Angiotensin II receptor type 1 (AT1R)

The equimolar comparison clearly identifies such compounds (TRV120026, TRV120034, TRV120045, TRV120044, and SGG) as β-arrestin-biased ligands, whereas the other compounds seem to be balanced (TRV120055, TRV120056, A1, and S1C4) (Fig. 6A; Supplemental Fig. S4). For example, the SGG and TRV120044 compounds are shifted to the left portion of the plot, whereas the balanced agonists AngII and TRV120055 both have similar hyperbolic shapes consistent with increased amplification in the IP1 assay compared with the β-arrestin recruitment assay (Fig. 6A). The plots for these two β-arrestin-biased compounds suggest that TRV120044 (red) has more β-arrestin bias than SGG (green), although it is difficult to ascertain in such a qualitative analysis. Bias factors for all of the compounds using the equiactive approach were then calculated (Fig. 6B). Consistent with the equimolar comparison, the TRV120026, TRV120034, TRV120044, TRV120045, and SGG compounds all had bias factors consistent with β-arrestin bias, although the large errors for a number of these compounds led to the differences being statistically insignificant. This was due to the poor fits of the IP1 concentration-response data, in which many of the compounds displayed little to no signaling activity [1].

---

Third, consistent with the prediction made for biased activation of Gq over βarr2 (Fig. 1C), the molecular efficacies of Gq-biased agonists TRV120055 and TRV120056 were 10-fold larger at the AT1R-Gq fusion protein compared with the AT1R-βarr2 fusion protein (Fig. 3C). The Gq molecular efficacies of TRV120055 and TRV120056 were, in fact, the largest of any ligand tested (Table 1). This is consistent with their extremely high efficacy values for activating Gq in cells (17). Fourth, as predicted (Fig. 1D), the antagonist telmisartan lacked detectable molecular efficacy at either transducer (Fig. 3D).[2]
A similar relationship was observed for βarr2. Efficacies corresponding to the abilities of AngII, TRV120055, TRV120056, TRV120023, and SII to promote AT1R internalization in cells (i.e. a classic βarr-dependent process (4); Fig. 5C) were significantly correlated with their βarr2 molecular efficacies calculated at the AT1R-βarr2 fusion protein (r2 = 0.7909, p = 0.0435 by two-tailed Pearson correlation; Fig. 5D). Because of the low level of amplification in the internalization assay, strong agreement was also observed between maximal cellular responses (Emax) and βarr2 molecular efficacies (r2 = 0.9140, p = 0.011 by two-tailed Pearson correlation). Rank orders of potency and efficacy (calculated as τ and Emax values) were conserved in a shorter internalization assay (30 min), alleviating concerns that cellular contributions over the 3-h incubation period confounded internalization measurements [2].

β-Arrestin Recruitment Assays.[1]
For the β2AR, β-arrestin recruitment to receptor was assessed by the Tango assay, as described previously by Barnea et al. (2008). In this assay, the C terminus of the human β2AR is replaced with the C-terminal tail of the V2 vasopressin receptor tail (to increase signal-to-noise ratio) followed by a Tobacco Etch Virus (TEV) protease cleavage site and a tTA transcription factor. This construct was stably transfected in HEK293 cells along with a construct encoding β-arrestin 2 fused to TEV protease. Upon ligand stimulation, the recruitment of β-arrestin to the receptor results in the cleavage tTA from the receptor. The tTA translocates to the nucleus, in which it transcribes a stably expressing luciferase reporter gene. HEK293 cells stably transfected with these constructs were seeded at 2.5 × 104 cells per well in a 96-well plate. The next day, compounds diluted in phosphate-buffered saline were added to the wells to their final concentration followed by incubation at 37°C for 14 to 20 h. The next day, the plate was cooled to room temperature, and an equal amount of Bright-Glo luciferase assay reagent was added to each well. After 5 min, luminescence was read in a NOVOstar microplate reader. To ensure that the results obtained using this technology were not an artifact of the overnight incubation with ligand or the V2R tail, we also used the PathHunter β-arrestin assay from DiscoveRx (see below), which uses the human β2AR (with a Prolink peptide added to the C terminus) with a shorter incubation time with ligand (∼30 min), the representative data of which are shown in Supplemental Fig. S5.[1]
For the AT1AR, we used the PathHunter β-arrestin assay from DiscoveRx and read for chemiluminescent signaling on a PheraStar reader as described previously (Violin et al., 2010). In brief, complementary halves of β-galactosidase were genetically fused to the carboxyl termini of the human AT1R and β-arrestin2. When cotransfected, the two fusion proteins serve as a proximity sensor; when β-arrestin 2 translocates to active receptor, the β-galactosidase fragments interact to form a functional enzyme, which is detected by a chemoluminescent substrate.

