125‐I‐Sarcosine(1), D‐Alanine(8) Angiotensin II as a Biased Radioligand for AT1 Angiotensin II Receptors

Abstract

The concept of biased agonism of G protein‐coupled receptors arose from studies of the AT1 subtype of angiotensin (Ang) II receptor. A putative antagonist ligand, sarcosine1, isoleucine4, isoleucine8 Ang II (SII) showed negligible ability to activate G protein signaling pathways of the AT1 receptor, but was able to cause AT1 receptor internalization and mitogen activated protein (MAP) kinase activation at micromolar concentrations. Subsequently, a different Ang II congener; sarcosine1, D‐alanine8 Ang II was developed as a potential therapeutic (TRV‐027; TRV120027) for treatment of acute heart failure. While clinical trials did not support a therapeutic benefit of sarcosine1, D‐alanine8 Ang II for treatment of heart failure, it still promises to be a better biased agonist than SII, which acts only at micromolar concentrations. To assess the potential use of sarcosine1, D‐alanine8 Ang II as a radioligand for quantification of Ang II receptor binding, we radiolabeled sarcosine1, D‐alanine8 Ang II with 125iodine and compared its binding to that of 125I‐sarcosine1 isoleucine8 Ang II in rat liver membranes, which express high levels of AT1 receptors and negligible levels of AT2 receptors. At concentrations up to 5.6 nM, 125I‐sarcosine1, D‐alanine8 Ang II showed little evidence of saturable binding to AT1 receptors, in contrast to 125I‐sarcosine1 isoleucine8 Ang II, which showed a KD of 0.6 nM. At 5.6 nM 125I‐sarcosine1 isoleucine8 Ang II binding to AT1 receptors was 8.5 times greater than 125I‐sarcosine1, D‐alanine8 Ang II. To verify that sarcosine1, D‐alanine8 Ang II binds to liver membrane AT1 receptors, we compared the ability of sarcosine1, D‐alanine8 Ang II and Ang II to compete for 125I‐sarcosine1 isoleucine8 Ang II binding. Sarcosine1 isoleucine8 Ang II and Ang II were similar in their ability to compete for AT1 receptor binding: 6.7±0.7 nM and 15.6±0.8 nM and 84.3±1.1 and 86.7±0.3 % of total binding (IC50, n=2±SD), respectively. This suggests that iodination of tyr4 of sarcosine1, D‐alanine8 Ang II alters its ability to bind to AT1 receptors and potentially act as a biased agonist. Continuing studies will examine the ability of higher concentrations of 125/127I‐sarcosine1, D‐alanine8 Ang II to interact with Ang II receptors.Support or Funding InformationNova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund