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BioAssay: AID 1489

Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium 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(212)
 
 
Active(31)
 
 
Inactive(90)
 
 
Inconclusive(92)
 
 
 Tested Substances
 Tested Substances
All(213)
 
 
Active(31)
 
 
Inactive(90)
 
 
Inconclusive(92)
 
 
AID: 1489
Data Source: NCGC (NPSR006)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-08
Modify Date: 2010-03-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 31
Depositor Specified Assays
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
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
434936Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal Transductionconfirmatory
2568Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for Probeconfirmatory
624054Robust Characterization of cAMP Signal Transduction for Antagonists of the Neuropeptide S Receptor: SARother
434931Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of Calcium Signal Transductionconfirmatory
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 assay with transfected NPS receptor. NPS can stimulate the production of cAMP as well as release intracellular calcium in Chinese hamster ovary cells stably expressing NPS receptor. This change in intracellular calcium can be detected using a calcium indicator dye on a calcium imaging plate reader system (FDSS).
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 on a FDSS-7000 kinetic plate reader in 1536-well format. For both agonist (1-220s) and antagonist (221-400s) phases, the maximums of kinetic fluorescence responses were converted separately into text files using the instrument's software data export utility. Data for agonist response were normalized to the controls for basal activity (DMSO only) and 100% activation (NPS). Data for antagonist response were normalized to the controls for basal activity (DMSO only) and 100% inhibition (No NPS control). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

NPS 1536-well FDSS assay protocol:
(1) Frozen CHO-NPSR cells were thaw, washed once with fresh media and resuspended in F-12 Kaighn's media supplemented with 10 % FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 3 ul/well (1200 cells) to black, clear-bottom, tissue-culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours.
(2) Add 3 ul of calcium dye (from High Performance PBX Calcium Assay Kit, BD Biosciences). The calcium dye was prepared according to the manufactory's instruction.
(3) Incubation at 37C, 5 % CO2 for 60 to 120 min.
(4) Load plates to FDSS-7000. The following steps were performed on FDSS-7000.
(5) Record fluorescent background (Ex 480 nm, Em 520-560 nm) for 10 s.
(6) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM. Record agonist response for 210 s.
(7) Add 2 ul of stimulation reagent (1X HBSS buffer, 0.1% BSA, 60 nM NPS). Record antagonist response for 140 s.

