Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assay, Set 2.
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assay, Set 2. ..more
BioActive Compound: 1
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_4XIC50_SET2
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assay, Set 2.
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.
Late stage, late stage AID, OPRK1, kappa, opioid, receptor, GPCR, beta-arrestin, U2OS, Tango trade mark, beta-lactamase, FRET, FRET-enabled substrate, TEV, TEV protease, beta-arrestin, EC80 challenge, SNC80, antagonist, inhibitor, 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 the potency of test synthesized compounds. This assay uses Tango OPRK1-bla U2OS cells which express 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 Beta-arrestin 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 agonist 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 either quadulplicat or octuplet using a 12-point, 1:3 dilution series starting at a nominal concentration of 10 uM.
The Tango OPRK1-U20S dividing cell line was routinely cultured in 150 mm dishes at 37 C, 5% CO2 and 95% relative humidity (RH). The growth medium consisted of McCoys 5A Media supplemented with 10% v/v dialyzed fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1X antibiotic mix (penicillin streptomycin). On day 1 of the assay, 16,000 cells in 10 uL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. On Day 2, 50 nl of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37 C, 5% CO2 and 95% RH. Next, 0.6 uL of 111 nM U50488 OPRK1 Agonist (EC80 Challenge; final concentration 6 nM) or DMSO in assay medium was added to appropriate wells and incubated 4 hours at 37 C, 5% CO2 and 95% RH. 2.5 uL of LiveBLazer trade mark FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 uL solution A, 60 uL Solution B, 904 uL Solution C, and 30 uL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 409 nm, Emission filters at 460 nm and 590 nm, bottom read.
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.
Low_Control (0% inhibition) is defined as wells containing U50488 EC80 challenge
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. In cases where the highest concentration tested (i.e. 50 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 50 uM.
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 100-100. There are no inactive compounds.
List of Reagents:
Tango trade mark OPRK1-bla U20S Cells (Invitrogen K1576)
McCoy's 5A Medium, (Invitrogen, part 16600-082)
Dialyzed Fetal Bovine Serum, (Invitrogen, part 26400-036)
Non Essential Amino Acids 100X (Invitrogen, part 11140-050)
HEPES (pH 7.3) 1M, (Invitrogen, part 15630-080)
Sodium Pyruvate 100X (Invitrogen, part 11360-070)
Penicillin Streptomycin, (Invitrogen, part 15640)
Trypsin 0.25% EDTA (Invitrogen, part 25200056)
DPBS without Calcium /Magnesium (Invitrogen, part 14190-136)
DMEM, High Glucose,GlutaMAX (Invitrogen, part 10569-010)
Fetal Bovine Serum, Charcoal Stripped (Invitrogen, part 12676-011)
DMSO Dry (Sigma, part D2650)
U50488 OPRK1 Agonist MW410.29 (Tocris 67198-19-0)
GNTI dihydrochloride OPRK1 Antagonist MW571.5 (Tocris 1282)
Nor-Binaltorphimine dihydrochloride MW770.75 (Tocris 0347)
384 black low profile, clear bottom, low volume plate (Greiner, part 788092)
Low profile plate lids (Greiner, part 656190)
LiveBLAzer trade mark-FRET/BG Loading Mix (Invitrogen, part K1030
This assay was performed by the SRIMSC with powder samples of synthesized test compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: = [Baseline Response] + ( [Maximal Response] - [Baseline Response] ) / ( 1 + 10 ^ ( ( [LogEC50] - 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)