Nonpeptide Ligands Research Articles

Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor, an Insect Ortholog of the Vertebrate Neuromedin U Receptor

The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.

Epunctanone, a New Benzophenone, and Further Secondary Metabolites from Garcinia epunctataStapf (Guttiferae)

AbstractA new polyprenylated benzophenone, named epunctanone (1), was isolated from the stem bark of Garcinia epunctata Stapf, together with eight known compounds, 7‐epiisogarcinol (2), 2,6‐dimethoxy‐p‐benzoquinone (3), friedelin (4), lupeol (5), 16β‐hydroxylupeol (6), betulin (7), stigmasterol (8), and rheediaxanthone A (9). The structure of epunctanone (1) was established by detailed analysis of its spectroscopic data, especially 1D‐ and 2D‐NMR, and HR‐MS data. All these compounds were evaluated for their antimicrobial and anti‐protozoan activities. They were also assayed to determine if any of the compounds were nonpeptide agonist ligands for nematodal G‐protein‐coupled receptors, which would be an indication of potential antinematodal activity. Among the isolated compounds, 7‐epiisogarcinol (2) was the most active against Candida albicans.

Hybrid Opioid/Non-Opioid Ligands in Pain Research

To address the different types of pain (e.g. acute, chronic, neuropathic) different classes of medications, mainly non-steroidal anti-inflammatory drugs and narcotics (opioids), are used. More specifically, the alleviation or treatment of moderate to severe pain states commonly invokes the use of opioids. Unfortunately, their chronic administration induces various undesirable side effects, such as for example physical dependence and tolerance. One strategy to overcome these major side effects and to prolong the antinociceptive efficiency of the applied drugs involves the creation of multifunctional compounds which contain hybridized structures. Combination of opioid agonist and antagonist pharmacophores in a single chemical entity has been considered and extensively investigated, but opioids have also been combined with other bioactive neurotransmitters and peptide hormones that are involved in pain perception (e.g. substance P, neurotensin, cholecystokinin, cannabinoids, melanocortin ligands, etc.). Such novel chimeras (also called designed multiple ligands or twin/triplet drugs), may interact independently with their respective receptors and potentially result in more effective antinociceptive properties. The designed multiple ligands presented in this work include opioid-non-opioid peptide dimer analogs, mixed peptidic- non-peptidic bifunctional ligands and dual non-peptidic dimers. The main focus herein is placed on the design and biological evaluation of these multiple opioid compounds, rather than the synthetic approach and preparation.

Nanofibrous scaffolds releasing a small molecule BDNF-mimetic for the re-direction of endogenous neuroblast migration in the brain

Brain tissue engineering has the potential to harness existing elements of neurogenesis within the adult brain to overcome a microenvironment that is otherwise inhibitory to regeneration, especially following severe tissue damage. This study investigates the ability of electrospun poly ε-caprolactone (PCL) to re-direct the migratory pathway of endogenous neuroblasts from the disrupted subventricular zone (SVZ). A small molecule non-peptide ligand (BDNF-mimetic) that mimicked the trophic properties of brain-derived neurotrophic factor (BDNF) was incorporated into electrospun PCL scaffolds to improve neuroblast survival and promote neuroblast migration towards the implant. PCL scaffolds were able to support neuroblast infiltration and migration along the implant tract. In the presence of the BDNF-mimetic, neuroblasts were able to migrate towards the implant via the parenchyma, and their persistence within the implants was prolonged. In addition, the BDNF-mimetic improved implant integration and increased local neuronal plasticity by increasing neurite sprouting at the tissue-implant interface. SMI32+ neurites were observed inside scaffolds at 21 days but not 8 days post implantation, indicating that at least some of the infiltrated neuroblasts had differentiated into neurons.

Neuropeptide Receptors in Pain Circuitries: Useful Targets for CNS Imaging with Non-Peptide Ligands Suitable for PET?

Neuropeptide receptors of the brain and spinal cord are parts of the pain circuits targeted by analgesic drugs. Some of these receptors have been found in the central nervous system as well as in intracranial vascular structures and achieved revival of attention because of their role in acute and chronic pain syndromes. A number of them are of high clinical relevance for e.g. migraine. Others participate in symptoms of rare diseases like amyotrophic lateral sclerosis. Here we will focus on five of the neuropeptide receptors and their non-peptide ligands potentially or already successfully used as PET probes. Opioid receptors and neurotensin receptors are known to mediate analgesic actions. Bradykinin and calcitonin gene-related peptide (CGRP) receptors are known to be involved in the regulation of vascular tone and inflammatory responses, and neurokinin receptors play a role in the occurrence of pain perception in a rather indirect manner. Most experiences as PET tracers have been gathered with opioid receptor ligands and neurokinin receptor ligands. The most innovative fields revealed by the studies summarized in this report are the ligands of κ opioid receptors and CGRP receptors for which a first PET tracer was presented recently.

