Compounds SID7969543 and SID7970631 were purchased from Life Chemicals (Kiev, Ukraine). Compound AC-45594 (Del Tredici et al., 2007
) was acquired from Sigma-Aldrich (Milwaukee, WI).
pGal4DBD_SF-1LBD and pGal4DBD_RORALBD were generated by cloning PCR fragments encoding either human SF-1 (aa 198–462) or mouse RORA (aa 266–523) LBD in frame with the DBD of the yeast transcriptional factor Gal4 encoded by the pFA-CMV vector (Stratagene, La Jolla, CA). SF-1 (aa 198–462) was amplified from an Invitrogen EST clone (San Diego, CA; clone# 5163875). BamHI and XbaI sites introduced by the primers GATCGGATCCCCGGAGCCTTATGCCAGCCC (forward) and GATCTCTAGATCAAGTCTGCTTGGCTTGCAGCATTTCGATGAG (reverse) were used for subcloning the amplicon into pFA-CMV. RORA (aa 266–523) was generated by PCR primers GCCGCCCCCGGGCCGAACTAGAACACCTTGCCC (forward) and TATATAAAGCTTTCCTTACCCATCGATTTGCATGG (reverse) from a mouse liver cDNA library from Clontech (Mountain View, CA) and subcloned through XmaI and HindIII restriction sites into pFA-CMV.
Cell culture and transient transfection conditions
Chinese Hamster Ovary (CHO) cells of the K1 subtype (ATCC, Manassas, VA) were grown in T-175 flasks (Corning, Lowell, MA) at 37°C, 5% CO2, 95% relative humidity in F12 media (Gibco, Carlsbad, CA) supplemented with 10% v/v fetal bovine serum (Gemini Bio-products, West Sacramento, CA) and 1% v/v penicillin-streptomycin-neomycin mix (Gibco, Carlsbad, CA). Cells were routinely cultured by splitting them from 1:4 to 1:8.
The day before transfection, cells were rinsed with PBS and trypsinized with a 0.25% trypsin-EDTA solution (Gibco, Carlsbad, CA), then 6 million CHO-K1 cells were seeded in T-175 flasks containing 20 mL of F12 media supplemented as mentioned above. Cells were allowed to incubate overnight at 37°C, 5% CO2 and 95% relative humidity (RH). On the following day, CHO-K1 cells were transiently co-transfected with either 250 ng of pGal4DBD_SF-1LBD plasmid or 125 ng of pGal4DBD_RORALBD in combination with 9 µg of pG5luc (Promega Corporation, Madison, WI) and 8.75 µg of empty pcDNA3.1 (Invitrogen, Carlsbad, CA), in 1.2 mL of F12 media containing 54 µL of TransIT®-CHO reagent and 9 µL of TransIT-CHO® Mojo reagent, according to the manufacturer's protocol (Mirus Bioproducts, Madison, WI).
For uHTS assays, cells transfected with the pG5-luc and empty pcDNA3.1 plasmids alone, designated “−NR” cells (as opposed to “+NR” cells, which are co-transfected with the Gal4-LBD encoding plasmid) were used as positive control for inhibition. Flasks were then placed back in the incubator at 37°C, 5%CO2 and 95% relative humidity. Four hours after transfection, cells were trypsinized and suspended to a concentration of 800,000 cells per milliliter in supplemented F12 media.
1536-well format SF-1 and RORA uHTS assays
The assay began by dispensing 5 µL of transfected cell suspension to each well (i.e. 4,000 cells/well) of a white solid-bottom 1536-well plate (Greiner, North Carolina, USA) using a Bottle Valve liquid dispenser (GNF/Kalypsys, San Diego, CA). Cells from flasks designated −NR were seeded in the first two columns of the 1536-well plate (Low Control) and the remaining 46 columns were filled with +NR cells. One hour after seeding, +NR cells were treated with 50 nL/well of test compounds or DMSO alone (High Control) using a 1536-well head PinTool unit (GNF/Kalypsys, San Diego, CA). Plates were then incubated at 37°C, 5%CO2
and 95% relative humidity. Twenty hours later, plates were equilibrated to room temperature for 20 minutes and a luciferase assay was performed by adding 5 µL per well of SteadyLite HTS™ reagent (PerkinElmer, Waltham, MA). After a 15 minutes incubation time, light emission was measured for 30 seconds with the ViewLux™ reader (PerkinElmer, Turku, Finland). Activity of each compound was calculated on a per-plate basis using the following equation:
where High Control represents wells containing +NR cells treated with DMSO (n=24) and Low Control represents wells containing −NR cells treated with DMSO (n=24).
Primary high-throughput screening and hit selection
A library of 64,908 compounds provided by the Molecular Library Screening Center Network (MLSCN) in fifty-two 1536-well plates was tested in the SF-1 assay as described above. The final nominal test concentration was 10 µM, in a final DMSO concentration of 1%.
The uHTS campaign was executed on the automated GNF/Kalypsys robotic platform of the Scripps Research Institute Molecular Screening Center (Jupiter, FL). Raw data from each primary campaign were uploaded and analyzed in our institutional HTS database (MDL Information Systems, San Ramon, CA). The percent inhibition of each tested compound was calculated on a per-plate basis as described in the previous section. A mathematical algorithm was used to determine active compounds (Hodder et al., 2003
). Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater percent inhibition than the cutoff parameter was declared active. Z’ value was calculated as previously described (Zhang et al., 1999
). Detailed information regarding this screen can be found in the MLSCN PubChem website (pubchem.ncbi.nlm.nih.gov, Bioassay AID 525).
