study, we mutated the positions of Tyr292 (Y292A) and Asn295 (N295A) in TM7. Since the Kd values of R781253 against Y292A and N295A were 95 nM and >10,000 nM, respectively, and since the binding affinities were very low, we did not confirm IP production or ERK activation using R781253 in the AT1-Y292A and -N295A receptors. Further study is needed to resolve these issues.
Activation of the Gq/11 pathway is associated with a specific conformational transition that is stabilized by the agonist. On the other hand, activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor [22]. Distinct conformations of the AT1 receptor are associated with distinct cell signaling pathways [23]. The activities of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. GRK2 and GRK5 are predominantly expressed in the heart, where they have both canonical and non-canonical functions. Biased ligands induce the recruitment of distinct subtypes of GRK to the receptor as compared with balanced ligands. β-arrestin induces sustained G protein-induced cell signaling by a ternary GPCR–G-protein–β-arrestin complex. The activation of the G protein triggers the initiation of the β-arrestin system by releasing free Gβγ subunits and activating GRK2/3. Moreover, while β-arrestin recruitment depends on both GRK5/6 and GRK2/3 upon the binding of Ang II, it depends solely on GRK5/6 upon the binding of the β-arrestin-biased ligand TRV027 [24]. In this study, although we did not analyze the conformational transition or the importance of the kinds of GRK of R239470, R781253, and R794847, the biased ligand R781253 may induce a distinct conformational transition and the recruitment of different GRKs.
AT1 receptor signaling has been implicated in Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection [25]. SARS-CoV-2 enters cells by binding to angiotensin converting enzyme 2 (ACE2), which lead to the downregulation of ACE2 activity. Ang II, the ligand for the AT1 receptor, activates a Gq signaling pathway, leading to vasoconstriction and adverse cellular responses in heart failure and acute lung injury. On the other hand, the action of Ang II is moderated by β-arrestin [25]. Cleavage of Ang II by ACE2 decreases Ang II levels and increases Ang(1-7) production, which is an endogenous β-arrestin-biased agonist of the AT1 receptor with potential therapeutically beneficial effects. Compared with ARBs, the biased agonist of AT1 receptor R781253, which can improve potency and pharmacodynamic properties compared with Ang(1-7), may be more advantageous in patients with SARS-CoV-2 infection because R781253 blocks Gq-dependent Ang II activity and induces β-arrestin signaling.
A limitation of this study is that it was conducted under limited conditions and in vitro, and no research has been conducted in vivo within the human body. However, it is considered to have sufficient reliability for verifying the binding mode between the ligand and receptor and subsequent specific intracellular signaling pathways. In addition, based on previous reports, mutated receptors were created and verified. Nonetheless, we performed experiments with only one type of cell. Each type of cell has different intracellular signal transduction systems, and the strength of the signals varies, so the results obtained in this study do not necessarily occur universally in all kinds of cells.

5. Conclusions

R239470, R781253, and R794847 were shown to be 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, were agonists with respect to ERK1/2 activation. Thus, we developed a non-peptide AT1 receptor compound as a biased ligand.

Author Contributions

Conceptualization, Y.M. and S.-i.M.; methodology, Y.M.; validation, Y.S. and H.M.; formal analysis, Y.M.; investigation, Y.M.; resources, S.-i.M.; data curation, Y.S. and Y.M.; writing—original draft preparation, Y.M.; writing—review and editing, S.-i.M.; supervision, S.-i.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a Grant-in-Aid to S.-i.M. for Scientific Research (No.19K08593) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available are available from the corresponding author on reasonable request.

