Primary cell based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)
Name: Primary cell based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2) ..more
BioActive Compounds: 1384
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scripps Florida
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number 1 R21 NS056950-01 (Fast Track)
PI: Claes Wahlestedt
External Assay ID: NPY-Y2_ANT_CNGC_1536_%INH
Name: Primary cell based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)
Neuropeptide Y (NPY) is a neurotransmitter with diverse physiologic roles including control of feeding behavior, regulation of cortical neural activity, heart neural activity, and emotional regulation. Importantly, NPY is implicated in human diseases such as obesity, depression and alcoholism.
NPY mediates its biological effects in part through activation of the NPY-Y2 receptor, a 381-amino acid Galphai protein coupled receptor (GPCR) which decreases cytosolic cAMP production. NPY Y2 is expressed in the periventricular nucleus, amygdala, hypothalamus, hippocampus, tractus solitarius, septum and paraventricular nucleus brain regions (1,2). Due to its expression profile and biological action, NPY Y2 is an attractive target for anxiolytic research. Additionally, Y2 is predicted to be a therapeutic target in alcoholism. Because Y2 receptors increase NPY transmission, Y2 antagonists may also mediate anxiolytic-like effects in animal models (3). Consistent with this hypothesis Y2 receptor mutant mice demonstrate reduced anxiety behavior compared with wild type controls (4). Moreover, use of the Y2 receptor antagonist BIIE0246 has been
shown to suppress ethanol self-administration in rats (5). It has been reported, however, that the complex structure and high molecular weight of BIIE0246 limit its usefulness as an in vivo pharmacological tool (6). Therefore, it is necessary to produce high affinity selective ligands for the Y2 receptor.
1.Wahlestedt C, Ekman R, Widerlov E. Neuropeptide Y (NPY) and the central nervous system: distribution effects and possible relationship to neurological and psychiatric disorders. Prog Neuropsychopharmacol Biol Psychiatry. 1989;13(1-2):31-54.
2.Redrobe JP, Dumont Y, Quirion R. Neuropeptide Y (NPY) and depression: from animal studies to the human condition. Life Sci. 2002 Nov 8;71(25):2921-37.
3.Wahlestedt C, Yanaihara N, Hakanson R. Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides. Regul Pept. 1986 Feb;13(3-4):307-18.
4.Redrobe JP, Dumont Y, Herzog H, Quirion R. Neuropeptide Y (NPY) Y2 receptors mediate behaviour in two animal models of anxiety: evidence from Y2 receptor knockout mice. Behav Brain Res. 2003 May 15;141(2):251-5.
5.Rimondini R, Thorsell A, Heilig M. Suppression of ethanol self-administration by the neuropeptide Y (NPY) Y2 receptor antagonist BIIE0246: evidence for sensitization in rats with a history of dependence. Neurosci Lett. 2005 Feb 28;375(2):129-33. Epub 2004 Nov 30.
6. Bonaventure P, Nepomuceno D, Mazur C, Lord B, Rudolph DA, Jablonowski JA, Carruthers NI, Lovenberg TW. Characterization of
a small molecule antagonist of the neuropeptide Y Y 2 receptor. J Pharmacol Exp Ther. 2004 Mar;308(3):1130-7.
NPY, Neuropeptide Y, NPY-Y2, NPY2R, neuropeptide Y receptor Y2, G protein coupled receptor, GPCR, Galphai,CNGC, cyclic nucleotide gated channel assay, ACTOne, membrane potential, HEK 293, HTS assay, primary screen, antagonist, inhibition, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps
A cell line transfected with the NPY-Y2 receptor and a cyclic-nucleotide gated channel (CNG) were used to measure receptor antagonism. As designed, an NPY-Y2 antagonist increases the concentration of agonist-abrogated cytosolic cyclic adenosine monophosphate (cAMP), and therefore causes the opening of the CNG channel. Once open, the CNG channel changes the cell membrane potential. A fluorescent probe can be used to measure this change in membrane potential; in this assay an increase or decrease in the probe's fluorescence correlates to an increase or decrease, respectively, in cAMP concentration.
