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 ..
BioActive Compounds: 70
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.
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
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)