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BioAssay: AID 2570

Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin 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(86)
 
 
Inactive(82)
 
 
Inconclusive(5)
 
 
 Tested Substances
 Tested Substances
All(88)
 
 
Inactive(83)
 
 
Inconclusive(5)
 
 
AID: 2570
Data Source: NCGC (NPSR009h)
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 ):           View All Data
Target
Tested Compounds:
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
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
2568Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for ProbeConfirmatory 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
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for ProbeConfirmatory same project related to Summary assay
2567Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transduction, 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.

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). To validate the specific action of compounds on the NPS receptor, a counterscreen was developed that tests compounds' antagonism of AVPR1B vasopressin receptor stimulation by vasopressin. Vasopressin shares 27% identity with the NPS receptor.
Protocol
NCGC Assay Protocol Summary:

A Chinese hamster ovary (CHO) cell line stably expressing human vasopressin receptor (AVPR1B) was 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. The maximums of kinetic fluorescence responses were converted into text files using the instrument's software data export utility. Data for antagonist response were normalized to the controls for basal activity (DMSO only) and 100% inhibition (no signal). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

CHO-V1B 1536-well FDSS assay protocol:
(1) Frozen CHO-V1B 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 min.
(4) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM.
(5) Load plates to FDSS-7000. The following steps were performed on FDSS-7000.
(6) Record fluorescent background (Ex 480 nm, Em 520-560 nm) for 10 s.
(7) Add 2 ul of stimulation reagent (1X HBSS buffer, 0.1% BSA, 60 nM vasopressin). Record antagonist response (Ex 480 nm, Em 520-560 nm) for 180 s.

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
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 significant, 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 0.0000034256 uM (3.42555e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000068511 uM (6.85111e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
20Activity at 0.0002192355 uM (0.000219235μM**)% Activity at given concentration.Float%
21Activity at 0.0004384710 uM (0.000438471μM**)% Activity at given concentration.Float%
22Activity at 0.0008769419 uM (0.000876942μM**)% Activity at given concentration.Float%
23Activity at 0.00175 uM (0.00175388μM**)% Activity at given concentration.Float%
24Activity at 0.00351 uM (0.00350777μM**)% Activity at given concentration.Float%
25Activity at 0.00702 uM (0.00701554μM**)% Activity at given concentration.Float%
26Activity at 0.014 uM (0.0140311μM**)% Activity at given concentration.Float%
27Activity at 0.028 uM (0.0280621μM**)% Activity at given concentration.Float%
28Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
29Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
30Activity at 0.224 uM (0.224497μM**)% Activity at given concentration.Float%
31Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
32Activity at 0.898 uM (0.897989μM**)% Activity at given concentration.Float%
33Activity at 1.796 uM (1.79598μM**)% Activity at given concentration.Float%
34Activity at 3.592 uM (3.59195μM**)% Activity at given concentration.Float%
35Activity at 7.184 uM (7.18391μM**)% Activity at given concentration.Float%
36Activity at 14.37 uM (14.3678μM**)% Activity at given concentration.Float%
37Activity at 28.74 uM (28.7356μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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