Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: 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 activators of OPRM1 and OPRD1 heterodimer formation: 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
Name: Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compounds.
Opiates such as morphine are the choice analgesic in the treatment of chronic pain due to their potent and rapid action. However, their long-term use is limited because of the development of tolerance and dependence, as well as respiratory suppression and constipation (1). Due to their clinical importance, various strategies have been considered for making opiates more effective while curbing liabilities such as addiction. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. The OPRM1 gene encodes the mu opioid receptor, which is the primary site of action for morphine (2) and other commonly used opioid such as heroin, fentanyl, and methadone. OPRM1 activation and subsequent dissociation of the Gi/Go G-proteins results in reduction of adenylyl cyclase-mediated cAMP production (3). There are at least two other types of opioid receptors: delta (OPRD1) and kappa (OPRK1), each with a distinct pharmacologic profile. In particular, delta (OPRD1) opioid receptor ligands have been useful in enhancing morphine's potency, but the underlying molecular basis is not understood (4). It has been shown that modulation of receptor function by physical association between mu and delta opioid receptors is a potential mechanism (5). The assay provider has previously found that a combination of OPRM1 agonist with OPRD1 antagonist selectively activates the OPRM1-OPRD1 heteromer (5) and recently showed that this could be blocked by antibodies that selectively recognize the heteromer (6). Since OPRD1 antagonist have anxiogenic effects, these are not ideal as therapies. Hence, the identification of compounds that selectively activate mu-delta opioid receptor heterodimerization may have potential in the treatment of pain and alleviate unwanted effects associated with opiate use.
1. Raehal KM, Bohn LM. Mu opioid receptor regulation and opiate responsiveness. AAPS J. 2005 Oct 19;7(3):E587-91.
2. Matthes H, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques B, Kieffer B (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819-823.
3. Maguea SD and Blendy JAOPRM1 SNP (A118G): Involvement in disease development, treatment response, and animal models. Drug and Alcohol Dependence. 2010 May 108 (3): 172-182.
4. Traynor J, Elliot J. Delta-opioid receptor subtypes and cross talk with mu-receptors. Trends Pharmacol Sci 1993 14:84-86.
5. Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, Devi LA. Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci. 2000 Nov 15;20(22):RC110.
6. Gupta, A., Mulder, J., Gomes, I., Rozenfeld, R., Bushlin, I., Ong, E., Lim, M., Maillet, E., Junek, M., Cahill, C.M., Harkany, T. Devi, L.A. Increased abundance of opioid receptor heteromers after chronic morphine administration. Science Signaling 3:ra54, 2010
Late stage, late stage AID, powders, OPRD1, delta, OPRM1, mu, heterodimer, opioid, receptor, GPCR, PathHunter, U2OS, luminescence, cytotoxicity, CellTitre-Glo, CC50, holoenzyme, agonist, activator, pain, Deltorphin B, 384, dose response, 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 powder compounds that activate heterodimer formation between the mu (OPRM1) and delta (OPRD1) opioid receptors. 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 13-point 1:3 dilution series starting at a nominal test concentration of 10 uM.
This assay was started by dispensing PathHunter trade mark OPRM1/D1-Beta-Arrestin 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. Thirteen 1:3 serial dilutions of compound (100 uM in growth media) were made. 5 uL of diluted compound or media were added to wells, giving final compound concentrations of 0-10 uM. 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 is 0-0. There are no active compounds.
List of Reagents:
PathHunter trade mark OPRM1/D1-Beta-Arrestin U20S Cell Line (Discover X, part 93-0558C3)
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)
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: U-2 OS
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)