125I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors

Recent genetic and pharmacological studies have implicated the α3, β4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4 and α4β2 nAChR transfected into HEK cells, as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at the human α3β4α5 nAChR transfected into HEK cells. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells. Binding studies with [3H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label α3β4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We demonstrate that [125I]AT-1012 is an excellent tool for labeling the α3β4 nAChR in the presence of other nAChR subtypes.

Transient Domain Interactions Enhance the Affinity of the Mitotic Regulator Pin1 toward Phosphorylated Peptide Ligands

The mitotic regulator Pin1 plays an important role in protein quality control and age-related medical conditions such as Alzheimer disease and Parkinson disease. Although its cellular role has been thoroughly investigated during the past decade, the molecular mechanisms underlying its function remain elusive. We provide evidence for interactions between the two domains of Pin1. Several residues displayed unequivocal peak splits in nuclear magnetic resonance spectra, indicative of two different conformational states in equilibrium. Pareto analysis of paramagnetic relaxation enhancement data demonstrates that the two domains approach each other upon addition of a nonpeptidic ligand. Titration experiments with phosphorylated peptides monitored by fluorescence anisotropy and chemical shift perturbation indicate that domain interactions increase Pin1's affinity toward peptide ligands. We propose this interplay of the domains and ligands to be a general mechanism for a large class of two-domain proteins.

Accidental Interaction between PDZ Domains and Diclofenac Revealed by NMR-Assisted Virtual Screening

In silico approaches have become indispensable for drug discovery as well as drug repositioning and adverse effect prediction. We have developed the eF-seek program to predict protein–ligand interactions based on the surface structure of proteins using a clique search algorithm. We have also developed a special protein structure prediction pipeline and accumulated predicted 3D models in the Structural Atlas of the Human Genome (SAHG) database. Using this database, genome-wide prediction of non-peptide ligands for proteins in the human genome was performed, and a subset of predicted interactions including 14 PDZ domains was then confirmed by NMR titration. Surprisingly, diclofenac, a non-steroidal anti-inflammatory drug, was found to be a non-peptide PDZ domain ligand, which bound to 5 of 15 tested PDZ domains. The critical residues for the PDZ–diclofenac interaction were also determined. Pharmacological implications of the accidental PDZ–diclofenac interaction are further discussed.

Structure of class B GPCR corticotropin-releasing factor receptor 1

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

Trial of zoledronic acid and interleukin-2 to expand tumoricidal γδ T cells in vivo in patients with refractory neuroblastoma.

TPS10073 Background: CD3+ γδ+ T cells comprise 2 to 5% of the circulating T cells. Vγ9Vδ2+ cells are the dominant circulating γδ T cell and recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of γδ T cells represent a promising means of harnessing the innate immune system to treat malignancies including neuroblastoma (NB). Indeed, γδ T cells from both healthy volunteers and NB patients exert a potent cytotoxic effect on NB cell lines and autologous NB in vitro following expansion and activation in culture. Vγ9Vδ2+ cells can also be induced to proliferate in vivo. By blocking farnesyl pyrophosphate synthase in the mevalonate pathway of isoprenoid synthesis in monocytes, the aminobisphosphonate zoledronic acid (ZOL) promotes the accumulation of isopentenyl pyrophosphate which is sensed by γδ T cells. IL-2 is also required for robust expansion γδ T cells. Methods: The trial is a prospective, non-randomized trial that assesses two dose levels of recombinant IL-2 (aldesleukin) in combination with ZOL. To be eligible for the study patients must be 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options. Patients must also have adequate organ function and performance status. ZOL is given intravenously on day 1, and aldesleukin is given subcutaneously on days 1 to 5 and 15 to 19 of every 28 day cycle. The single-institution study is being conducted at the University of Alabama at Birmingham. Correlative studies include evaluating the absolute count, phenotype, activation, and effector/memory progression of γδ T cells by flow cytometry and the biological function of autologous expanded/activated γδ T cells by in vitro cytotoxicity assays employing established human NB cell lines. In order to determine the ability of in vivo expanded/activated γδ T cells to infiltrate NB tissue, bone marrow core biopsies obtained before and after the first course of therapy from patients with bone marrow metastasis are assayed for T cell infiltration by immunohistochemistry. The study has currently enrolled 1 of the planned 6 patients. Clinical trial information: NCT01404702.

Structures composing protein domains

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships.

Three-Dimensional Structural Diversity-Oriented Peptidomimetics Based on the Cyclopropylic Strain

Conformationally restricted peptidomimetics comprising eight stereoisomeric scaffolds with three-dimensional structural diversity were designed based on the structural features of cyclopropane, that is, cyclopropylic strain, which mimic wide-ranging tetrapeptide conformations covering β-turns through β-strands. Stereoselective synthesis of the designed peptidomimetics led to the identification of nonpeptidic melanocortin-4 receptor ligands.