---

cAMP Assay.[1]
The GloSensor cAMP biosensor (Promega) uses a modified form of firefly luciferase containing a cAMP-binding motif (Fan et al., 2008). Upon cAMP binding, a conformational change leads to enzyme complementation and incubation with a luciferase substrate results in a luminescence readout. Analysis of cAMP accumulation was performed in HEK293 cells stably transfected with the Glosensor construct and the human β2AR. Cells were seeded in 96-well white, clear-bottomed plates at 8 × 104 cells/well, in minimal essential medium supplemented with 10% fetal bovine serum [10% (v/v)]. The next day, the GloSensor reagent [4% (v/v)] was incubated at room temperature for 2 h. Cells were then stimulated with a range of β2 AR agonists for 5 min, and increases in luminescence were read on a NOVOstar microplate reader. These assays were repeated in the Tango cell lines used for the β-arrestin recruitment assays with transient transfection of the Glosensor construct, which demonstrated the same behavior, albeit with poorer signal-to-noise ratio (Supplemental Fig. S6).

---

Inositol 1-Phosphate Assay.[1]
Inositol 1-phosphate (IP1), a downstream metabolite of inositol trisphosphate, which itself is downstream of signaling by Gq, was detected by the IP-One Tb HTRF kit as described previously (Violin et al., 2010). Plates were read on a PheraStar reader using a time-resolved fluorescence ratio (665/620 nm).

---

Angiotensin II Type IA Receptor Competition Membrane Radioligand Binding Assays.[1]
HEK293 cells with stable expression of the rat (r) AT1 receptor were harvested by centrifugation at 400g for 30 min at 4°C, washed once with a balanced salt solution, repelleted, and the pellet was flash-frozen in liquid nitrogen. The cell pellets were stored at −80°C until processed for membranes. Pellets were resuspended in buffer (50 mM HEPES, 2 mM EDTA, pH 7.4) containing fresh protease inhibitors, Complete Brand protease tablets from Roche Diagnostics, and subjected to nitrogen cavitation with a Parr Cell Disruption Bomb at 1000 psi for 20 min on ice. Ruptured cells were sedimented at 500g for 10 min at 4°C, and the supernatant containing cellular membranes was washed twice at 48,000g for 15 min. cell pellets were resuspended at 4°C in 10 volumes of ice-cold buffer A and cavitation, placed on ice. To remove large particles, a low-speed centrifugation (500g for 30 min at 4°C) was performed, followed by high-speed centrifugation (48,000g for 45 min at 4°C), resuspension in buffer plus protease inhibitor cocktail, and a final high-speed centrifugation at (48,000g for 45 min at 4°C). A Dounce homogenizer was used to resuspend the final pellet using ice-cold buffer. The membrane suspension was passed through a 23-gauge needle, and aliquots were made and stored at −80°C. Total protein concentration of the membrane preparation was determined with a Coomassie Plus Reagent Kit from Pierce Biotechnology using bovine serum albumin as the standard.[1]
Membranes were diluted in assay buffer [50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, Gpp(NH)p 10 μM, pH 7.2, at 23°C] to a concentration of 1 to 3 μg of protein/well. Assays were initiated by the addition of 94 μl of membrane suspension to 200 μl of [125I]Sar1Ile8-angiotensin II (specific activity, 2200 Ci/mmol), at 0.4 to 1 times Kd and various concentrations of inhibitors in buffer plus a cocktail of protease inhibitors and 0.02% bovine serum albumin to reduce nonspecific radioligand binding. Compounds were diluted in dimethyl sulfoxide and tested at a final concentration of 1% dimethyl sulfoxide (determined to be nondetrimental to the assay). Competition binding with compounds (11-point concentrations) was performed in polypropylene 96-well plates. Nonspecific binding was defined in the presence of 10 μM losartan. Competition assays were performed at 23°C for 4 h to allow adequate time for compounds and radioligand to reach equilibrium for binding. The separation of bound from free radioligand was accomplished by rapid vacuum filtration of the incubation mixture over GF/B unifilter (polyethylenimine-treated) plates using a Brandel cell harvester. Filters were washed two times with 0.3 ml of ice-cold phosphate-buffered saline, pH 7.0, containing 0.01% Triton X-100. Radioactivity on the filters was quantified using a MicroBeta TriLux Liquid Scintillation Counter.

