Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V₂ GPCR: Application To Ligand Screening and Oligomerization Assays.

A series of fluorescent benzazepine ligands for the arginine–vasopressin V₂ receptor (AVP V₂R) was synthesized using “Click” chemistry. Their in vitro pharmacological profile at AVP V₂R, V_1aR, V_1bR, and oxytocin receptor was measured by binding assay and functional studies. Compound <b>9p</b>, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V₂R (4.0 nM), an excellent selectivity toward V₂R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes <b>44</b> and <b>47</b> still display a high affinity for V₂R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V₂R. They enabled the development of V₂R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V_1aR–V₂R dimerization on cell surface

Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V₂ GPCR: Application To Ligand Screening and Oligomerization Assays.

A series of fluorescent benzazepine ligands for the arginine–vasopressin V₂ receptor (AVP V₂R) was synthesized using “Click” chemistry. Their in vitro pharmacological profile at AVP V₂R, V_1aR, V_1bR, and oxytocin receptor was measured by binding assay and functional studies. Compound <b>9p</b>, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V₂R (4.0 nM), an excellent selectivity toward V₂R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes <b>44</b> and <b>47</b> still display a high affinity for V₂R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V₂R. They enabled the development of V₂R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V_1aR–V₂R dimerization on cell surface

Pharmacological properties of the R137C and R137L V2 receptors compared to the those of the wild-type and D136A receptor.

*<p>: the values cannot be determined since the curves do not reach a plateau.</p>**<p>: values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008383#pone.0008383-Morin2" target="_blank">[19]</a>.</p

β-arrestin 1 recruitment to V2R studied by BRET.

<p><b>a</b>, For BRET measurements, V2 receptors and β-arrestin 1 were fused to Rluc and YFP proteins, respectively, and co-expressed in COS-7 cells treated or not with AVP for 45 minutes. <b>b</b>, AVP-induced β-arrestin 1 recruitment to either V2 wild-type, R137C, R137L or D136A mutants. Data are means±S.E.M of three independent experiments. <b>c</b>, Expression levels of BRET partners determined by Rluc luminescence and YFP fluorescence <b>d</b>, Dose-response of AVP-induced BRET after AVP stimulation for 45 minutes. <b>e</b>, BRET time-course: BRET increase between V2 receptors and β-arrestin 1 after AVP stimulation (1 µM) at the indicated time. Data are means±S.E.M of three independent experiments.</p

Coupling properties of the wild-type and mutants receptors.

<p><b>a</b>, Basal, agonist induced and antagonist-inhibited cAMP accumulation was measured on cos 7 cells expressing wild-type or mutants receptors. Values of cAMP accumulation were normalized to the number of receptors expressed at the surface of the cells determined by ligand binding [3H]AVP. <b>b</b>, AVP dose-response experiments performed on cells expressing wild-type, R137C or R137L V2 receptor. <b>c</b>, effect of an inverse agonist, SR121463, on AVP-induced stimulation.</p