Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compounds
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compounds. ..more
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: U2OSCTYOX_INH_LUMI_384_4XEC50_OPRK1
Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compounds.
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, Tango, U2OS, luminescence, cytotoxicity, CellTitre-Glo, CC50, 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 determine cytotoxicity of a powder compound that inhibits the kappa (OPRK1) opioid receptor. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 12-point 1:3 dilution series starting at a nominal test concentration of 10 uM.
This assay was started by dispensing Tangotrade mark OPRK1-bla U20S cells in McCoy's 5A medium plus 10% FBS, penicillin 100 U/mL and streptomycin 100 ug/mL (20 uL, 4 x 10E3 cells/well) into the wells of a 384-well plate. Twelve 1:3 serial dilutions of compound (100 uM in growth media) were made. 5 uL of diluted compound or media were added to wells. The plate was incubated at 37 C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 uL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader.
The % Cell Viability for each well was then calculated as follows:
%_Cell_Viability = 1 - ( MedianRFU_High_Control - RFU_Test_Compound ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100
Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).
Percent Cell Viability was plotted against the log of the compound concentration. The CC50 is reported as ">X uM" (where X = the highest concentration tested for which > 50% Cell Viability was observed).
PubChem Activity Outcome and Score:
Compounds with a CC50 value equal to or less than of less than 10 uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 uM were considered inactive (non-cytotoxic).
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 inactive compounds 0-0. There are no active compounds.
List of Reagents:
Tangotrade mark OPRK1-bla U2OS Cells (Invitrogen K1576)
McCoy's 5A Medium (Invitrogen, part 16600-082)
FBS (Invitrogen, part # 26140-079)
Penicillin / Streptomycin (Invitrogen, part # 15140-122)
Cell Titer-Glo (Promega, part G7572)
384-well plates (Corning 3570)
This assay was performed by the SRIMSC with powder samples of synthesized test compounds.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: = 100 / ( 1 + 10^( ( [LogCC50] - Log( [Concentration] * 10^-6 ) * [Hill Slope] ) )
* Activity Concentration. ** Test Concentration.
Data Table (Concise)