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BioAssay: AID 1006

Counter Screen for Luciferase-based Primary Inhibition Assays

This functional assay was developed for detection of compounds inhibiting luciferase. These compounds would be observed as false positives of assays employing luciferase-based detection. ..more
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 Tested Compounds
 Tested Compounds
All(195564)
 
 
Active(2976)
 
 
Inactive(192588)
 
 
 Tested Substances
 Tested Substances
All(195634)
 
 
Active(2978)
 
 
Inactive(192656)
 
 
 Related BioAssays
 Related BioAssays
AID: 1006
Data Source: Burnham Center for Chemical Genomics (Counter Screen for Luciferase-based Primary Assays)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-01-03
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2976
Related Experiments
AIDNameTypeComment
411qHTS Assay for Inhibitors of Firefly LuciferaseConfirmatorydepositor-specified cross reference
618Luminescent HTS for small molecule inhibitors of MT1-MMP transcriptionConfirmatorydepositor-specified cross reference
620Fluorescent HTS Cytotoxicity/Cell viability assay (HT1080 cells)Confirmatorydepositor-specified cross reference
773Counter Screen for Luciferase-based Assay PositivesConfirmatorydepositor-specified cross reference
1027Counter Screen for Luciferase-based Primary Stimulation AssaysScreeningdepositor-specified cross reference
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: None


This functional assay was developed for detection of compounds inhibiting luciferase. These compounds would be observed as false positives of assays employing luciferase-based detection.
Protocol
Luciferase HTS protocol:
1) 2.5 uL of PBS dispensed into to columns 1-2 of 1536-well white Corning plates (cat #3725) using the Thermo WellMate dispenser
2) 2.5 uL of 10.24 uM ATP solution in PBS buffer added to columns 3-48
3) 20 nL of DMSO added to columns 1-4
4) 20 nL of 2 mM compounds in 100% DMSO added to columns 5-48
2) 1.5 uL ATPLite solution (PerkinElmer) containing luciferase and luciferin were added to the whole plate
3) Luminescence is measured after 10 min at room temperature on a ViewLux plate reader (Perkin Elmer).
4) Data analysis was performed using CBIS software
Comment
Compounds with >50% inhibition at 10 uM concentration are defines as active.
Activity Scoring
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration different information available from screening, and in this case include compound potency, the screening stage of the data and compound behavior in the assay. Details of the Scoring System will be published elsewhere.
Briefly, the outline of the scoring system utilized for the Luciferase assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % displacement in the assay demonstrated by a compound at 10 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data and their analogues and is not applicable in this assay.
3) Third tier (81-100 range) is reserved for resynthesized confirmed positives and their analogues and is not applicable in this assay.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 10 uM (10μM**)% inhibition of luciferase in primary screeningFloat
2Mean HighMean luminescence signal of negative controls in the corresponding plateFloatcps
3STD Deviation HighStandard deviation (n=16) of negative controls in the corresponding plateFloatcps
4Mean LowMean luminescence signal of positive controls in the corresponding plateFloatcps
5STD Deviation LowStandard deviation (n=16) of positive controls in the corresponding plateFloatcps

** Test Concentration.

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