Cell line and materials
HEK 293-MOR cell line (HEK-MOR), kindly provided by Dr. Horace Loh (Department of Pharmacology, Medical School at University of Minnesota), stably express mu opioid receptor-1. These cells were maintained in advanced DMEM (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM glutaMAX (Invitrogen), 100 units/ml penicillin/100 µg/ml streptomycin (Invitrogen) and 0.2 mg/ml G418 (Invitrogen) - complete culture medium. Ro20-1724, 3-isobutyl methyl xanthine (IBMX), naloxone hydrochloride dihydrate and morphine sulfate pentahydrate were from Sigma-Aldrich St-Louis, MO, USA. NKH 477 was from Tocris Bioscience (Ellisville, MO, USA).
Morphine tolerance cell model and cAMP assay
For a 384-well plate format, HEK-MOR cells were suspended in the complete culture medium and were dispensed in 10 µl aliquots in white/solid bottom 384 well plates (Greiner Bio-One North America, NC, USA). These were incubated for 18 hr at 37°C in 5% CO2 and 95 % air atmosphere. cAMP levels were measured in presence of NKH477 and phosphodiesterase inhibitors. Final concentrations of Ro20-1724 and IBMX were 0.5mM. Ro20-1724 and IBMX stock solutions were prepared in DMSO and diluted with complete culture media to a final concentration of 0.3 % DMSO in the assay. NKH477, morphine and naloxone solutions were prepared in complete culture media.
The cAMP production from cells was measured using cAMP dynamic HTRF kits (Cisbio Bioassays, Bedford, MA, USA) according to manufacturer instructions. Using the HTRF technology, the assay is based on competition between native cAMP produced by cells and cAMP labelled with the dye d2. The tracer binding is visualized by an anti-cAMP antibody labeled with cryptate. Briefly, after the compound treatment, 12.5 µl each of d2 and cryptate were added into the assay plates. The plates were sealed and incubated at room temperature (RT) in the dark for 1 hr. The fluorescence intensity of the assay plates was measured at 314 nm excitation, and 668 and 620 nm emission in a FlexStation 3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Data was expressed as the ratio of 668nm/620nm emissions, and then was converted into cAMP levels according to cAMP standard curve. The concentration response curves were fitted using the non-linear regression analysis program, GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
For a 1536-well plate format, HEK-MOR cells were suspended in the complete culture medium and dispensed at 1000 cells per 4 µl volume per well in 1536-well tissue culture treated white/solid bottom plates (Aurora Biotechnologies, Carlsbad, CA) using a Flying Reagent Dispenser (FRD, Aurora Discovery, Carlsbad, CA). After the cells were incubated at 37°C with 5% CO2 for 6 hrs, 1 µl of 1 µM morphine (final concentration) was added, followed by addition of 23 nL of compound or DMSO into the assay plates using a pin tool (Kalypsys, San Diege, CA). The plates were incubated at 37°C with 5% CO2 overnight (18 hrs). The next day 1 µl of stimulation mixture containing 0.5 mM Ro 20-1724, 0.5 mM IBMX, 0.1 mM naloxone and 25 nM NKH 477 (final concentration) was added into the plates. After the assay plates were incubated at 37°C for 10 min, 2 µl of 5.2X d2 and cryptate was added. The plates were incubated at RT in the dark for 1 hr. The fluorescence intensity of the assay plates was measured at 340 nm excitation and 665 and 620 nm emission by an Envision plate reader (Perkin Elmer, Boston, MA). Data was expressed as the ratio of 665nm/620nm emissions in the primary screen.
qHTS and Data Analysis
The Library of Pharmacologically Active Compounds (LOPAC, Sigma-Aldrich) which contains 1280 compounds was screened in this study. Compound plates were prepared as inter-plate titrations of seven concentrations with the first four columns in each plate reserved for controls. Pin tool transfer of compounds to assay plates resulted in a 217-fold dilution. The final compound concentration in the 5 µl assay volume ranged from 3 nM to 46 µM. Analysis of the primary screening data was performed as previously described (Xia et al., 2009a
). Briefly, raw plate reads for each titration point were first normalized relative to the morphine control (1 µM, 100%) in the presence of 25 nM NKH 477 and DMSO without morphine in the presence of 25 nM NKH 477 wells (basal, 0%), and then corrected by applying a pattern correction algorithm using compound-free control plates (DMSO plates). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half maximal inhibition (IC50
) and maximal response (efficacy) values.
Cell viability assay
In order to exclude the compounds which attenuate morphine tolerance due to the cytotoxic effect of the compound, the LOPAC library was also tested in a cell viability assay that measures intracellular ATP content using a luciferase-coupled ATP quantitation assay (CellTiter-Glo®, Promega, Madison, WI). HEK-MOR cells were dispensed at 1000 cells per well in 1536-well white/solid bottom assay plates (Greiner Bio-One North America, Monroe, NC) using a FRD. The assay plates were incubated for 5 hr and followed by addition of compounds via a pin tool. The assay plates were incubated for 18 hr at 37°C. At the end of the incubation period, 5 µl of CellTiter-Glo® reagent was added, plates were incubated at RT for 30 minutes, and luminescence intensity determined using a ViewLux plate reader (PerkinElmer).