Acknowledgments

Three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) were provided by Daiichi Sankyo Co., Ltd., Tokyo, Japan. We are grateful to Sayo Tomita for her expert technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Karnik, S.S.; Unal, H.; Kemp, J.R.; Tirupula, K.C.; Eguchi, S.; Vanderheyden, P.M.; Thomas, W.G. International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli [corrected]. Pharmacol. Rev. 2015, 67, 754–819, Erratum in Pharmacol. Rev. 2015, 67, 820. [Google Scholar] [CrossRef] [PubMed]
  2. Forrester, S.J.; Booz, G.W.; Sigmund, C.D.; Coffman, T.M.; Kawai, T.; Rizzo, V.; Scalia, R.; Eguchi, S. Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology. Physiol. Rev. 2018, 98, 1627–1738. [Google Scholar] [CrossRef] [PubMed]
  3. Balakumar, P.; Jagadeesh, G. A century old renin-angiotensin system still grows with endless possibilities: AT1 receptor signaling cascades in cardiovascular physiopathology. Cell Signal. 2014, 26, 2147–2160. [Google Scholar] [CrossRef] [PubMed]
  4. Bonde, M.M.; Hansen, J.T.; Sanni, S.J.; Haunsø, S.; Gammeltoft, S.; Lyngsø, C.; Hansen, J.L. Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms. PLoS ONE 2010, 5, e14135. [Google Scholar] [CrossRef]
  5. Miura, S.; Zhang, J.; Matsuo, Y.; Saku, K.; Karnik, S.S. Activation of extracellular signal-activated kinase by angiotensin II-induced Gq-independent epidermal growth factor receptor transactivation. Hypertens. Res. 2004, 27, 765–770. [Google Scholar] [CrossRef]
  6. Burnier, M.; Brunner, H.R. Angiotensin II receptor antagonists. Lancet 2000, 355, 637–645. [Google Scholar] [CrossRef]
  7. Miura, S.; Fujino, M.; Saku, K. Angiotensin II receptor blocker as an inverse agonist: A current perspective. Curr. Hypertens. Rev. 2005, 1, 115–121. [Google Scholar] [CrossRef]
  8. Kim, J.; Ahn, S.; Ren, X.R.; Whalen, E.J.; Reiter, E.; Wei, H.; Lefkowitz, R.J. Functional antagonism of different G protein-coupled receptor kinases for b-arrestin-mediated angiotensin II receptor signaling. Proc. Natl. Acad. Sci. USA 2005, 102, 1442–1447. [Google Scholar] [CrossRef]
  9. Miura, S.; Okabe, A.; Matsuo, Y.; Karnik, S.S.; Saku, K. Unique binding behavior of the recently approved angiotensin II receptor blocker azilsartan compared with that of candesartan. Hypertens. Res. 2013, 36, 134–139, Erratum in Hypertens Res. 2013, 36, 476. [Google Scholar] [CrossRef]
  10. Violin, J.D.; DeWire, S.M.; Yamashita, D.; Rominger, D.H.; Nguyen, L.; Schiller, K.; Whalen, E.J.; Gowen, M.; Lark, M.W. Selectively engaging β-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance. J. Pharmacol. Exp. Ther. 2010, 335, 572–579. [Google Scholar] [CrossRef]
  11. Tarigopula, M.; Davis, R.T., 3rd; Mungai, P.T.; Ryba, D.M.; Wieczorek, D.F.; Cowan, C.L.; Violin, J.D.; Wolska, B.M.; Solaro, R.J. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model. Cardiovasc. Res. 2015, 107, 226–234. [Google Scholar] [CrossRef]
  12. Ryba, D.M.; Li, J.; Cowan, C.L.; Russell, B.; Wolska, B.M.; Solaro, R.J. Long-Term Biased β-Arrestin Signaling Improves Cardiac Structure and Function in Dilated Cardiomyopathy. Circulation 2017, 135, 1056–1070. [Google Scholar] [CrossRef]