To measure NPY-Y2 antagonism, adenylate cyclase activity was activated by the beta adrenergic receptor agonist isoproterenol.
Isoproterenol increased the concentration of cAMP, and therefore probe fluorescence via the activated CNG channel. The addition of the agonist NPY peptide counteracted the accumulation of cAMP induced by isoproterenol. Effective NPY-Y2 receptor antagonists therefore would reverse this reduction in NPY-mediated fluorescence, yielding higher fluorescence units.
Note: a 1uM concentration of isoproterenol (EC100) and a 100nM concentration of NPY (EC90) were used to challenge the cells in this assay. At these concentrations, the IC50 for a known NPY-Y2 receptor antagonist, BIIE0246, was determined to be 3 nM.
A total of 3,750 neuropeptide Y receptor Y2 HEK293-CNG cells (BD Biosciences) in a 5uL volume per well were dispensed into a 1536-well black clear bottom tissue culture-treated microtiter plate. Plates were incubated for 24 hours at 37 degrees C, 5% CO2 and 95% RH. Two ul per well of 4.5x concentrated probe loading dye was then dispensed into all wells. Plates were then incubated at room temperature for 3 hours. Following incubation, the first fluorescence measurement (plate read) was performed (510-545nm excitation and 565-625nm emission). Next, 25 nl of test compound (i.e. compound from the MLSCN compound library; final nominal concentration of 2.7 uM, 0.27% final DMSO concentration) or positive or negative control (1 uM BIIE0246 and DMSO, respectively) was then added to the appropriate wells. The cells were then challenged by dispensing 2 microliters of 4.5 uM isoproterenol and 500 nM NPY in 1x phosphate buffer saline containing 112.5 uM of the phosphodiesterase inhibitor Ro 20-1724 (final concentration of 111nM NPY and 1 uM Isoproterenol). Plates were then incubated 30 minutes at room temperature before the final read using the same instrument settings.
The following mathematical expression was used to normalize data:
Ratio = (T30 / T0)
Where T0 represents the measured fluorescence emission intensity before the addition of compounds and challenge and T30 represents the measured fluorescence emission intensity 30 minutes post addition of compounds and challenge.
The percent inhibition for each compound was calculated as follows:
% Inhibition = (1-((Ratio TestCompound - Median_Ratio_HighControl)/(Median_Ratio_LowControl - Median_Ratio_HighControl)))*100
TestCompound= Well containing test compound and challenge
LowControl: Wells containing DMSO and challenge
HighControl: Wells containing 1 uM BIIE0246 in the presence of challenge.
A mathematical algorithm was used to determine nominally inhibitory compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %inhibition than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
List of reagents:
Neuropeptide Y receptor Y2 HEK293-CNG cells (BD Biosciences)
10x ACTOne membrane potential Assay Kit (BD Biosciences cat# BD354663)
Dulbecco's Phosphate Buffered Saline (Invitrogen, cat# 14190-144)
DMEM high glucose with glutamine (Invitrogen, cat# 11965-092)
Fetal Bovine Serum (Invitrogen, cat# 16140-071)
Trypsin-EDTA solution (Invitrogen, cat# 25200-056)
Geneticin (Invitrogen, Cat# 10131-027)
Puromycin (Sigma, cat# P9620)
Phosphodiesterase inhibitor: Ro 20-1724 (Sigma, cat# B8279)
Isoproterenol (Sigma, cat # I6504)
Neuropeptide Y (American peptide, cat# 60-1-11B)
BIIE0246 (Tocris, cat# 1700)
Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on the microtiter plate, compounds that non-specifically modulate cAMP & CNG activity or membrane potential, and compounds that quench or emit fluorescence within the well.
Categorized Comment - additional comments and annotations
Data Table (Concise)