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BioAssay: AID 1464

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 ..
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 Tested Compounds
 Tested Compounds
All(11)
 
 
Probe(2)
 
 
Active(11)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Probe(2)
 
 
Active(11)
 
 
AID: 1464
Data Source: NCGC (NPSR004)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2008-12-30
Modify Date: 2010-10-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: Chemical Probe: 2    Active: 11
Depositor Specified Assays
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AIDNameTypeComment
1461qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal TransductionconfirmatorycAMP-based assay primary screen.
1491Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal TransductionconfirmatorycAMP-based assay confirmation screen.
1489Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal TransductionconfirmatoryCa++ signaling confirmation screen.
1492Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Muscarinic Receptor Calcium Signal Transduction.confirmatoryMuscarinic counter screen.
1493Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand DisplacementconfirmatoryRadioligand displacement confirmation assay.
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for ProbeconfirmatoryRadioligand displacement confirmation assay for second probe series.
2567Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transduction, SAR for ProbeconfirmatoryCa++ signaling confirmation screen for second probe series.
2568Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for ProbeconfirmatorycAMP-based assay confirmation screen for second probe series.
2570Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin Receptor Calcium Signal TransductionconfirmatoryVasopressin receptor counter screen for second probe series.
434931Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of Calcium Signal TransductionconfirmatoryCa++ signaling confirmation screen in very high density concentration-response.
434936Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal TransductionconfirmatorycAMP-based assay confirmation screen in very high density concentration-response.
624005Extended Characterization of Antagonists of the Neuropeptide S Receptor: Summarysummary
624052Robust Characterization of Calcium Signal Transduction Antagonists of the Neuropeptide S Receptor: SARother
624053qHTS for Antagonists of the Neuropeptide S Receptor: SAR in ERKother
624054Robust Characterization of cAMP Signal Transduction for Antagonists of the Neuropeptide S Receptor: SARother
Description:
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.
Protocol
Please refer to other AIDs (1461,1489,1491,1492,1493,2566,2567,2568,2570,434931,434936) for detailed assay protocols.
Comment
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity SummaryNature of interaction of the sample with the receptor.String
2Neuropeptide S Receptor Ca++ PotencyThe concentration of sample yielding half-maximal inhibition of the neuropeptide S receptor stimulated with native ligand.FloatμM
3Neuropeptide S Receptor cAMP PotencyFloatμM
4Neuropeptide S Receptor Radioligand Displacement PotencyFloatμM
5Muscarinic Receptor PotencyFloatμM
6Vasopressin Receptor PotencyFloatμM
Additional Information
Grant Number: X01-DA026210-01

Data Table (Concise)
Classification
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