ChemInform Abstract: Neuropeptides and Neuropeptide Receptors: Drug Targets, and Peptide and Non‐Peptide Ligands: A Tribute to Prof. Dieter Seebach

AbstractReview: ca. 50 refs.

Non-peptide ligands in the characterization of peptide receptors at the interface between neuroendocrine and mental diseases

Hypothalamic receptors for neuropeptide Y, melaninconcentrating hormone, melanocortins and orexins/ hypocretins as well as for the downstream signaling corticotrophic factor have been discussed broadly for their influence on food intake and reward but also on several psychiatric disorders. For the development of non-peptide ligands for the in vivo detection of alterations in density and affinity of such G-protein coupled (GPCRs) peptide receptors the requirements to affinity and pharmacokinetics have been shifted to thresholds markedly distict from classical GPCRs to dissociation constants ported originally as potential therapeutics in the treatment of obesity among which some are suitable candidates for labeling as PET or SPECT-tracers providing receptor affinities even below 0.1 nM. These could be unique tools not only for better understanding of the mechanism of obesity but also for investigations of extrahypothalamic role of “feeding receptors” at the interface between neuroendocrine and mental diseases.

Comparative functional properties of two structurally similar selective nonpeptide drug-like ligands for the angiotensin II type-2 (AT2) receptor. Effects on neurite outgrowth in NG108-15 cells

There is increasing evidence that angiotensin II (Ang II), through binding to the type 2 (AT2) receptor may have beneficial effects in various physiological and pathological situations. However, specific action presumably mediated by the angiotensin AT2 receptor has been hampered by the absence of appropriate selective ligands. The aim of this study was to compare the biological properties of two related and selective drug-like nonpeptide AT2 ligands, namely an agonist called M024 (also known as Compound 21) and a new ligand, presumably an antagonist, C38/M132, (originally called C38). Properties of the compounds were investigated in NG108-15 cells expressing angiotensin AT2 receptor and known to develop neurite outgrowth upon Ang II stimulation. NG108-15 cells stimulated for three days with C21/M024 (0.1 or 100nM) exhibited the same neurite outgrowth as cells stimulated with Ang II (100nM) while co-incubation of Ang II or C21/M024 with C38/M132 (10 or 100nM) inhibited their effects, similarly to the angiotensin AT2 receptor antagonist, PD123319 (10μM). As Ang II, C21/M024 induced a Rap1-dependent activation of p42/p44mapk whereas preincubation of cells with C38/M132 inhibited p42/p44mapk and Rap1 activation induced by Ang II. Three-day treatment with C21/M024 or Ang II decreased cell number in culture, an effect that was rescued by preincubation with C38/M132. Taken together, these results indicate that the nonpeptide ligand C21/M024 is a potent angiotensin AT2 receptor agonist while C38/M132 acts as an antagonist. These selective nonpeptide angiotensin AT2 ligands may represent unique and long-awaited tools for the pursuit of in vivo studies.

Potent Inhibitors of Malarial Aspartic Proteases, the Plasmepsins, by Hydroformylation of Substituted 7‐Azanorbornenes

The increasing prevalence of multidrug-resistant strains of the malarial parasite Plasmodium falciparum requires the urgent development of new therapeutic agents with novel modes of action. The vacuolar malarial aspartic proteases plasmepsin (PM) I, II, and IV are involved in hemoglobin degradation and play a central role in the growth and maturation of the parasite in the human host. We report the structure-based design, synthesis, and in vitro evaluation of a new generation of PM inhibitors featuring a highly decorated 7-azabicyclo[2.2.1]heptane core. While this protonated central core addresses the catalytic Asp dyad, three substituents bind to the flap, the S1/S3, and the S1' pockets of the enzymes. A hydroformylation reaction is the key synthetic step for the introduction of the new vector reaching into the S1' pocket. The configuration of the racemic ligands was confirmed by extensive NMR and X-ray crystallographic analysis. In vitro biological assays revealed high potency of the new inhibitors against the three plasmepsins (IC(50) values down to 6 nM) and good selectivity towards the closely related human cathepsins D and E. The occupancy of the S1' pocket makes an essential contribution to the gain in binding affinity and selectivity, which is particularly large in the case of the PM IV enzyme. Designing non-peptidic ligands for PM II is a valid route to generate compounds that inhibit the entire family of vacuolar plasmepsins.

