Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen. ..more
BioActive Compounds: 8
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: OPRM1_ANT_LUMI_384_4XIC50
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen.
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, OPRM1, mu, opioid, receptor, GPCR, beta-arrestin, U2OS, lumi, luminescence, antagonist, antagonism, inhibit, inhibitor, inhibition, decrease, DiscoverRx, beta-arrestin, beta-galactosidase, fragment complementation, EC80 challenge, DAMGO, pain, analgesic, dynorphin, neuropathic pain, drug addiction, addiction, 384, counterscreen, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRM1 receptor. The assay monitors GPCR-Beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). The reconstituted holoenzyme catalyzes the hydrolysis of a substrate which yields a chemiluminescent signal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compounds and an agonist DAMGO (EC80 challenge), followed by measurement of well luminescence. As designed, compounds that inhibit OPRM1 will decrease the level of beta-arrestin recruitment elicited by DAMGO, resulting in a decrease in the level of reconstitution of the beta-gal holoenzyme, and decreased well luminescence. Compounds were tested in quadruplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.
The PathHunter(R) DiscoverX OPRM1-U20S cell line was routinely cultured in 150 mm dishes at 37 C, 5% CO2 and 95% relative humidity (RH). The growth medium consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1x antibiotic mix (penicillin streptomycin). On Day 1 of the assay, 5000 cells in 20 uL of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384-well plate, and incubated 16-24 hours at 37 C, 5% CO2 and 95% RH. On Day 2, 100 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, 2.2 uL of DAMGO OPRMu1 agonist (EC80 Challenge; 1.8 uL of 3.7 uM DAMGO and 0.4 uL assay buffer; final assay concentration 303 nM) or DMSO in assay media were added. After incubation for 3 hours at 37 C, 5% CO2 and 95% RH, 10 uL of Path Hunter Detection Mix prepared according to manufacturer's protocol; 1 part Galacton Star:5 parts Emerald II:19 parts PH Cell Assay Buffer) was added to each well, and plates were incubated at room temperature in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.
Percent Inhibition was calculated from the median ratio as follows:
%_Inhibition = 1 - ( ( FI_Test_Compound - Median_FI_High_Control ) / ( 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 DAMGO challenge (303 nM final).
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.
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 100-83, and for inactive compounds 76-0.
List of Reagents:
OPRM1-U20S cell line (PathHunter(R) DiscoverX, part 93-0213E3CPOL))
DMEM Medium (Invitrogen, part 11965)
F12 Medium (Invitrogen, part 11765)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082147)
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)
DMSO Dry (Sigma, part D2650 )
DAMGO OPRMu1 Agonist MW513.19 (Sigma, part E7384-5MG)
B-Funaltrexamine Hydrochloride OPRM1 Antagonist MW (Sigma, part O003-2MG)
PathHunter Cell Plating 5 Reagent (Discover X, part 93-0563R5A)
Standard 384 well white plate with lid (Corning, part 3750)
PathHunter Detection Mix (DiscoverX, part 93-0001)
This assay was performed by the SRIMSC with powder samples of synthesized test compounds.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: = [Baseline Response] + ( [Maximal Response] - [Baseline Response] ) / ( 1 + 10 ^ ( ( [LogEC50] - Log( [Concentration] * 10^-6) ) * [Hill Slope] ) )
* Activity Concentration. ** Test Concentration.
Data Table (Concise)