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BioAssay: AID 434931

Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of 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(96)
 
 
Active(72)
 
 
Inactive(9)
 
 
Inconclusive(15)
 
 
 Tested Substances
 Tested Substances
All(97)
 
 
Active(73)
 
 
Inactive(9)
 
 
Inconclusive(15)
 
 
AID: 434931
Data Source: NCGC (NPSR006tr)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-05-20
Modify Date: 2010-10-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 72
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
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
434936Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal TransductionConfirmatory 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
624054Robust Characterization of cAMP Signal Transduction for Antagonists of the Neuropeptide S Receptor: SAROther 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). This particular assay uses a high density of sampling across concentration-response space to establish more confidence estimates of potency.
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 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 0.00000308304 uM (3.08304e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000034256 uM (3.4256e-06μM**)% Activity at given concentration.Float%
17Activity at 0.00000376816 uM (3.76816e-06μM**)% Activity at given concentration.Float%
18Activity at 0.00000616599 uM (6.16599e-06μM**)% Activity at given concentration.Float%
19Activity at 0.0000068511 uM (6.8511e-06μM**)% Activity at given concentration.Float%
20Activity at 0.00000753621 uM (7.53621e-06μM**)% Activity at given concentration.Float%
21Activity at 0.00001233198 uM (1.2332e-05μM**)% Activity at given concentration.Float%
22Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
23Activity at 0.00001507242 uM (1.50724e-05μM**)% Activity at given concentration.Float%
24Activity at 0.00002466396 uM (2.4664e-05μM**)% Activity at given concentration.Float%
25Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
26Activity at 0.00003014484 uM (3.01448e-05μM**)% Activity at given concentration.Float%
27Activity at 0.00004932801 uM (4.9328e-05μM**)% Activity at given concentration.Float%
28Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
29Activity at 0.00006028979 uM (6.02898e-05μM**)% Activity at given concentration.Float%
30Activity at 0.00009865593 uM (9.86559e-05μM**)% Activity at given concentration.Float%
31Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
32Activity at 0.00012057947 uM (0.000120579μM**)% Activity at given concentration.Float%
33Activity at 0.00019731195 uM (0.000197312μM**)% Activity at given concentration.Float%
34Activity at 0.0002192355 uM (0.000219236μM**)% Activity at given concentration.Float%
35Activity at 0.00024115905 uM (0.000241159μM**)% Activity at given concentration.Float%
36Activity at 0.00039462390 uM (0.000394624μM**)% Activity at given concentration.Float%
37Activity at 0.0004384710 uM (0.000438471μM**)% Activity at given concentration.Float%
38Activity at 0.00048231810 uM (0.000482318μM**)% Activity at given concentration.Float%
39Activity at 0.00078924771 uM (0.000789248μM**)% Activity at given concentration.Float%
40Activity at 0.0008769419 uM (0.000876942μM**)% Activity at given concentration.Float%
41Activity at 0.00096463609 uM (0.000964636μM**)% Activity at given concentration.Float%
42Activity at 0.001575 uM (0.001575μM**)% Activity at given concentration.Float%
43Activity at 0.00175 uM (0.00175μM**)% Activity at given concentration.Float%
44Activity at 0.001925 uM (0.001925μM**)% Activity at given concentration.Float%
45Activity at 0.003159 uM (0.003159μM**)% Activity at given concentration.Float%
46Activity at 0.00351 uM (0.00351μM**)% Activity at given concentration.Float%
47Activity at 0.003861 uM (0.003861μM**)% Activity at given concentration.Float%
48Activity at 0.006318 uM (0.006318μM**)% Activity at given concentration.Float%
49Activity at 0.00702 uM (0.00702μM**)% Activity at given concentration.Float%
50Activity at 0.007722 uM (0.007722μM**)% Activity at given concentration.Float%
51Activity at 0.0126 uM (0.0126μM**)% Activity at given concentration.Float%
52Activity at 0.014 uM (0.014μM**)% Activity at given concentration.Float%
53Activity at 0.0154 uM (0.0154μM**)% Activity at given concentration.Float%
54Activity at 0.0252 uM (0.0252μM**)% Activity at given concentration.Float%
55Activity at 0.028 uM (0.028μM**)% Activity at given concentration.Float%
56Activity at 0.0308 uM (0.0308μM**)% Activity at given concentration.Float%
57Activity at 0.0504 uM (0.0504μM**)% Activity at given concentration.Float%
58Activity at 0.056 uM (0.056μM**)% Activity at given concentration.Float%
59Activity at 0.0616 uM (0.0616μM**)% Activity at given concentration.Float%
60Activity at 0.1008 uM (0.1008μM**)% Activity at given concentration.Float%
61Activity at 0.112 uM (0.112μM**)% Activity at given concentration.Float%
62Activity at 0.1232 uM (0.1232μM**)% Activity at given concentration.Float%
63Activity at 0.2016 uM (0.2016μM**)% Activity at given concentration.Float%
64Activity at 0.224 uM (0.224μM**)% Activity at given concentration.Float%
65Activity at 0.2464 uM (0.2464μM**)% Activity at given concentration.Float%
66Activity at 0.4041 uM (0.4041μM**)% Activity at given concentration.Float%
67Activity at 0.449 uM (0.449μM**)% Activity at given concentration.Float%
68Activity at 0.4939 uM (0.4939μM**)% Activity at given concentration.Float%
69Activity at 0.8082 uM (0.8082μM**)% Activity at given concentration.Float%
70Activity at 0.898 uM (0.898μM**)% Activity at given concentration.Float%
71Activity at 0.9878 uM (0.9878μM**)% Activity at given concentration.Float%
72Activity at 1.6164 uM (1.6164μM**)% Activity at given concentration.Float%
73Activity at 1.796 uM (1.796μM**)% Activity at given concentration.Float%
74Activity at 1.9756 uM (1.9756μM**)% Activity at given concentration.Float%
75Activity at 3.2328 uM (3.2328μM**)% Activity at given concentration.Float%
76Activity at 3.592 uM (3.592μM**)% Activity at given concentration.Float%
77Activity at 3.9512 uM (3.9512μM**)% Activity at given concentration.Float%
78Activity at 6.4656 uM (6.4656μM**)% Activity at given concentration.Float%
79Activity at 7.184 uM (7.184μM**)% Activity at given concentration.Float%
80Activity at 7.9024 uM (7.9024μM**)% Activity at given concentration.Float%
81Activity at 12.933 uM (12.933μM**)% Activity at given concentration.Float%
82Activity at 14.37 uM (14.37μM**)% Activity at given concentration.Float%
83Activity at 15.807 uM (15.807μM**)% Activity at given concentration.Float%
84Activity at 25.866 uM (25.866μM**)% Activity at given concentration.Float%
85Activity at 28.74 uM (28.74μM**)% Activity at given concentration.Float%
86Activity at 31.614 uM (31.614μM**)% Activity at given concentration.Float%

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

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