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 signficant 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
Show more
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 signficant, 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 0.0000022837 uM (2.2837e-06μM**)% Activity at given concentration.Float%
15Agonist-Activity at 0.0000045674 uM (4.56741e-06μM**)% Activity at given concentration.Float%
16Agonist-Activity at 0.0000091348 uM (9.13481e-06μM**)% Activity at given concentration.Float%
17Agonist-Activity at 0.0000182696 uM (1.82696e-05μM**)% Activity at given concentration.Float%
18Agonist-Activity at 0.0000365392 uM (3.65392e-05μM**)% Activity at given concentration.Float%
19Agonist-Activity at 0.0000730785 uM (7.30785e-05μM**)% Activity at given concentration.Float%
20Agonist-Activity at 0.0001461570 uM (0.000146157μM**)% Activity at given concentration.Float%
21Agonist-Activity at 0.0002923140 uM (0.000292314μM**)% Activity at given concentration.Float%
22Agonist-Activity at 0.0005846279 uM (0.000584628μM**)% Activity at given concentration.Float%
23Agonist-Activity at 0.00117 uM (0.00116926μM**)% Activity at given concentration.Float%
24Agonist-Activity at 0.00234 uM (0.00233851μM**)% Activity at given concentration.Float%
25Agonist-Activity at 0.00468 uM (0.00467702μM**)% Activity at given concentration.Float%
26Agonist-Activity at 0.00935 uM (0.00935405μM**)% Activity at given concentration.Float%
27Agonist-Activity at 0.019 uM (0.0187081μM**)% Activity at given concentration.Float%
28Agonist-Activity at 0.037 uM (0.0374162μM**)% Activity at given concentration.Float%
29Agonist-Activity at 0.075 uM (0.0748324μM**)% Activity at given concentration.Float%
30Agonist-Activity at 0.150 uM (0.149665μM**)% Activity at given concentration.Float%
31Agonist-Activity at 0.299 uM (0.29933μM**)% Activity at given concentration.Float%
32Agonist-Activity at 0.599 uM (0.598659μM**)% Activity at given concentration.Float%
33Agonist-Activity at 1.197 uM (1.19732μM**)% Activity at given concentration.Float%
34Agonist-Activity at 2.395 uM (2.39464μM**)% Activity at given concentration.Float%
35Agonist-Activity at 4.789 uM (4.78927μM**)% Activity at given concentration.Float%
36Agonist-Activity at 9.579 uM (9.57854μM**)% Activity at given concentration.Float%
37Agonist-Activity at 19.16 uM (19.1571μM**)% Activity at given concentration.Float%
38Agonist-Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
39Antagonist-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
40Antagonist-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
41Antagonist-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
42Antagonist-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
43Antagonist-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
44Antagonist-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
45Antagonist-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
46Antagonist-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
47Antagonist-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
48Antagonist-Activity at 0.0000000739 uM (7.39192e-08μM**)% Activity at given concentration.Float%
49Antagonist-Activity at 0.0000002218 uM (2.21757e-07μM**)% Activity at given concentration.Float%
50Antagonist-Activity at 0.0000004704 uM (4.7042e-07μM**)% Activity at given concentration.Float%
51Antagonist-Activity at 0.0000015822 uM (1.58224e-06μM**)% Activity at given concentration.Float%
52Antagonist-Activity at 0.0000034306 uM (3.43061e-06μM**)% Activity at given concentration.Float%
53Antagonist-Activity at 0.0000069922 uM (6.99223e-06μM**)% Activity at given concentration.Float%
54Antagonist-Activity at 0.0000191532 uM (1.91532e-05μM**)% Activity at given concentration.Float%
55Antagonist-Activity at 0.0000524684 uM (5.24684e-05μM**)% Activity at given concentration.Float%
56Antagonist-Activity at 0.0001151172 uM (0.000115117μM**)% Activity at given concentration.Float%
57Antagonist-Activity at 0.0003047708 uM (0.000304771μM**)% Activity at given concentration.Float%
58Antagonist-Activity at 0.0008388002 uM (0.0008388μM**)% Activity at given concentration.Float%
59Antagonist-Activity at 0.00186 uM (0.00186447μM**)% Activity at given concentration.Float%
60Antagonist-Activity at 0.00490 uM (0.00489834μM**)% Activity at given concentration.Float%
61Antagonist-Activity at 0.014 uM (0.0135091μM**)% Activity at given concentration.Float%
62Antagonist-Activity at 0.030 uM (0.0296664μM**)% Activity at given concentration.Float%
63Antagonist-Activity at 0.078 uM (0.0777275μM**)% Activity at given concentration.Float%
64Antagonist-Activity at 0.213 uM (0.21273μM**)% Activity at given concentration.Float%
65Antagonist-Activity at 0.475 uM (0.47526μM**)% Activity at given concentration.Float%
66Antagonist-Activity at 1.247 uM (1.24727μM**)% Activity at given concentration.Float%
67Antagonist-Activity at 3.416 uM (3.41635μM**)% Activity at given concentration.Float%
68Antagonist-Activity at 7.604 uM (7.60416μM**)% Activity at given concentration.Float%
69Antagonist-Activity at 19.27 uM (19.2699μM**)% Activity at given concentration.Float%
70Antagonist-Activity at 50.88 uM (50.8754μM**)% Activity at given concentration.Float%
71Antagonist-Activity at 142.9 uM (142.898μM**)% Activity at given concentration.Float%
72Antagonist-Activity at 287.4 uM (287.356μM**)% Activity at given concentration.Float%
73Antagonist-Activity at 574.7 uM (574.713μM**)% Activity at given concentration.Float%
74Compound 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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