  13. Pang, P.S.; Butler, J.; Collins, S.P.; Cotter, G.; Davison, B.A.; Ezekowitz, J.A.; Filippatos, G.; Levy, P.D.; Metra, M.; Ponikowski, P.; et al. Biased ligand of the angiotensin II type 1 receptor in patients with acute heart failure: A randomized, double-blind, placebo-controlled, phase IIB, dose ranging trial (BLAST-AHF). Eur. Heart J. 2017, 38, 2364–2373. [Google Scholar] [CrossRef]
  14. Miura, S.; Karnik, S.S.; Saku, K. Review: Angiotensin II type 1 receptor blockers: Class effects versus molecular effects. J. Renin-Angiotensin-Aldosterone Syst. 2011, 12, 1–7. [Google Scholar] [CrossRef]
  15. Miura, S.; Kiya, Y.; Hanzawa, H.; Nakao, N.; Fujino, M.; Imaizumi, S.; Matsuo, Y.; Yanagisawa, H.; Koike, H.; Komuro, I.; et al. Small molecules with similar structures exhibit agonist, neutral antagonist or inverse agonist activity toward angiotensin II type 1 receptor. PLoS ONE 2012, 7, e37974, Erratum in PLoS ONE 2013, 8. [Google Scholar] [CrossRef]
  16. Zhang, H.; Unal, H.; Desnoyer, R.; Han, G.W.; Patel, N.; Katritch, V.; Karnik, S.S.; Cherezov, V.; Stevens, R.C. Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor. J. Biol. Chem. 2015, 290, 29127–29139. [Google Scholar] [CrossRef]
  17. Modestia, S.M.; Malta de Sá, M.; Auger, E.; Trossini, G.H.G.; Krieger, J.E.; Rangel-Yagui, C.O. Biased Agonist TRV027 Determinants in AT1R by Molecular Dynamics Simulations. J. Chem. Inf. Model. 2019, 59, 797–808. [Google Scholar] [CrossRef]
  18. Miura, S.; Fujino, M.; Hanzawa, H.; Kiya, Y.; Imaizumi, S.; Matsuo, Y.; Tomita, S.; Uehara, Y.; Karnik, S.S.; Yanagisawa, H.; et al. Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor. J. Biol. Chem. 2006, 281, 19288–19295. [Google Scholar] [CrossRef]
  19. Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9, 671–675. [Google Scholar] [CrossRef]
  20. Suomivuori, C.M.; Latorraca, N.R.; Wingler, L.M.; Eismann, S.; King, M.C.; Kleinhenz, A.L.W.; Skiba, M.A.; Staus, D.P.; Kruse, A.C.; Lefkowitz, R.J.; et al. Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor. Science 2020, 367, 881–887. [Google Scholar] [CrossRef]
  21. Wingler, L.M.; Skiba, M.A.; McMahon, C.; Staus, D.P.; Kleinhenz, A.L.W.; Suomivuori, C.M.; Latorraca, N.R.; Dror, R.O.; Lefkowitz, R.J.; Kruse, A.C. Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR. Science 2020, 367, 888–892. [Google Scholar] [CrossRef] [PubMed]
  22. Cabana, J.; Holleran, B.; Leduc, R.; Escher, E.; Guillemette, G.; Lavigne, P. Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations. J. Biol. Chem. 2015, 290, 15835–15854. [Google Scholar] [CrossRef] [PubMed]
  23. Wingler, L.M.; Elgeti, M.; Hilger, D.; Latorraca, N.R.; Lerch, M.T.; Staus, D.P.; Dror, R.O.; Kobilka, B.K.; Hubbell, W.L.; Lefkowitz, R.J. Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations. Cell 2019, 176, 468–478. [Google Scholar] [CrossRef] [PubMed]
  24. Kawakami, K.; Yanagawa, M.; Hiratsuka, S.; Yoshida, M.; Ono, Y.; Hiroshima, M.; Ueda, M.; Aoki, J.; Sako, Y.; Inoue, A. Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying β-arrestin transducer bias. Nat. Commun. 2022, 13, 487. [Google Scholar] [CrossRef]
  25. Manglik, A.; Wingler, L.M.; Rockman, H.A.; Lefkowitz, R.J. β-Arrestin-Biased Angiotensin II Receptor Agonists for COVID-19. Circulation 2020, 142, 318–320. [Google Scholar] [CrossRef]
