Comparative evaluation of biased agonists Sarcosine1 , d-Alanine8 -Angiotensin (Ang) II (SD Ang II) and Sarcosine1 , Isoleucine8 -Ang II (SI Ang II) and their radioiodinated congeners binding to rat liver membrane AT1 receptors

Abstract

Angiotensin II analogue and β-arrestin biased agonist TRV027 (Sarcosine¹ , d-Alanine⁸ -Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β-arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT₁ receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using ¹²⁵ I-Sarcosine¹ , Isoleucine⁸ -Ang II (¹²⁵ I-SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT₁ receptors. We also compared radioiodinated TRV027 (¹²⁵ I-SD Ang II) binding affinity for liver AT₁ receptors with ¹²⁵ I-SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and ¹²⁵ I-SD Ang II had reduced affinity for the AT₁ receptor compared with SI Ang II and ¹²⁵ I-SI Ang II. Additionally, male-female comparisons showed that females have a higher AT₁ receptor density, potentially attributed to tissue-dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT₁ R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT₁ R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism.

Keywords: AT1R; GPCR; Sarcosine1, Isoleucine8-Angiotensin II; TRV027; binding assay; d-Alanine8-Angiotensin II.

FIGURE 1

Classic Ang II/AT₁R signaling pathway. Binding of Ang II to the extracellular domain of the AT₁R causes G protein to bind to the receptor intracellularly. Hydrolysis of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) by the α_q/11 subunit activates the protein, resulting in the dissociation of the α_q/11 from the βγ subunit. The α_q/11 subunit is then free to initiate the signaling pathway, activating secondary messengers, and leading to an increase in intracellular calcium and the activation of protein kinase C (PKC). At the AT₁R, G‐protein‐coupled receptor kinases 2/3 (GRK2/3), phosphorylate its intracellular domain, making it possible for β‐arrestin to bind to it, initiating receptor internalization, degradation, and recycling. Clathrin is a structural protein of the plasma membrane that plays a role in creating a “template” of rounded vesicles, such as endosomes, in the cytoplasm to be used in intracellular trafficking. Abbreviations: AKT, Ak strain transforming (also known as protein kinase B); DAG, diacylglycerol; ERK 1/2, extracellular signal‐regulated kinase 1/2; GRK 2/3, G‐protein‐coupled receptor kinase 2/3; GTP, guanosine triphosphate; IP₃, inositol trisphosphate; JNK, c‐Jun N‐terminal kinase; P, phosphate; PDK1, phosphoinositide‐dependent kinase‐1; PI3K, phosphoinositide 3‐kinase; MEK, mitogen‐activated protein kinase; MKK, mitogen‐activated protein kinase kinase; MNK1, mitogen‐activated protein kinase–interacting kinase 1; NF‐κB, nuclear factor kappa B; PIP₂, phospholipase C; PKC, protein kinase C; PLC, phospholipase C; Raf, rapidly accelerated fibrosarcoma; Src, SRC proto‐oncogene, non‐receptor tyrosine kinase.

FIGURE 2

SI Ang II and SD Ang II signaling pathway. Binding of biased agonists TRV027 (SD Ang II) or SI Ang II to the extracellular domain of the AT₁R causes the activation of the signaling pathway through β‐arrestin2. Before it can bind, the intracellular domain of the receptor needs to be phosphorylated. Studies have shown that binding of these ligands shift the preference toward GRK5/6, as opposed to GRK2/3, which is preferred by Ang II. Upon binding, proto‐oncogene tyrosine‐protein kinase Src, is recruited to bind to β‐arrestin2, initiating a variety of signaling pathways. Abbreviations as in Figure 1.

FIGURE 3

¹²⁵I‐SI Ang II saturation binding assay for female and male rat liver AT₁R. Representative binding in the female and male rat liver indicates saturable binding of ¹²⁵I‐SI Ang II, as analyzed by GraphPad Prism 9.0 using a one site specific binding linear regression. The pink and blue curves are representatives of one of five female and one of eight male rat liver membranes used, respectively.

FIGURE 4

¹²⁵I‐SI Ang II vs. ^125/127I‐SD Ang II saturation binding assays in female rat liver membranes. Representative figure of three saturation analyses using female rat liver membrane. B_max and K_D values were 35.1 fmol and 0.329 nM, respectively, for ¹²⁵I‐SI Ang II, and 5.09 fmol and 4.00 nM, respectively, for ¹²⁵I‐SD Ang II. Values were obtained by GraphPad Prism 9.0 using a one site specific binding linear regression.

FIGURE 5

Comparison of SD Ang II and SI Ang II competition binding to male and female rat liver AT₁R. Representative competition binding for ¹²⁵ I‐SI Ang II by SD Ang II and SI Ang II in the male rat liver (n = 6; A) show IC₅₀ values of 225 and 7.27 nM, respectively. As for competition binding in the female liver rat (n = 3; B), these values are 181 and 9.40 nM, respectively. Analyses were made by GraphPad Prism 9.0 using a one site competition binding algorithm. The values indicated in the y‐axis are the amount bound in the absence of any competing ligand.

FIGURE 6

Comparison of SD Ang II and SI Ang II K_i values for binding to male and female rat liver membrane AT₁Rs. Comparison by two‐way ANOVA showed that there is a significant difference in K_i values between the two competing ligands, F(1, 7) = 111.6, p < .0001. There was a less significant difference between males (n = 6) and females (n = 3) (p = .0258).

FIGURE 7

Comparison of ¹²⁵I‐SI Ang II binding to AT₁Rs in male and female rat liver membranes. Comparisons done using unpaired t‐tests showed that (A) There is a significantly greater (t = 7.01, p < .0001) AT₁R density in females (n = 5) compared to males (n = 8), and (B) There is no difference in K_D values (t = 0.800, p = .442).