Primary cell-based high-throughput screening assay for potentiators or agonists of 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 more ..
BioActive Compounds: 969
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center(SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Patricia McDonald, Scripps Florida
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R21 NS056950-01
Grant Proposal PI: Claes Wahlestedt
External Assay ID: NPY-Y2_POT_CNGC_1536_%ACT
Name: Primary cell-based high-throughput screening assay for potentiators or agonists of 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, the use of Y2 modulators may help to elucidate the anxiolytic-like effects of NPY in animal models (3). Consistent with this hypothesis Y2 receptor mutant mice demonstrate reduced anxiety behavior compared with wild type controls (4). Therefore, the identification and characterization of small molecule modulators of NPY signaling and high affinity selective ligands for the Y2 receptor may prove useful for understanding NPY-associated human disease.
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.
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, potentiator, activator, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Screening Center Network, MLPCN, 1536.
The purpose of this assay is to determine the ability of test compounds to act as potentiators or agonists of the NPY-Y2 receptor (Y2). A cell line transfected with Y2 and a cyclic-nucleotide gated channel (CNGC) is used to measure potentiation of the NPY response or direct agonism by test compound of the Y2 receptor. The cells are treated with isoproterenol to activate adenylate cyclase, therefore increasing cytosolic cyclic adenosine monophosphate (cAMP) concentrations, and as a consequence CNGC activity. Changes in CNGC activity also change the cell membrane potential, which is measured using a fluorescent probe. As designed, a test compound that acts as an Y2 potentiator or agonist will increase Y2 activity, leading to decreased cAMP levels, reduced CNG channel opening and therefore reduced probe fluorescence.
The Y2 HEK293-CNG cells were routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 250 micrograms/mL Geneticin, 1 microgram/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). Prior to the start of the assay, 3600 Y2 HEK293-CNG cells in a 4 microliter volume of assay media (growth media as above) were dispensed into each well of 1536-well black clear bottom tissue culture-treated microtiter plates. Next, the plates were incubated for 23 hours at 37 degrees C, 5% CO2 and 95% RH. The assay was started by dispensing 2 microliters per well of 4.5x concentrated probe loading dye into all wells, and the plates were incubated at room temperature for 3 hours. Following incubation, the cells were challenged by dispensing 2 microliters of NPY (200 pM final concentration) in 0.1% BSA. Next, 32 nL of test compound (3.6 micromolar final nominal concentration) in DMSO (0.4% final concentration) or DMSO alone was added to the appropriate wells. The plates were then incubated for 60 minutes at room temperature, followed by challenge with 1 microliter of a solution containing isoproterenol at its EC100 (1 micromolar final concentration) and the phosphodiesterase inhibitor, Ro 20-1724, in PBS (25 micromolar final concentration). The plates were then incubated for 45 minutes at room temperature before a fluorescence measurement was performed (510-545 nm excitation and 565-625 nm emission) on the FLIPR Tetra (Molecular Devices).
The percent activation for each compound was calculated using well fluorescence as follows:
% Activation = (Median_Test_Compound - Median_Low_Control)/ (Median_High_Control - Median_Low_Control)*100
Test_Compound is defined as wells containing test compound, NPY and isoproterenol.
High_Control is defined as wells with DMSO, 40 nM NPY (EC100) and isoproterenol.
Low_Control is defined as wells with DMSO, 200 pM NPY (~EC25) and isoproterenol.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % activation than that particular plate's cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary activation value. Negative % activation values are reported as activity score zero.
List of Reagents:
Neuropeptide Y receptor Y2 HEK293-CNG cells (BD Biosciences, part 344870)
10x ACTOne Membrane Potential Assay Kit (BD Biosciences, part BD354663)
Phosphate Buffered Saline (Invitrogen, part 10010-023)
Fatty Acid-Free Bovine Serum Albumin (Calbiochem, part 126609)
DMEM (Invitrogen, part 11965-092)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Geneticin (Invitrogen, part 10131-027)
Puromycin (Sigma, part P9620)
Ro 20-1724 (Sigma, part B8279)
Isoproterenol (Sigma, part I6504)
Neuropeptide Y (American Peptide, part 60-1-11B)
1536-well plates (Greiner, part 789072)
T-175 tissue culture flasks (Corning, part 431080)
At the isoproterenol concentration used in this assay, the EC50 for NPY in 0.1% BSA was approximately 772 pM. Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate cAMP and CNG activity or membrane potential, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)