Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1)
Name: Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1). ..more
BioActive Compounds: 7
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Lakshmi A. Devi, Mount Sinai School of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03NS053751
Grant Proposal PI: Lakshmi A. Devi, Mount Sinai School of Medicine
External Assay ID: OPRK1_ANT_FRET_384_3X%IC50
Name: Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1).
Potent and selective OPRK antagonists will be useful for studying the mechanisms involved in OPRK-mediated analgesia and may have therapeutic value as novel analgesics with an improved side effect profile to currently available drugs. Studies have identified a role for dynorphin and OPRK stimulation in neuropathic pain (1). The dynorphins act as endogenous agonists at the opioid receptors, including OPRK (2), and the increased dynorphin expression in neuropathic pain also leads to a sustained activation of OPRK (1, 3). The mechanisms and neural circuits in OPRK-mediated analgesia are active areas of study; it is hoped those studies will assist in the development of novel analgesics that bypass OPRK-mediated depression (4-5). A role for dynorphin/OPRK in modulating drug addiction has been proposed (for review, see (6-7)). The function of dynorphin/OPRK systems in addiction appears to be diverse, and may modulate drug-seeking behavior depending on factors such as drug history, pattern of intake, and stress (for review, see (6)). The availability of potent and selective OPRK ligands may help unravel these mechanisms, as well as prove to be of therapeutic utility. Evidence from preclinical studies indicates that the dynorphin/OPRK system may be dysregulated in affective psychiatric disorders (for review, see (6, 8)). However, solid evidence from clinical studies is lacking. There is increasing evidence for a potential involvement of dynorphin/OPRK in schizophrenia; OPRK agonists appear to induce symptoms in humans and animals that are present in schizophrenia (8-10). Thus, the availability of new research tools such as potent and selective OPRK antagonists will facilitate understanding the physiological and pathophysiological mechanisms of dynorphin/OPRK systems and their roles in psychiatric disease in humans.
1. Xu, M., et al., Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J Neurosci, 2004. 24(19): p. 4576-84.
2. Chavkin, C., I.F. James, and A. Goldstein, Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science, 1982. 215(4531): p. 413-5.
3. Xu, M., et al., Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase. J Neurosci, 2007. 27(10): p. 2570-81.
4. Al-Hasani, R. and M.R. Bruchas, Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology, 2011. 115(6): p. 1363-81.
5. Muschamp, J.W., A. Van't Veer, and W.A. Carlezon, Jr., Tracking down the molecular substrates of stress: new roles for p38alpha MAPK and kappa-opioid receptors. Neuron, 2011. 71(3): p. 383-5.
6. Tejeda, H.A., T.S. Shippenberg, and R. Henriksson, The dynorphin/kappa-opioid receptor system and its role in psychiatric disorders. Cell Mol Life Sci, 2012. 69(6): p. 857-96.
7. Yoo, J.H., I. Kitchen, and A. Bailey, The endogenous opioid system in cocaine addiction: what lessons have opioid peptide and receptor knockout mice taught us? Br J Pharmacol, 2012. 166(7): p. 1993-2014.
8. Schwarzer, C., 30 years of dynorphins--new insights on their functions in neuropsychiatric diseases. Pharmacol Ther, 2009. 123(3): p. 353-70.
9. Bortolato, M. and M.V. Solbrig, The price of seizure control: dynorphins in interictal and postictal psychosis. Psychiatry Res, 2007. 151(1-2): p. 139-43.
10. Sheffler, D.J. and B.L. Roth, Salvinorin A: the "magic mint" hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci, 2003. 24(3): p. 107-9.
Maybridge Library, Maybridge, OPRK1, kappa, opioid, receptor, GPCR, beta-arrestin, U2OS, Tangotrade mark, beta-lactamase, FRET, FRET-enabled substrate, TEV, TEV protease, beta-arrestin, EC80 challenge, U-50488, antagonist, inhibitor, dose response, inhibit, pain, analgesic, dynorphin, neuropathic pain, drug addiction, addiction, 384, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to confirm activity of compounds from the Maybridge Library that act as antagonists of OPRK1. This assay uses Tango OPRK1-BLA U2OS cells which contain the human opiod kappa receptor (OPRK1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRK1 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRK1 antagonists will inhibit OPRK1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.
U2OS cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, 200 ug/mL Zeocin, 50 ug/mL Hygromycin, and 100 ug/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (McCoy's 5A Medium supplemented with 10% v/v charcoal dextran stripped fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomyci). The assay was started by dispensing 10 ul of cell suspension to each well, followed by overnight incubation at 37 C in 5% CO2 and 95% RH. The next day, 50 nL of test compound (9 uM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, U-50488 in Assay Medium (8 nM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37 C in 5% CO2 for 4 hrs. After the incubation, 2.2 ul/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm.
Percent Inhibition was calculated from the median ratio as follows:
%_Inhibition = 1 - ( ( FI_Test_Compound - Median_FI_HighControl ) / ( Median_FI_Low_Control - Median_FI_High_Control ) ) * 100
FI is defined as Fluorescence Intensity at 460 nm/Fluorescence Intensity at 530 nm.
Test_Compound is defined as wells containing test compound and U-50488
Low_Control is defined as wells containing U-50488
High Control (100% inhibition) is defined as wells containing DMSO
For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated IC50 values were reported.
PubChem Activity Outcome and Score:
Compounds with an IC50 of 10 uM or less were considered active. Compounds with an IC50 of greater than 10 uM were considered inactive.
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 of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-80, and for inactive compounds 58-0.
List of Reagents:
Tango trade mark OPRK1-bla U2OS cells (Invitrogen, part K1576)
GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (Invitrogen, part K1025)
Probenecid (Sigma, part P8761)
McCoy's 5A Medium (modified) (1X) (Invitrogen, 16600-082)
Fetal Bovine Serum, dialyzed (Invitrogen, part 26400-036)
Fetal Bovine Serum, charcoal (Invitrogen, part 12676-029)
NEAA (Invitrogen, part 1114-050)
Penicillin-Streptomycin antibiotic mix (Invitrogen, part 15140-122)
Sodium Pyruvate (Invitrogen, part 11360-070)
PBS without calcium or magnesium (Invitrogen, part 14190-136)
HEPES (Invitrogen, part 15630-080)
Trypsin/EDTA (Invitrogen, part 25300-054)
U-50488 (Sigma, D8040)
Zeocin (Invitrogen, part R250-01)
Hygromycin (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
384-well plates (Greiner, part 788092)
T175 tissue culture flasks (Corning, part 431080)
This assay was performed by the SRIMSC with powder samples of purchased test compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: = 100 / ( 1 + 10^( ( [LogIC50] - Log( [Concentration] * 10^-6 ) * [Hill Slope] ) )
Assay: Dictionary: Version: 0.1
Assay Format: Cell-based
Assay Cell Type: U-2 OS
* Activity Concentration. ** Test Concentration.
Data Table (Concise)