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BioAssay: AID 1493

Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement

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(15)
 
 
Active(11)
 
 
Inconclusive(4)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Active(12)
 
 
Inconclusive(4)
 
 
AID: 1493
Data Source: NCGC (NPSR008)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-09
Modify Date: 2009-06-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
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AIDNameTypeProbeComment
1461qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal TransductionConfirmatory depositor-specified cross reference
1464Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: SummarySummary2 depositor-specified cross reference
2567Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transduction, SAR for ProbeConfirmatory depositor-specified cross reference
2568Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for ProbeConfirmatory depositor-specified cross reference
2570Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin Receptor Calcium Signal TransductionConfirmatory depositor-specified cross reference
434931Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of Calcium Signal TransductionConfirmatory depositor-specified cross reference
434936Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal TransductionConfirmatory depositor-specified cross reference
624054Robust Characterization of cAMP Signal Transduction for Antagonists of the Neuropeptide S Receptor: SAROther depositor-specified cross reference
1489Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal TransductionConfirmatory same project related to Summary assay
1491Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal TransductionConfirmatory same project related to Summary assay
1492Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Muscarinic Receptor Calcium Signal Transduction.Confirmatory same project related to Summary assay
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for ProbeConfirmatory same project related to Summary assay
624005Extended Characterization of Antagonists of the Neuropeptide S Receptor: SummarySummary same project related to Summary assay
624052Robust Characterization of Calcium Signal Transduction Antagonists of the Neuropeptide S Receptor: SAROther same project related to Summary assay
624053qHTS for Antagonists of the Neuropeptide S Receptor: SAR in ERKOther same project related to Summary assay
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.

As one method of confirming the specific activity of compounds active in the primary screen (AID 1461), we developed an assay to measure the displacement of radio-labeled native ligand, NPS, from its receptor in vitro. The lack of displacement does not necessarily indicate that a compound is non-specific for the NPS receptor, rather, it indicates that the binding site is unlikely to be the same as the native ligand.
Protocol
The assay was carried out as described (Xu et al. (2004) Neuron, 43: 487-497) with minor modification.
Y10-NPS labeled with 125I was bought from NEN Perkin Elmer (Boston, MA). CHO cells stably expressing human NPSR were seeded into 24-well plates and cultured until reaching 90-95% confluency. Cells were washed with 1ml PBS once and then incubated with radioligand with or without compounds or in DMEM medium containing 0.1% bovine serum albumin at 20C for 1.5 hr. Increasing concentrations of compounds or unlabeled human NPS were used to compete with 0.15 nM [125I] Y10-NPS. Nonspecific binding was determined in the presence of 1 uM unlabeled human NPS. Cells were washed twice with cold PBS and lysed with 1 N NaOH. Bound radioactivity was counted in a liquid scintillation counter.
Comment
Compounds displacing radio-labeled ligand were scored 50. Other compounds are inconclusive and are scored 10.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
2Hill SlopeThe Hill slope from a fit of the data to the Hill equation.Float
3Counts at 2.15E-7 uM (2.14575e-07μM**)Radioactive counts at given concentration.Float
4Counts at 8.58E-7 uM (8.58302e-07μM**)Radioactive counts at given concentration.Float
5Counts at 3.43E-6 uM (3.43321e-06μM**)Radioactive counts at given concentration.Float
6Counts at 1.37E-5 uM (1.37328e-05μM**)Radioactive counts at given concentration.Float
7Counts at 5.49E-5 uM (5.49313e-05μM**)Radioactive counts at given concentration.Float
8Counts at 6.78E-5 uM (6.78063e-05μM**)Radioactive counts at given concentration.Float
9Counts at 8.58E-5 uM (8.58302e-05μM**)Radioactive counts at given concentration.Float
10Counts at 2.44E-4 uM (0.000244141μM**)Radioactive counts at given concentration.Float
11Counts at 2.71E-4 uM (0.000271225μM**)Radioactive counts at given concentration.Float
12Counts at 3.43E-4 uM (0.000343323μM**)Radioactive counts at given concentration.Float
13Counts at 6.87E-4 uM (0.000686646μM**)Radioactive counts at given concentration.Float
14Counts at 1.08E-3 uM (0.0010849μM**)Radioactive counts at given concentration.Float
15Counts at 1.37E-3 uM (0.00137329μM**)Radioactive counts at given concentration.Float
16Counts at 2.75E-3 uM (0.00274658μM**)Radioactive counts at given concentration.Float
17Counts at 4.34E-3 uM (0.00433959μM**)Radioactive counts at given concentration.Float
18Counts at 5.49E-3 uM (0.00549316μM**)Radioactive counts at given concentration.Float
19Counts at 0.011 uM (0.0109863μM**)Radioactive counts at given concentration.Float
20Counts at 0.017 uM (0.0173584μM**)Radioactive counts at given concentration.Float
21Counts at 0.022 uM (0.0219727μM**)Radioactive counts at given concentration.Float
22Counts at 0.044 uM (0.0439453μM**)Radioactive counts at given concentration.Float
23Counts at 0.069 uM (0.0694335μM**)Radioactive counts at given concentration.Float
24Counts at 0.088 uM (0.0878907μM**)Radioactive counts at given concentration.Float
25Counts at 0.176 uM (0.175781μM**)Radioactive counts at given concentration.Float
26Counts at 0.278 uM (0.277735μM**)Radioactive counts at given concentration.Float
27Counts at 0.352 uM (0.351562μM**)Radioactive counts at given concentration.Float
28Counts at 0.703 uM (0.703126μM**)Radioactive counts at given concentration.Float
29Counts at 1.111 uM (1.11094μM**)Radioactive counts at given concentration.Float
30Counts at 1.406 uM (1.40625μM**)Radioactive counts at given concentration.Float
31Counts at 2.812 uM (2.8125μM**)Radioactive counts at given concentration.Float
32Counts at 4.444 uM (4.44374μM**)Radioactive counts at given concentration.Float
33Counts at 5.625 uM (5.62501μM**)Radioactive counts at given concentration.Float
34Counts at 11.250 uM (11.25μM**)Radioactive counts at given concentration.Float
35Counts at 17.775 uM (17.775μM**)Radioactive counts at given concentration.Float
36Counts at 22.500 uM (22.5μM**)Radioactive counts at given concentration.Float
37Counts at 45.000 uM (45.0001μM**)Radioactive counts at given concentration.Float
38Counts at 71.100 uM (71.1001μM**)Radioactive counts at given concentration.Float
39Counts at 90.000 uM (90.0001μM**)Radioactive counts at given concentration.Float
40Counts at 180.000 uM (180μM**)Radioactive counts at given concentration.Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01-DA026210-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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