Selective Nonpeptidic Fluorescent Ligands for Oxytocin Receptor: Design, Synthesis, and Application to Time-Resolved FRET Binding Assay

The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor

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

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

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

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

From the Promiscuous Asenapine to Potent Fluorescent Ligands Acting at a Series of Aminergic G‑Protein-Coupled Receptors

Monoamine neurotransmitters such as serotonin, dopamine, histamine, and noradrenaline have important and varied physiological functions and similar chemical structures. Representing important pharmaceutical drug targets, the corresponding G-protein-coupled receptors (termed aminergic GPCRs) belong to the class of cell membrane receptors and share many levels of similarity as well. Given their pharmacological and structural closeness, one could hypothesize the possibility to derivatize a ubiquitous ligand to afford rapidly fluorescent probes for a large set of GPCRs to be used for instance in FRET-based binding assays. Here we report fluorescent derivatives of the nonselective agent asenapine which were designed, synthesized, and evaluated as ligands of 34 serotonin, dopamine, histamine, melatonin, acetylcholine, and adrenergic receptors. It appears that this strategy led rapidly to the discovery and development of nanomolar affinity fluorescent probes for 14 aminergic GPCRs. Selected probes were tested in competition binding assays with unlabeled competitors in order to demonstrate their suitability for drug discovery purposes

Time-Resolved FRET Binding Assay to Investigate Hetero-Oligomer Binding Properties: Proof of Concept with Dopamine D₁/D₃ Heterodimer

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D₁ and D₃ receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D₁/D₃ heteromer. Based on the high-resolution structure of the dopamine D₃ receptor (D₃R), six fluorescent probes derived from a known D₃R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D₃/D₂ receptors, and for other dopamine receptor subtypes. The highest affinity ligand <b>21</b> was then employed in the development of the D₁/D₃ heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D₁ receptor (D₁R) and a fluorescent acceptor D₃R ligand <b>21</b>. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands