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BioAssay: AID 2774

LOPAC Circadian Assay

To dissect the molecular mechanisms underlying the mammalian circadian clock by using a chemical biology approach, we analyzed the effect of 1,280 pharmacologically active compounds (LOPAC, Sigma) on the circadian period length that is indicative of the core clock mechanism. We utilized the circadian luciferase reporter Bmal1-dLuc for monitoring circadian rhythms in cultured human cells and more ..
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 Tested Compounds
 Tested Compounds
All(1274)
 
 
Active(13)
 
 
Inactive(1261)
 
 
 Tested Substances
 Tested Substances
All(1279)
 
 
Active(13)
 
 
Inactive(1266)
 
 
 Related BioAssays
 Related BioAssays
AID: 2774
Data Source: Circadian Research, Kay Laboratory, University of California at San Diego (UCSD) (LOPAC Circadian Assay 1)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
BioAssay Version:
Deposit Date: 2010-04-23
Modify Date: 2011-10-20

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 13
Related Experiments
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493019Longdaysin Circadian AssayConfirmatorydepositor-specified cross reference
Description:
To dissect the molecular mechanisms underlying the mammalian circadian clock by using a chemical biology approach, we analyzed the effect of 1,280 pharmacologically active compounds (LOPAC, Sigma) on the circadian period length that is indicative of the core clock mechanism. We utilized the circadian luciferase reporter Bmal1-dLuc for monitoring circadian rhythms in cultured human cells and developed a 384-well plate-based assay system to screen compound libraries. Among the 1,280 compounds tested, we identified 11 compounds causing reproducible period changes. Many of them are previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype. This work was done in collaboration with Genomics Institute of the Novartis Research Foundation and The Scripps Research Institute.
Protocol
Human osteosarcoma U2OS cells stably expressing Bmal1-dLuc reporter were suspended in the culture medium (DMEM supplemented with 10% fetal bovine serum, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin) and plated onto 384-well white solid-bottom plates (Greiner) at 20 ul (2,000 cells) per well. They were cultured for 2 days to reach confluence. Then, 50 ul of the explant medium [DMEM supplemented with 2% B27 (Invitrogen), 10 mM Hepes (pH 7.2), 0.38 mg/ml sodium bicarbonate, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin, 0.1 mg/ml gentamicin, and 1 mM luciferin (Promega)] was dispensed to each well, followed by the application of 500 nl of compounds (arrayed on 384-well plates at 1 mM in DMSO) by using 384-well head PinTool (GNF Systems). The plate was covered by an optically clear film (USA Scientific) and set to a GNF automated robotic system (GNF Systems) equipped with a CCD imager (ViewLux, Perkin Elmer) for luminescence recording. The settings are as follows: incubator temperature, 37C; exposure time, 120 sec; interval time, 120 min. Raw luminescence data were fit to a damped cosine curve using nonlinear least squares with CellulaRhythm algorithm, and period parameter was calculated. Each period data was normalized by subtracting the mean period of DMSO control (included in each plate) and indicated in columns Exp1_Period and Exp2_Period. Positive value means long period, and negative value means short period. The quality of the curve fitting was inspected manually, and the data with poor fitting were filtered out (indicated by blank in columns Exp1_Period and Exp2_Period). The screening was repeated twice (Exp1 and Exp2), and 13 compounds causing more than 0.5-h period change in both experiments were labeled as active. The 13 primary hits were tested in confirmation assay with 8-point dilution series, and the effects of 11 compounds were confirmed.
Comment
The data represent primary screening result and contain plate and well position (columns Plate and Well) and normalized period parameter from 2 independent experiments (columns Exp1_Period and Exp2_Period). Note that S(-)-Pindolol (SID 92303745) was duplicated in the library, and representative data (from Plate 4, Well N15) is shown. The other well (Plate 2, Well F19) also showed minor period effect (Exp1_Period, -0.24; Exp2_Period, -0.17). Among 13 primary hits, Ethamivan (SID 92303969) and 2-Methoxyestradiol (SID 92304296) did not show period effect in confirmation assay.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1PlatePlate number (total 4 plates)Integer
2WellWell positionString
3Exp1_Period (7μM**)Normalized period length of experiment 1Floathour
4Exp2_Period (7μM**)Normalized period length of experiment 2Floathour
5General FunctionalityString

** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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