Nanobody-Mediated Dualsteric Engagement of the Angiotensin Receptor Broadens Biased Ligand Pharmacology

Abstract

Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or β-arrestin (TRV027). While the nanobody itself acts as a probe-specific neutral or negative allosteric ligand of N-terminally 6e-tagged AT1R, nanobody conjugation to orthosteric ligands had varying effects on Gq dissociation and β-arrestin plasma membrane recruitment. The potency of certain AngII analogs was enhanced up to 100-fold, and some conjugates behaved as partial agonists, with up to a 5-fold decrease in maximal efficacy. Nanobody conjugation also biased the signaling of TRV055 and TRV056 toward Gq, suggesting that Gq bias at AT1R can be modulated through molecular mechanisms distinct from those previously elucidated. Both competition radioligand binding experiments and functional assays demonstrated that orthosteric antagonists (angiotensin receptor blockers) act as non-competitive inhibitors of all these nanobody-peptide conjugates. This proof-of-principle study illustrates the array of pharmacological patterns that can be achieved by incorporating neutral or negative allosteric pharmacophores into dualsteric ligands. Nanobodies directed toward linear epitopes could provide a rich source of allosteric reagents for this purpose. SIGNIFICANCE STATEMENT: Here we engineer bitopic (dualsteric) ligands for epitope-tagged angiotensin II type 1 receptor by conjugating angiotensin II or its biased analogs to an epitope-specific nanobody (antibody fragment). Our data demonstrate that nanobody-mediated interactions with the receptor N-terminus endow angiotensin analogs with properties of allosteric modulators and provide a novel mechanism to increase the potency, modulate the maximal effect, or alter the bias of ligands.

Graphical abstract

Fig. 1.

Design of dualsteric AT1R ligands. (A) Schematic of 6e-tagged AT1R construct. (B,C) Comparison of AngII-induced activation of (B) Gq dissociation and (C) β-arrestin2 recruitment to the plasma membrane by transiently transfected wild-type AT1R (FLAG-AT1R lacking the 6e epitope) or the 6e-AT1R construct shown in panel A. (D) Effect of a 15-minute pre-incubation with 1 μM control nanobody or unconjugated Nb6e on β-arrestin2 plasma membrane recruitment by the indicated ligands at 6e-AT1R. Curve fit parameters are provided in Supplemental Table 3. Data in panels B–D are normalized to the response induced by 1 μM AngII and represent the mean ± S.D. of three independent experiments each performed with two technical replicates. (E) Synthesis of Nb6e-AT1R ligand conjugates by sortase A (Srt A)-mediated ligation. AT1R ligands with the indicated pharmacological profiles and peptide sequences were modified at their N-termini with a triglycine motif (G₃) and one of the linkers shown. The N-terminal G₃ moiety enables ligands to be ligated to Nb6e with a C-terminal “LPETGG” tag, followed by a hexahistidine (H6) epitope tag that is removed upon ligation.

Fig. 2.

Functional activity of Nb6e-Ahx-AT1R ligand conjugates. (A–P) Activity of the parental AT1R ligands, the G₃-Ahx-modified ligands, and the Nb6e-Ahx-ligand conjugates in assays for Gq dissociation (left half of figure) and β-arrestin2 recruitment to the plasma membrane (right half of figure) in cells expressing either 6e-AT1R or wild-type AT1R as indicated. Data are normalized to the response induced by 1 μM AngII and represent the mean ± S.D. of three independent experiments each performed with two technical replicates. Curve fit parameters are provided in Supplemental Table 1.

Fig. 3.

Bias plots of Nb6e-Ahx-AT1R ligand conjugates. (A–D) Using the data shown in Fig. 2, the normalized β-arrestin2 recruitment response is plotted versus the normalized Gq dissociation response for the same concentration of ligand. Data represent the mean ± S.D. of three independent experiments each performed with two technical replicates.

Fig. 4.

Interaction of Nb6e-Ahx-AT1R ligand conjugates with the orthosteric ligand-binding site. (A–D) Competition of ligands with [³H]-olmesartan in cell membranes overexpressing 6e-AT1R. Data from each individual experiment are normalized to the top and bottom of the competition curve of the parental ligands. Data for in panel D were normalized to the TRV055 competition curve due to the low affinity of TRV056. Data represent the mean ± S.D. of three independent experiments performed with single technical replicates. Curve fit parameters are provided in Supplemental Table 1. (E) Model of competition between the orthosteric [³H]-olmesartan RL and dualsteric Nb-peptide conjugates. The cooperativity factor α describes the effect that nanobody binding to the allosteric site only has on radioligand binding. (F) Simulation of competition binding curves for dualsteric Nb-peptide conjugates with varying values of K_Nb+pep based on the model shown in panel E, eq. 2, the indicated values of α and K_Nb, and the experimentally determined values of K_RL and [RL] (see main text).

Fig. 5.

Characterization of Nb6e-AngII conjugates with varying linker lengths. (A–C) Activity of the parental AT1R ligands, the G₃-linker-modified ligands, and the Nb6e-linker-AngII conjugates in assays for Gq dissociation (left panels) and β-arrestin2 recruitment to the plasma membrane (middle panels) in cells expressing 6e-AT1R. Data are normalized to the response induced by 1 μM AngII and represent the mean ± S.D. of three independent experiments each performed with two technical replicates. Competition of ligands with [³H]-olmesartan in cell membranes overexpressing 6e-AT1R is shown in the right panels. Data from each individual experiment are normalized to the top and bottom of the AngII competition curve. Data represent the mean ± S.D. of three independent experiments performed with single technical replicates. Curve fit parameters for all experiments are provided in Supplemental Table 1.

Fig. 6.

Characterization of Nb6e-TRV027 conjugates with varying linker lengths. (A–C) Activity of the parental AT1R ligands, the G₃-linker-modified ligands, and the Nb6e-linker-TRV027 conjugates in β-arrestin2 recruitment to the plasma membrane (left panels) in cells expressing 6e-AT1R. Data are normalized to the response induced by 1 μM AngII and represent the mean ± S.D. of three independent experiments each performed with two technical replicates. Competition of ligands with [³H]-olmesartan in cell membranes overexpressing 6e-AT1R is shown in the right panels. Data from each individual experiment are normalized to the top and bottom of the TRV027 competition curve. Data represent the mean ± S.D. of three independent experiments performed with single technical replicates. Curve fit parameters for all experiments are provided in Supplemental Table 1.

Fig. 7.

Inhibition of β-arrestin2 plasma membrane recruitment induced by orthosteric and dualsteric ligands. (A–C) Cells overexpressing 6e-AT1R were pre-treated with varying concentrations of the orthosteric antagonist losartan (A,B) or unconjugated Nb6e (C) for 15 minutes before stimulation with parental AT1R ligands (A) or Nb6e-Ahx-AT1R ligand conjugates (B,C) for 20 minutes. Data are normalized to the response induced by 1 μM AngII in the absence of antagonists and represent the mean ± S.D. of three independent experiments each performed with two technical replicates. Parameters derived from (A) Gaddum/Schild or (B) operational model of allosterism curve fits are provided in Supplemental Tables 5 and 6, respectively. (D) Summary of the varying effects that conjugation of Nb6e has on the pharmacological properties of AT1R ligands.