Fluorescence-based counterscreen for agonists of NPY-Y2: cell-based high-throughput dose response assay for agonists of NPY-Y1.
- Deposit:2009-12-17
- Modify:2009-12-17
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Claes Wahlestedt, Scripps Florida
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056950-01
Grant Proposal PI: Claes Wahlestedt
External Assay ID: NPY-Y1_AG_CNGC_1536_3XEC50 CSDRUN
Name: Fluorescence-based counterscreen for agonists of NPY-Y2: cell-based high-throughput dose response assay for agonists of NPY-Y1.
Description:
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.
References:
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.
6. Bonaventure P, Nepomuceno D, Mazur C, Lord B, Rudolph DA, Jablonowski JA, Carruthers NI, Lovenberg TW. Characterization of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]acrylamide (JNJ-5207787), a small molecule antagonist of the neuropeptide Y Y 2 receptor. J Pharmacol Exp Ther. 2004 Mar;308(3):1130-7.
Keywords:
NPY, Neuropeptide Y, NPY-Y2, NPY2R, neuropeptide Y receptor Y2, G protein coupled receptor, GPCR, Galphai, CNGC, cyclic nucleotide gated channel assay, 1536, ACTOne, membrane potential, HEK 293, HTS assay, counterscreen, dose response, NPY1R, activator, activation, agonist, agonism, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Library Probe Production Centers Network, MLPCN.
Assay Overview:
The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay for potentiators or agonists of NPY-Y2" (AID 1359), "Fluorescence-based counterscreen assay for potentiators of NPY-Y2: cell-based high-throughput screening assay to identify agonists of NPY-Y2" (AID 1710),
and inactive in a set of experiments entitled,
"Primary cell-based high-throughput screening assay for potentiators or agonists of NPY-Y1" (AID 1304), and "Fluorescence-based counterscreen assay for potentiators or agonists of NPY-Y2: cell-based high-throughput screening assay to identify inhibitors of cyclic nucleotide gated ion channel (CNGC) activity" (PubChem AID 1703), were nonselective due to activation of NPY-Y1.
In this assay, a cell line transfected with Y1 and a cyclic-nucleotide gated channel (CNGC) is used to measure direct agonism of the Y1 receptor by test compound in the absence of the agonist, NPY. 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 agonist of NPY-Y1 will increase Y1 activity, leading to decreased cAMP levels, reduced CNG channel opening and probe fluorescence, thus leading to reduced well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 35 micromolar.
Protocol Summary:
The Y1 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 Y1 HEK293-CNG cells in a 4 ul 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 ul 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 first fluorescence measurement was performed (510-545 nm excitation and 565-625 nm emission) on the FLIPR Tetra (Molecular Devices), and then 2 ul of NPY in 0.1% BSA or 0.1% BSA alone was dispensed to the appropriate wells. Next, 32 nL of test compound in DMSO (0.4% final DMSO 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 ul of a solution containing isoproterenol at its EC100 (1 uM final concentration) and the phosphodiesterase inhibitor, Ro 20-1724, in PBS (25 uM final concentration). The plates were then incubated for 45 minutes at room temperature before the final fluorescence measurement with the same instrument settings.
The following mathematical expression was used to normalize data:
Ratio = T45 / T0
Where:
T0 represents the measured fluorescence emission intensity before the addition of compounds and challenge and;
T45 represents the measured fluorescence emission intensity 45 minutes post addition of compounds and challenge.
The percent activation for each compound was calculated using:
% Activation = ( MedianRatio_Test_Compound - MedianRatio_Low_Control ) / ( MedianRatio_High_Control - MedianRatio_Low_Control ) * 100
Where:
Test_Compound is defined as wells containing test compound, 0.1% BSA and isoproterenol.
High_Control is defined as wells with DMSO, 40 nM NPY (EC100) and isoproterenol.
Low_Control is defined as wells with DMSO, 0.1% BSA and isoproterenol.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 35 micromolar) did not result in greater than 50% activation, the EC50 was determined manually as greater than 35 uM. Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The activity score range for active compounds is 100-78, for inactive compounds 76-0.
List of Reagents:
Neuropeptide Y receptor Y1 HEK293-CNG cells (BD Biosciences, part 344869)
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)
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