Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: Summary
Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acids peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. more ..
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: X01-DA026210-01
Assay Submitter (PI): Heilig, Markus Alexander
NCGC Assay Overview:
Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acids peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. Specifically they are highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders.
To identify NPSR antagonists, we developed a cell-based cAMP assay. NPS can stimulate the production of cAMP in Chinese hamster ovary cells stably expressing NPS receptor. This change in intracellular cAMP level can be detected by a homogeneous LANCE cAMP assay based on the TR-FRET (time resolved fluorescence resonance energy transfer) between a europium-labeled cAMP tracer complex and a cAMP-specific antibody labeled with Alexa Fluor 647. The europium-labeled cAMP tracer complex is formed by the tight interaction between Biotin-cAMP (b-cAMP) and streptavidin labeled with Europium-W8044 chelate (Eu-SA). Light pulse at 320 nm excites the europium of the cAMP tracer and the energy emitted is transferred to the Alexa molecule bound to the cAMP antibody, generating a TR-FRET signal at 665 nm. Residual energy from the europium will produce a light at 620 nm. The native unlabeled cAMP from cell lysates competes with the europium-cAMP tracer for antibody binding and reversely reduces the emission signal of Alexa by interrupting FRET between the two labeled molecules. Both emission signals from the FRET donor (620 nm) and the acceptor (665 nm) can be detected by a plate reader in the TRF mode. Expression of result in fluorescence ratio (665 nm/620 nm) helps to normalize differences due to cell density and reagent dispensing. This assay was successfully optimized to a 1536-well plate format.
Please refer to other AIDs (1461,1489,1491,1492,1493,2566,2567,2568,2570,434931,434936) for detailed assay protocols.
This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive analogues from these series are given a score of 0. ML079 (SID 56431705) and ML154 (SID 87796314) have been declared as probes from this project.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML154
PubChem Substance ID (SID) for probe 1: 87796314
PubChem Compound ID (CID) for probe 1: 46930969
Probe type for probe 1: Antagonist
IC50/EC50 (nM) for probe 1: 1
Target for probe 1: NPSR (gi: 46395496)
Disease relevance for probe 1: Reasearch Tool for Addiction
Anti-target for probe 1: V1B Vasopressin Receptor
Fold selectivity for probe 1: > 100
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK51966/ (ID: 2412132)
Grant number for probe 1: DA026210-01
NCBI Book chapter title for probe 1: Identification of Small Molecule Antagonists of the Neuropeptide-S Receptor
Data Table (Concise)