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

The concept of biased agonism of G protein‐coupled receptors arose from studies of the AT 1 subtype of angiotensin (Ang) II receptor. A putative antagonist ligand, sarcosine 1 , isoleucine 4 , isoleucine 8 Ang II (SII) showed negligible ability to activate G protein signaling pathways of the AT 1 receptor, but was able to cause AT 1 receptor internalization and mitogen activated protein (MAP) kinase activation at micromolar concentrations. Subsequently, a different Ang II congener; sarcosine 1 , D‐alanine 8 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 sarcosine 1 , D‐alanine 8 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 sarcosine 1 , D‐alanine 8 Ang II as a radioligand for quantification of Ang II receptor binding, we radiolabeled sarcosine 1 , D‐alanine 8 Ang II with 125 iodine and compared its binding to that of 125 I‐sarcosine 1 isoleucine 8 Ang II in rat liver membranes, which express high levels of AT 1 receptors and negligible levels of AT 2 receptors. At concentrations up to 5.6 nM, 125 I‐sarcosine 1 , D‐alanine 8 Ang II showed little evidence of saturable binding to AT 1 receptors, in contrast to 125 I‐sarcosine 1 isoleucine 8 Ang II, which showed a K D of 0.6 nM. At 5.6 nM 125 I‐sarcosine 1 isoleucine 8 Ang II binding to AT 1 receptors was 8.5 times greater than 125 I‐sarcosine 1 , D‐alanine 8 Ang II. To verify that sarcosine 1 , D‐alanine 8 Ang II binds to liver membrane AT 1 receptors, we compared the ability of sarcosine 1 , D‐alanine 8 Ang II and Ang II to compete for 125 I‐sarcosine 1 isoleucine 8 Ang II binding. Sarcosine 1 isoleucine 8 Ang II and Ang II were similar in their ability to compete for AT 1 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 tyr 4 of sarcosine 1 , D‐alanine 8 Ang II alters its ability to bind to AT 1 receptors and potentially act as a biased agonist. Continuing studies will examine the ability of higher concentrations of 125/127 I‐sarcosine 1 , D‐alanine 8 Ang II to interact with Ang II receptors. Support or Funding Information Nova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund