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BioAssay: AID 1461

qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction

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(220868)
 
 
Active(2309)
 
 
Inactive(206040)
 
 
Inconclusive(12614)
 
 
 Tested Substances
 Tested Substances
All(221370)
 
 
Active(2314)
 
 
Inactive(206429)
 
 
Inconclusive(12627)
 
 
AID: 1461
Data Source: NCGC (NPSR003)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2008-12-29
Modify Date: 2009-06-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2309
Related Experiments
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AIDNameTypeProbeComment
1464Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: SummarySummary2 depositor-specified cross reference
1489Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal TransductionConfirmatory depositor-specified cross reference
1491Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal TransductionConfirmatory depositor-specified cross reference
1492Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Muscarinic Receptor Calcium Signal Transduction.Confirmatory depositor-specified cross reference
1493Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand DisplacementConfirmatory depositor-specified cross reference
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for ProbeConfirmatory 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
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.

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
NCGC Assay Protocol Summary:
A Chinese hamster ovary (CHO) cell line stably expressing the NPS receptor (CHO-NPSR) was obtained from Dr. Heilig lab at NIAAA and maintained in F-12 Kaighn's media (Invitrogen, Carlsbad, CA, 21127) supplemented with 10 % FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 250 ug/ml geneticin at 37C, 5% CO2 in a humidified atmosphere. Before the assay, aliquots of cells were frozen and stored at -135C. The assay was performed in 1536-well format. Data are reported for both the ratio of the two emission wavelengths, and also for the component 'donor' channel, Em2=620. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (No NPS control). IC50 values were determined from concentration-response data modeled with the standard Hill equation.
NPS 1536-well assay protocol:
(1) Fresh or frozen CHO-NPSR cells were suspended in F-12 Kaighn's media supplemented with 10 % FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 4 ul/well (1200 cells) to white, tissue culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours.
(2) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM.
(3) Add 1 ul of stimulation reagent (1X HBSS buffer, 5 mM HEPES, 0.1% BSA, 500 uM RO-201724 (Sigma-Aldrich, B8279), 1.5% Alexa 647-labeled anti-cAMP antibody (from Perkin Elmer), 100 nM NPS)
(4) Incubation at 37C, 5 % CO2 for 60 min.
(5) Add 1 ul/well detection reagent. Detection reagent was prepared by adding biotin labeled cAMP (4%), streptavidin labeled with Europium-W8044 chelate (1.33%) and TritonX-100 (1%) to detection buffer. Biotin labeled cAMP, streptavidin labeled with Europium-W8044 chelate and detection buffer all came from the LANCE cAMP kit (Perkin Elmer).
(6) Incubate at room temperature for 2 hours.
(7) Detect the assay plate (Ex = 320, Em1 =665 and Em2 620) in a ViewLux plate reader.
Keywords: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, NPS, Neuropeptide S Antagonists
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML079
PubChem Substance ID (SID) for probe 1: 56431681,56431665
PubChem Compound ID (CID) for probe 1: 3719993
Probe type for probe 1: Antagonist
IC50/EC50 (nM) for probe 1: 1585
Target for probe 1: neuropeptide S receptor (gi: 46395496)
Anti-target for probe 1: Muscarinic acetylcholine receptor M1
Fold selectivity for probe 1: >30
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47358/ (ID: 2360070)
Grant number for probe 1: DA026210-01
NCBI Book chapter title for probe 1: Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*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
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0005382686 uM (0.000538269μM**)% Activity at given concentration.Float%
15Activity at 0.00269 uM (0.00269008μM**)% Activity at given concentration.Float%
16Activity at 0.013 uM (0.0134504μM**)% Activity at given concentration.Float%
17Activity at 0.067 uM (0.0672474μM**)% Activity at given concentration.Float%
18Activity at 0.307 uM (0.307336μM**)% Activity at given concentration.Float%
19Activity at 1.532 uM (1.53174μM**)% Activity at given concentration.Float%
20Activity at 7.669 uM (7.66872μM**)% Activity at given concentration.Float%
21Activity at 38.30 uM (38.3001μM**)% Activity at given concentration.Float%
22Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
23Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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