5 research outputs found
Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V<sub>2</sub> GPCR: Application To Ligand Screening and Oligomerization Assays.
A series of fluorescent benzazepine ligands for the arginineāvasopressin
V<sub>2</sub> receptor (AVP V<sub>2</sub>R) was synthesized using
āClickā chemistry. Their in vitro pharmacological profile
at AVP V<sub>2</sub>R, V<sub>1a</sub>R, V<sub>1b</sub>R, 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 V<sub>2</sub>R (4.0 nM), an excellent selectivity toward V<sub>2</sub>R
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 V<sub>2</sub>R (5.6 and 5.8 nM, respectively).
These antagonists constitute the first high-affinity selective nonpeptidic
fluorescent ligands for V<sub>2</sub>R. They enabled the development
of V<sub>2</sub>R 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<sub>1a</sub>RāV<sub>2</sub>R dimerization on cell surface
Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V<sub>2</sub> GPCR: Application To Ligand Screening and Oligomerization Assays.
A series of fluorescent benzazepine ligands for the arginineāvasopressin
V<sub>2</sub> receptor (AVP V<sub>2</sub>R) was synthesized using
āClickā chemistry. Their in vitro pharmacological profile
at AVP V<sub>2</sub>R, V<sub>1a</sub>R, V<sub>1b</sub>R, 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 V<sub>2</sub>R (4.0 nM), an excellent selectivity toward V<sub>2</sub>R
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 V<sub>2</sub>R (5.6 and 5.8 nM, respectively).
These antagonists constitute the first high-affinity selective nonpeptidic
fluorescent ligands for V<sub>2</sub>R. They enabled the development
of V<sub>2</sub>R 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<sub>1a</sub>RāV<sub>2</sub>R 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
Ī²-arrestin 1 recruitment to V2R studied by BRET.
<p><b>a</b>, For BRET measurements, V2 receptors and Ī²-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 Ī²-arrestin 1 recruitment to either V2 wild-type, R137C, R137L or D136A mutants. Data are meansĀ±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 Ī²-arrestin 1 after AVP stimulation (1 ĀµM) at the indicated time. Data are meansĀ±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