Fluorescence-based primary cell-based high throughput screening assay to identify potentiators of Oxytocin Receptor (OXTR)
Name: Fluorescence-based primary cell-based high throughput screening assay to identify potentiators of Oxytocin Receptor (OXTR). ..more
BioActive Compounds: 199
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Jarstfer, University of North Carolina at Chapel Hill (UNC)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085678-01A1
Grant Proposal PI: Michael Jarstfer, UNC
External Assay ID: OTR_POT_FLUO8_1536_1X%ACT PRUNS
Name: Fluorescence-based primary cell-based high throughput screening assay to identify potentiators of Oxytocin Receptor (OXTR).
Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, disease-associated mutations (1-4). Certain GPCRs are involved in signaling pathways for neuropeptides such as oxytocin, serotonin, and vasopressin, and influence complex social behaviors such as trust, anxiety, and parenting (5). Oxytocin receptor (OXTR: OTR) activity is mediated by G proteins (Gαq, Gαi, and Gβγ) which activate a phosphatidylinositol-calcium second messenger system (6, 7). OTR belongs to the class A (rhodopsin-like) GPCR family shared with the vasopressin receptors (8), and is expressed in the brain, uterus, and mammary glands. Studies showing a role for OTR in autism (9), defective lactation and nurturing behavior (10), and altered social behavior and aggression (10) suggest that the identification of small molecules that act as potentiators of OTR activity may serve as useful tools in OTR biology and as potential therapeutic agents for human behavioral disorders.
1. Pan, HL, Wu, ZZ, Zhou, HY, Chen, SR, Zhang, HM and Li, DP, Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
2. Lagerstrom, MC and Schioth, HB, Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4): p. 339-57.
3. Thompson, MD, Cole, DE and Jose, PA, Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods Mol Biol, 2008. 448: p. 77-107.
4. Bosier, B and Hermans, E, Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci, 2007. 28(8): p. 438-46.
5. Bakermans-Kranenburg, MJ and van Ijzendoorn, MH, Oxytocin receptor (OXTR) and serotonin transporter (5-HTT) genes associated with observed parenting. Soc Cogn Affect Neurosci, 2008. 3(2): p. 128-34.
6. Ku, CY, Qian, A, Wen, Y, Anwer, K and Sanborn, BM, Oxytocin stimulates myometrial guanosine triphosphatase and phospholipase-C activities via coupling to G alpha q/11. Endocrinology, 1995. 136(4): p. 1509-15.
7. Strakova, Z and Soloff, MS, Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction. Am J Physiol, 1997. 272(5 Pt 1): p. E870-6.
8. Gimpl, G, Reitz, J, Brauer, S and Trossen, C, Oxytocin receptors: ligand binding, signalling and cholesterol dependence. Prog Brain Res, 2008. 170: p. 193-204.
9. Lerer, E, Levi, S, Salomon, S, Darvasi, A, Yirmiya, N and Ebstein, RP, Association between the oxytocin receptor (OXTR) gene and autism: relationship to Vineland Adaptive Behavior Scales and cognition. Mol Psychiatry, 2008. 13(10): p. 980-8.
10. Takayanagi, Y, Yoshida, M, Bielsky, IF, Ross, HE, Kawamata, M, Onaka, T, Yanagisawa, T, Kimura, T, Matzuk, MM, Young, LJ and Nishimori, K, Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice. Proc Natl Acad Sci U S A, 2005. 102(44): p. 16096-101.
oxytocin, oxytocin receptor, OTR, OXTR, autism, social behavior, GPCR, receptor, potentiation, potentiator, activation, activator, Fluo-8, FLUO8, dye, fluorescence, primary, primary screen, HTS, high throughput screen, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as potentiators of the human OTR. In this assay a CHO cell line that stably expresses OTR is incubated with a fluorescent, cell permeable calcium indicator dye (such as Fluo-8), followed by the addition of test compounds and agonist. The dye serves to monitor levels of intracellular calcium in the CHO-OTR cells. As designed, compounds that act as OTR potentiators will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and increased well fluorescence. Compounds were tested in singlicate at a final nominal concentration of 3.0 micromolar.
The CHO-OTR cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 200 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day before the assay 2000 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 microliters of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. The plates were then incubated for 1 hour at 37 degrees C, 5% CO2, and 95 % RH; followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells. Following a pre-incubation of 6 minutes at room temperature, 15 nL of oxytocin (corresponding to an EC15 average response throughout the screen) in DMSO, oxytocin (75 nM final concentration, EC100) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Then a real time fluorescence measurement was immediately performed for the remaining 95 seconds of the assay.
A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I_Max / I_Min
I_Max represents the maximum measured fluorescence emission intensity over the 100 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
Percent activation was calculated from the median ratio as follows:
% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100
Test_Compound is defined as wells containing Oxytocin (EC15 average response) and test compound.
Low_Control is defined as wells containing Oxytocin (EC15 average response) and DMSO.
High_Control is defined as wells containing Oxytocin (EC100) and DMSO.
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% observed primary activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-23, for inactive 61-0.
List of Reagents:
CHO-OTR cells (provided by Assay provider)
Oxytocin (Sigma-Aldrich, part 06379)
Fluo-8 No Wash Calcium Assay Kit (ABD Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
F-12 (Ham) (Invitrogen, part 11765)
Geneticin (Invitrogen, part 10131-027)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN 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. All data reported were normalized on a per-plate basis. 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 modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)