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BioAssay: AID 2568

Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for Probe

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(186)
 
 
Active(70)
 
 
Inactive(78)
 
 
Inconclusive(38)
 
 
 Tested Substances
 Tested Substances
All(186)
 
 
Active(70)
 
 
Inactive(78)
 
 
Inconclusive(38)
 
 
AID: 2568
Data Source: NCGC (NPSR005h)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-16
Hold-until Date: 2010-09-24
Modify Date: 2010-09-24

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 70
Depositor Specified Assays
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AIDNameTypeProbeComment
1461qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transductionconfirmatory
1464Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: Summarysummary2
1491Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transductionconfirmatory
1489Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transductionconfirmatory
1493Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacementconfirmatory
1492Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Muscarinic Receptor Calcium Signal Transduction.confirmatory
2567Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transduction, SAR for Probeconfirmatory
2570Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin Receptor Calcium Signal Transductionconfirmatory
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for Probeconfirmatory
434931Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of Calcium Signal Transductionconfirmatory
434936Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal Transductionconfirmatory
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.

Select compounds from the primary screen (AID 1461) were chosen for confirmation based on potency, efficacy, SAR, and activity across other assays in PubChem and at NCGC. Other compounds are analogs of active compounds from the primary screen.
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 were assayed in agonist (without prior NPS stimulation) and antagonist (with NPS stimulation; protocol step 3) screening mode. Antagonists were those compounds that had no response in agonist screening mode, and give a significant response in antagonist screening mode.
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Antagonist-Curve Description". For this assay, apparent antagonists are ranked higher than compounds that showed apparent agonism.
3. 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.
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
5Agonist-Curve_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 significant, but below 80% of control.String
6Agonist-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Agonist-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Agonist-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Agonist-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Agonist-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Agonist-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Agonist-Excluded_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
13Agonist-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Agonist-Activity at 0.0000411067 uM (4.11067e-05μM**)% Activity at given concentration.Float%
15Agonist-Activity at 0.0001644266 uM (0.000164427μM**)% Activity at given concentration.Float%
16Agonist-Activity at 0.0003288532 uM (0.000328853μM**)% Activity at given concentration.Float%
17Agonist-Activity at 0.0006577064 uM (0.000657706μM**)% Activity at given concentration.Float%
18Agonist-Activity at 0.00132 uM (0.00131541μM**)% Activity at given concentration.Float%
19Agonist-Activity at 0.00263 uM (0.00263083μM**)% Activity at given concentration.Float%
20Agonist-Activity at 0.00526 uM (0.00526165μM**)% Activity at given concentration.Float%
21Agonist-Activity at 0.011 uM (0.0105233μM**)% Activity at given concentration.Float%
22Agonist-Activity at 0.021 uM (0.0210466μM**)% Activity at given concentration.Float%
23Agonist-Activity at 0.042 uM (0.0420932μM**)% Activity at given concentration.Float%
24Agonist-Activity at 0.084 uM (0.0841864μM**)% Activity at given concentration.Float%
25Agonist-Activity at 0.168 uM (0.168373μM**)% Activity at given concentration.Float%
26Agonist-Activity at 0.337 uM (0.336746μM**)% Activity at given concentration.Float%
27Agonist-Activity at 1.347 uM (1.34698μM**)% Activity at given concentration.Float%
28Agonist-Activity at 2.694 uM (2.69397μM**)% Activity at given concentration.Float%
29Agonist-Activity at 5.388 uM (5.38793μM**)% Activity at given concentration.Float%
30Agonist-Activity at 10.78 uM (10.7759μM**)% Activity at given concentration.Float%
31Agonist-Activity at 21.55 uM (21.5517μM**)% Activity at given concentration.Float%
32Agonist-Activity at 43.10 uM (43.1034μM**)% Activity at given concentration.Float%
33Agonist-Activity at 86.21 uM (86.2069μM**)% Activity at given concentration.Float%
34Antagonist-Curve_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
35Antagonist-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
36Antagonist-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
37Antagonist-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
38Antagonist-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
39Antagonist-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
40Antagonist-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
41Antagonist-Excluded_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
42Antagonist-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
43Antagonist-Activity at 0.0000001185 uM (1.18543e-07μM**)% Activity at given concentration.Float%
44Antagonist-Activity at 0.0000003556 uM (3.55629e-07μM**)% Activity at given concentration.Float%
45Antagonist-Activity at 0.0000007544 uM (7.54406e-07μM**)% Activity at given concentration.Float%
46Antagonist-Activity at 0.0000018479 uM (1.84791e-06μM**)% Activity at given concentration.Float%
47Antagonist-Activity at 0.0000055398 uM (5.53984e-06μM**)% Activity at given concentration.Float%
48Antagonist-Activity at 0.0000110662 uM (1.10662e-05μM**)% Activity at given concentration.Float%
49Antagonist-Activity at 0.0000307410 uM (3.0741e-05μM**)% Activity at given concentration.Float%
50Antagonist-Activity at 0.0000817167 uM (8.17167e-05μM**)% Activity at given concentration.Float%
51Antagonist-Activity at 0.0001695316 uM (0.000169532μM**)% Activity at given concentration.Float%
52Antagonist-Activity at 0.0003669120 uM (0.000366912μM**)% Activity at given concentration.Float%
53Antagonist-Activity at 0.0009936476 uM (0.000993648μM**)% Activity at given concentration.Float%
54Antagonist-Activity at 0.00271 uM (0.00271185μM**)% Activity at given concentration.Float%
55Antagonist-Activity at 0.00577 uM (0.00576728μM**)% Activity at given concentration.Float%
56Antagonist-Activity at 0.016 uM (0.0156184μM**)% Activity at given concentration.Float%
57Antagonist-Activity at 0.043 uM (0.0431331μM**)% Activity at given concentration.Float%
58Antagonist-Activity at 0.094 uM (0.0940718μM**)% Activity at given concentration.Float%
59Antagonist-Activity at 0.188 uM (0.188425μM**)% Activity at given concentration.Float%
60Antagonist-Activity at 0.504 uM (0.5037μM**)% Activity at given concentration.Float%
61Antagonist-Activity at 1.392 uM (1.39179μM**)% Activity at given concentration.Float%
62Antagonist-Activity at 2.985 uM (2.98512μM**)% Activity at given concentration.Float%
63Antagonist-Activity at 8.059 uM (8.0592μM**)% Activity at given concentration.Float%
64Antagonist-Activity at 22.27 uM (22.2686μM**)% Activity at given concentration.Float%
65Antagonist-Activity at 47.76 uM (47.762μM**)% Activity at given concentration.Float%
66Compound 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)
Classification
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