Design, Synthesis, and Pharmacological Characterization of Fluorescent Peptides for Imaging Human V1b Vasopressin or Oxytocin Receptors

Abstract

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described due to the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist, [deamino-Cys1, Leu4, Lys8]vasopressin (d[Leu4,Lys8]VP) synthesized earlier, we added blue, green or red fluorophores to the lysine residue at position 8, either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu4, Lys (Alexa 647)8]VP (3) and d[Leu4, Lys (11-aminoundecanoyl-Alexa 647)8]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localisation of V1b or OT receptor isoforms in native tissues.

Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems

“…These pharmacological properties of terlipressin seem beneficial for the treatment of septic shock. Compared with the defined clinical applications of AVP agonists directed to V1a and V2 receptors, the clinical utility of V1b receptor agonists remains to be clarified, although synthetic ligands against V1b receptors have been developed recently and knowledge is accumulating (98,323).…”

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…Although many different fluorophores have been effectively used to generate useful fluorescent ligands the main classes of fluorophores used have been the BODIPY series (Allen et al, 2000;Baker et al, 2011;Daly et al, 2010;Tomasch et al, 2012b;Vernall et al, 2012), the Alexa series (Arttamangkul et al, 2000;Brand et al, 2008;Corbani et al, 2011;Morishima et al, 2010) and rhodamine derivatives (Castro et al, 2005;Daly et al, 2010;Guo et al, 2012). Fluorescent ligands containing these fluorophores have been effectively used in many of the techniques detailed below with the exception of certain time-resolved fluorescence resonance energy transfer (TR-FRET) studies in which europium linked fluorescent ligands were developed to provide a signal with a long fluorescence lifetime (Albizu et al, 2010).…”

“…To perform useful microscopy studies, a fluorescent ligand for a GPCR needs to show discrete plasma membrane binding which can be blocked by an unlabelled ligand, and low levels of non-specific binding (including intracellular fluorescence). There are many examples in the literature of fluorescent ligands with good imaging properties, including those for the vasopressin and oxytocin receptors (Corbani et al, 2011), kappa opioid receptor (Houghten et al, 2004), b 2 adrenergic receptor (Baker et al, 2011), adenosine-A 3 receptor and the leukotriene B 4 receptor (Sabirsh et al, 2005). Recent work in our laboratory has focused on the development of fluorescent ligands for the adenosine receptors with improved imaging properties.…”

V1b and CRHR1 Receptor Heterodimerization Mediates Synergistic Biological Actions of Vasopressin and CRH

“…First, the use of a specific antibody directed against tyrosine hydroxylase (TH) showed that the amount of positively stained cells reached 93 Ϯ 5% (three distinct cultures) (data not shown), suggesting that our primary culture mainly consists in chromaffin cells. We then took advantage of our recently characterized V1b-specific fluorescent ligand, d[Leu 4 -(Lys-Alexa647) 8 ]VP, to quantify the V1b-expressing cells (24). We observed that 58 Ϯ 2% of TH immunostained cells were labeled with d[Leu 4 -(Lys-Alexa647) 8 (Fig.…”

“…After 2 d of culture, the adherent cells were incubated with the fluorescent agonist d[Leu 4 -(Lys-Alexa647) 8 ]VP at 150 nM in the presence of 100 nM the selective OT antagonist SR126768A for 2 h at 12 C as previously described (24). The incubations were realized in the presence or absence of an excess of AVP to determine nonspecific labeling.…”