---

Radioligand Binding Assays [2]
Competition radioligand binding assays using unfused and transducer-fused AT1R-expressing membranes were conducted in parallel to facilitate rigorous analysis (see “Experimental Procedures”). Competition binding assays measuring Gq molecular efficacy were performed in a Gq assay buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 12.5 mm MgCl2, and 0.2% BSA) containing 50 pm 125I-Sar1,Ile8-AngII, AT1R (0.5 μg) or AT1R-Gq (0.25 μg) membranes, and a serial dilution of test ligand. Binding assays measuring βarr2 molecular efficacy were performed in βarr2 assay buffer (50 mm Tris-HCl, pH 7.4, 50 mm potassium acetate, 150 mm NaCl, 5 mm MgCl2, and 0.2% BSA) containing 50 pm 125I-Sar1,Ile8-AngII, AT1R + CAAX GRK2 (1 μg) or AT1R-βarr2 + CAAX GRK2 (0.4 μg) membranes, and a serial dilution of test ligand. Nonspecific binding was determined in the presence of 10 μm telmisartan, whereas total binding was determined in the absence of a competitor. To ensure accurate low affinity determination, binding assays on unfused AT1R membranes included a non-hydrolyzable GTP analog 5′-guanylyl imidodiphosphate (GPP(NH)P) at 1 μm for Gq assay and 100 μm for βarr2 assay. After a 1.5-h incubation at 25 °C, bound radioactivity was collected on 0.3% polyethyleneimine-treated GF/C filters using cold Gq wash buffer (50 mm Tris-HCl, pH 7.4) or cold βarr2 wash buffer (50 mm Tris-HCl, pH 7.4, and 50 mm potassium acetate). Bound radioactivity was quantified on a Packard Cobra gamma counter. Saturation binding assays were performed on AT1R-Gq and AT1R-βarr2 membranes as described above except that serial dilutions of 125I-AngII (5 pm to 1 nm) and 125I-Sar1,Ile8-AngII (5 pm to 2 nm) were used to label transducer-coupled and total AT1R populations, respectively.

---

IP1 Accumulation [2]
IP1 accumulation was measured using the IP-One Tb HTRF kit as described previously (18).

---

AT1R Internalization Assays [2]
βarr-dependent AT1R internalization was measured using the activated endocytosis assay. Briefly, the human AT1R was transiently expressed in U2OS cells stably expressing an Enzyme Acceptor-tagged βarr2 and an endosome-localized ProLink tag. Cells (20,000 cells/well) were incubated in 96-well plates for 24 h and stimulated with test ligand (100 μm to 0.1 nm) for 30 min or 3 h at 37 °C. AT1R internalization was detected as luminescence resulting from the complementation of β-galactosidase fragments (Enzyme Acceptor and ProLink) within endosomes. Luminescence was detected on a NOVOstar plate reader using the PathHunter Detection kit.

---

βarr2 Recruitment Assays [2]
The PathHunter βarr assay from DiscoveRx measured recruitment of βarr2 to the AT1R for cellular bias calculations as described previously.

---

ERK1/2 Phosphorylation Assays [2]
ERK1/2 phosphorylation was measured using the Cellul'erk HTRF kit. HEK 293 cells (50,000 cells/well) transiently expressing the unfused AT1R, AT1R-Gq, or AT1R-βarr2 fusion proteins were stimulated with 1 μm AngII for 10 min at 37 °C. Plates were read on a PheraStar reader using a time-resolved fluorescence ratio of 665 nm/615 nm. ERK1/2 phosphorylation was reported as -fold over base-line response.

Co-immunoprecipitation [Nat Commun. 2017 Feb 9;8:14335.]
Co-immunoprecipitation was performed as previously described. HEK293 cells were co-transfected with Flag-AT1R-cherry, TRPC3-GFP (WT/Truncations) and HA-β-arrestin1 (WT/Truncations/Mutants). Forty-eight hours after transfection, the cells were starved for 12 h and then stimulated with Ang II (100 nM), TRV120055 (30 nM) or TRV120027 (100 nM) for 1 min. Subsequently, the cells were washed with cold PBS and then collected in cold lysis buffer. The cell lysates were subjected to immunoprecipitation using different antibody-conjugated beads (Flag or HA-conjugated beads), which were incubated overnight at 4 °C. Immune complexes containing AT1R or arrestin were extensively washed for at least five times with cold lysis buffer and analysed by western blotting with specific antibodies.

[1]. Quantifying ligand bias at seven-transmembrane receptors. Mol Pharmacol. 2011 Sep;80(3):367-77.

[2]. Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR). J Biol Chem. 2014 May 16; 289(20): 14211-24.

Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, β-arrestins. Biased agonists, which signal with different efficacies to a receptor's multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the β2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs. [1]

---

The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR. [2]

C34H54N12O7

742.868766307831

742.423

2410957-04-7

154573825

Sar-Arg-Val-Tyr-Val-His-NH2; H-Sar-Arg-Val-Tyr-Val-His-NH2; sarcosyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidinamide

GRVYVH; {Sar}-RVYVH-NH2

White to off-white solid powder

-0.8

11

10

22

53

1250

5

CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC2=CN=CN2)C(=O)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC

JZYGWEQFALQYNB-OQWKOXFXSA-N

InChI=1S/C34H54N12O7/c1-18(2)27(32(52)43-24(29(35)49)14-21-15-39-17-41-21)46-31(51)25(13-20-8-10-22(47)11-9-20)44-33(53)28(19(3)4)45-30(50)23(42-26(48)16-38-5)7-6-12-40-34(36)37/h8-11,15,17-19,23-25,27-28,38,47H,6-7,12-14,16H2,1-5H3,(H2,35,49)(H,39,41)(H,42,48)(H,43,52)(H,44,53)(H,45,50)(H,46,51)(H4,36,37,40)/t23-,24-,25-,27-,28-/m0/s1

(2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-5-(diaminomethylideneamino)-2-[[2-(methylamino)acetyl]amino]pentanamide

TRV120055; 2410957-04-7; CHEMBL4588946; (2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-5-(diaminomethylideneamino)-2-[[2-(methylamino)acetyl]amino]pentanamide; Sar-Arg-Val-Tyr-Val-His-NH2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 25 mg/mL (33.65 mM)

Note: Please refer to the "Guidelines for Dissolving Peptides" section in the 4th page of the "Instructions for use" file (upper-right section of this webpage) for how to dissolve peptides.

---

Solubility in Formulation 1: ≥ 0.62 mg/mL (0.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.2 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 0.62 mg/mL (0.83 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.2 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 0.62 mg/mL (0.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)