Neuropeptides and Neuropeptide Receptors: Drug Targets, and Peptide and Non‐Peptide Ligands: a Tribute to Prof. Dieter Seebach

The number of neuropeptides and their corresponding receptors has increased steadily over the last fourty years: initially, peptides were isolated from gut or brain (e.g., Substance P, somatostatin), then by targeted mining in specific regions (e.g., cortistatin, orexin in the brain), or by deorphanization of G-protein-coupled receptors (GPCRs; orexin, ghrelin receptors) and through the completion the Human Genome Project. Neuropeptides (and their receptors) have regionally restricted distributions in the central and peripheral nervous system. The neuropeptide signaling is somewhat more distinct spatially than signaling with classical, low-molecular-weight neurotransmitters that are more widely expressed, and, therefore, one assumes that drugs acting at neuropeptide receptors may have more selective pharmacological actions with possibly fewer side effects than drugs acting on glutamatergic, GABAergic, monoaminergic, or cholinergic systems. Neuropeptide receptors, which may have a few or multiple subtypes and splice variants, belong almost exclusively to the GPCR family also known as seven-transmembrane receptors (7TM), a favorite class of drug targets in the pharmaceutical industry. Most neuropeptides are co-stored and co-released with classic neurotransmitters, albeit often only at higher frequencies of stimulation or at bursting activity, thus restricting the neuropeptide signaling to specific circumstances, another reason to assume that neuropeptide drug mimics may have less side effects. Neuropeptides possess a wide spectrum of functions from neurohormone, neurotransmitter to growth factor, but also as key inflammatory mediators. Neuropeptides become 'active' when the nervous system is challenged, e.g., by stress, injury, drug abuse, or neuropsychiatric disorders with genetic, epigenetic, and/or environmental components. The unsuspected number of true neuropeptides and their cognate receptors provides opportunities to identify novel targets for the treatment of both central and peripheral nervous system disorders. Both, receptor subtype-selective antagonists and agonists are being developed, as illustrated by the success of somatostatin agonists, angiotensin, and endothelin antagonists, and the expected clinical applications of NK-1/2/3 (substance P) receptor antagonists, CRF, vasopressin, NPY, neurotensin, orexin antagonists, or neuropeptide receptor modulators; such ligands have efficacy in preclinical or clinical models of pain and neuropsychiatric diseases, such as migraine, chronic/neuropathic pain, anxiety, sleep disorders, depression, and schizophrenia. In addition, both positive and negative allosteric modulators have been described with interesting in vivo activities (e.g., at galanin receptors). The field has become more complex now that an increasing number of heteromeric neuropeptide receptors are described, e.g., ghrelin receptors with 5-HT(2C) or dopamine D(1), D(2) receptors. At long last, structure-based drug discovery can now be envisaged with confidence, since crystal or solution structure of GPCRs and GPCR-ligand complexes, including peptide receptors, are published almost on a monthly basis. Finally, although most compounds acting at peptide receptors are still peptidomimetics, the last decade has seen the emergence of low-molecular-weight nonpeptide ligands (e.g., for orexin, ghrelin, or neurokinin receptors), and surprising progress has been made with β- and γ-peptides as very stable and potent mimetics of, e.g., somatostatin (SRIF), where the native SRIF has a half-life limited to 2-3 min. This last point will be illustrated more specifically, as we have had a long-standing collaboration with Prof. D. Seebach to whom this review is dedicated at the occasion of his 75th birthday.

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. 3: Synthesis of novel triplet drugs with the bis(epoxymethano) or bis(dimethylepoxymethano) structure (double-capped triplet)

Novel double-capped triplet drugs, which have one pharmacophore unit and two epoxymethano or dimethylepoxymethano structures (termed cap or diMe-cap structures, respectively) were synthesized. Key intermediate oxazoline 16 derived from acetone enabled the effective synthesis of double-capped triplets. SYK-134 (7a) and SYK-135 (8a) with N-cyclopropylmethyl substituent and cap structures showed selectivities for the κ opioid receptor. On the other hand, the N-Me series exhibited selectivities for the μ opioid receptor. The double-capped triplet drugs with diMe-cap structures preferred the μ receptor independently of their N-substituents. SYK-385 (19b), one of the μ-selective double-capped triplet drugs, showed the highest selectivity for the μ receptor among the reported μ-selective nonpeptide ligands.

Structure of the agonist-bound neurotensin receptor

SummaryNeurotensin (NT) is a 13 amino acid peptide that functions as both a neurotransmitter and a hormone through activation of the neurotensin receptor NTS1, a G protein-coupled receptor (GPCR). In the brain, NT modulates activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake, and in the gut NT regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of NTS1 in an active-like state, bound to NT8-13, the C terminal portion of NT responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTS1 in an extended conformation nearly perpendicular to the membrane plane with the C-terminus oriented towards the receptor core. Our findings provide the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V2GPCR: 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(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, 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 44 and 47 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(1a)R-V₂R dimerization on cell surface.