HTS dose-response experiments
Compounds found active during primary screens were selected from the NIH’s Molecular Libraries Small Molecule Repository (MLSMR, San Francisco, CA) and 10-point, 1:3 serial dilutions starting from a nominal 10 mM solution were prepared using an automated liquid handler (Beckman-Coulter, Fullerton, CA). Titration experiments were performed as mentioned above, by transferring 50 nL of the compound solutions in the titration plate into 3 different assay plates. For each compound, triplicate percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL® Information Systems). IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept. For compounds where no curve was fitted by the algorithm, an IC50 was determined manually. Detailed information regarding the SF-1 titration assay and the RORA counterscreen can be found at the MLSCN PubChem website (Bioassays AID 600 and 599, respectively).
VP16 promiscuity assay
This assay used the same protocol as the uHTS titration assays described in the previous section, apart that 125 ng of pGal4DBD
plasmid (a kind gift from Dr. Michael Conkright, Dept. of Cancer Biology, Scripps Florida) was used for the transfection step (Amelio et al., 2007
Prior to the assay, an 11 point, 1:3 serial dilution of compound starting at 0.4 mM (40X of final assay concentration) was prepared in PBS containing 5% DMSO using a liquid handling robot (PlateMate® Plus, Matrix Technologies, Hudson, NH). Human Embryonic Kidney (HEK) cells of the 293T subtype (ATCC, Manassas, VA) were grown in DMEM (Gibco, Carlsbad, CA) supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin-streptomycin-glutamine mix (Gibco, Carlsbad, CA). Cells were co-transfected in white 384-well plate (5,000 cells per well in 40uL) using a mix of 25 ng of pCMV-SF-1 or pCMV-LRH-1 (Open Biosystems, Huntsville, AL), 25 ng of p5×SFRE (a kind gift of Dr. Donald McDonnell, Duke University Medical Center, Durnham, NC) and a 3:1 lipid to DNA ratio of FuGENE® 6 transfection reagent (Roche, Indianapolis, IN). Plates were then incubated at 37°C, 5% CO2, and 95% RH. Twenty-four hours later, cells were treated with 1 µL of the intermediate 40× serial dilution (n=6), giving a final DMSO concentration of 0.125%. Cells were then returned to standard incubation conditions for twenty four hours. Plates were then allowed to equilibrate for 15 minutes at room temperature and a luciferase assay was performed by adding 25 µL/well of BriteLite™ (PerkinElmer, Waltham, MA). After a 2 minutes incubation time, plates were read using the EnVision™ Multilabel Plate Reader (PerkinElmer, Shelton, CT).
Cell viability assay
CHO-K1 cells were plated at 500 cells per well in 1536-well plates in 5 µL of media (F12 supplemented with 10% FBS and 1% Pen/Strep/Neo). Compounds (50nL of 100× DMSO solution per well) were prepared as 10-point, 1:3 serial dilutions starting at 10 mM, then added to the cells using the pin tool. Plates were then incubated 20 hours at 37°C, 5%CO2 and 95% RH. After incubation, five microliters of CellTiter-Glo® (Promega, Madison, WI) were added to each well, and plates were allowed to incubate for 15 minutes at room temperature. Luminescence was recorded for 30 seconds using the ViewLux™ reader (PerkinElmer, Turku, Finland). Viability was expressed as a percentage relative to wells containing media only (0%) and wells containing cells treated with DMSO only (100%).
Hepatic microsomal stability
Microsome stability was evaluated by incubating 1 µM compound with 2 mg/ml hepatic microsomes from either human, monkey, rat, dog, or mouse in 100 mM potassium phosphate buffer, pH 7.4. The reactions were held at 37° C with continuous shaking. The reaction was initiated by adding NADPH (1mM final concentration). The final incubation volume was 300 µl and 40 µl aliquots were removed at 0, 1, 3, 5, 8, and 10 minutes for rapidly metabolized compounds, or at 0, 5, 10, 20, 40, and 60 minutes for more stable compounds. The removed aliquot was added to 160 µl acetonitrile to stop the reaction and precipitate the protein. NADPH dependence of the reaction is evaluated with parallel incubations without NADPH. At the end of the assay, the samples were centrifuged through a 0.45 micron filter plate (Millipore, Billerica, MA) and analyzed by LC-MS/MS. The data was log transformed and results are reported in units of half-life.
In a glass test tube, 1–2 mg of probe compound was added to 1 mL of either pH 7.4 or pH 3.5 potassium phosphate buffer. The samples were allowed to invert for 24 hours at room temperature. The samples were centrifuged and the supernatant was analyzed by HPLC against a known reference.
Parallel Artificial Membrane Permeability Assay (PAMPA)
An assessment of permeability was done using a commercial PAMPA kit (BD Biosciences, Franklin Lakes, NJ). Compounds were evaluated over a range of concentrations by addition of 300 µl of PBS containing the compound to the bottom donor plate. Compounds were from DMSO stocks and the final DMSO concentration in the donor wells was one percent. 200 µl of blank PBS was added to the top receiver plate. The plates were allowed to incubate at room temperature. After 5 hours, aliquots were taken from the donor and receiver plates and the concentration of drug was determined. Compound permeability was calculated using the equation
is expressed in units of cm/s, CA
) is drug concentration in the acceptor at time t, VD
is donor well volume, VA
is acceptor well volume, A is the area of the filter (0.3 cm2
), t is time in seconds, and
All data representations have been done with GraphPad Prism version 4.03 (GraphPad Software, San Diego, CA). Curve fitting and IC50 determination were performed using the variable slope sigmoidal dose-response analysis tool of GraphPad Prism.
analyses were performed using tools and screening data available in the Pubchem website (http://pubchem.ncbi.nlm.nih.gov/
). For determining whether compounds were luciferase assay artefact, data existing in PubChem was used (Bioassay AID 411).