Biomedicines 11 01486 g001 550
Figure 1. (A) Construction of olmesartan and its related compounds. (B) Secondary structure of the angiotensin II type 1 (AT1) receptor.
Figure 1. (A) Construction of olmesartan and its related compounds. (B) Secondary structure of the angiotensin II type 1 (AT1) receptor.
Biomedicines 11 01486 g001
Biomedicines 11 01486 g002 550
Figure 2. Levels of IP production using various ligands in AT1 wild-type, -L112A, and Q257A receptors. IP, inositol phosphate; AT1, angiotensin II type 1; A.U. arbitrary unit. n = 5. * p < 0.05 vs. (-).
Figure 2. Levels of IP production using various ligands in AT1 wild-type, -L112A, and Q257A receptors. IP, inositol phosphate; AT1, angiotensin II type 1; A.U. arbitrary unit. n = 5. * p < 0.05 vs. (-).
Biomedicines 11 01486 g002
Biomedicines 11 01486 g003 550
Figure 3. Levels of ERK activities using various ligands in AT1 wild-type, -L112A, and Q257A receptors. t-ERK, total extracellular signal-regulated kinase; p-ERK, phospho-ERK; AT1, angiotensin II type 1; A.U. arbitrary unit. n = 5. * p < 0.05 vs. (-).
Figure 3. Levels of ERK activities using various ligands in AT1 wild-type, -L112A, and Q257A receptors. t-ERK, total extracellular signal-regulated kinase; p-ERK, phospho-ERK; AT1, angiotensin II type 1; A.U. arbitrary unit. n = 5. * p < 0.05 vs. (-).
Biomedicines 11 01486 g003
Biomedicines 11 01486 g004 550
Figure 4. Summary of IP production and ERK activities by olmesartan and its related compounds in AT1 wild-type, -L112A, and Q257A receptors. IP, inositol phosphate; ERK, extracellular signal-regulated kinase.
Figure 4. Summary of IP production and ERK activities by olmesartan and its related compounds in AT1 wild-type, -L112A, and Q257A receptors. IP, inositol phosphate; ERK, extracellular signal-regulated kinase.
Biomedicines 11 01486 g004
Table
Table 1. Binding affinities (Kd, nM) of [Sar1,Ile8]Ang II, olmesartan, R239470, R781253, and R794847 to AT1 wild-type and mutant receptors (n = 3).
Table 1. Binding affinities (Kd, nM) of [Sar1,Ile8]Ang II, olmesartan, R239470, R781253, and R794847 to AT1 wild-type and mutant receptors (n = 3).
ReceptorsWild-TypeL112AQ257AY292AN295A
Ligands
[Sar1,Ile8]Ang II0.8 ± 0.41.3 ± 0.33.3 ± 1.52.1 ± 0.9 7.0 ± 2.0
Olmesartan2.3 ± 0.827 ± 7228 ± 3847 ± 15 334 ± 105
R2394700.8 ± 0.337 ± 1220 ± 2 6.3 ± 3.869 ± 8
R78125321 ± 1023 ± 714 ± 6 95 ± 7>10,000
R794847 48 ± 1231 ± 5 2.7 ± 0.8 30 ± 8>10,000
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Matsuo, Y.; Suematsu, Y.; Morita, H.; Miura, S.-i. Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist. Biomedicines 2023, 11, 1486. https://doi.org/10.3390/biomedicines11051486

AMA Style

Matsuo Y, Suematsu Y, Morita H, Miura S-i. Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist. Biomedicines. 2023; 11(5):1486. https://doi.org/10.3390/biomedicines11051486

Chicago/Turabian Style

Matsuo, Yoshino, Yasunori Suematsu, Hidetaka Morita, and Shin-ichiro Miura. 2023. "Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist" Biomedicines 11, no. 5: 1486. https://doi.org/10.3390/biomedicines11051486

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop