Counter Screen for Luciferase-based Assay Positives
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
BioActive Compounds: 237
Depositor Specified Assays
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: XO1 MH78949-01
Assay Provider: Dr. Alex Strongin, Sanford-Burnham Medical Research Institute
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.
This screening was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN).
1) 4 uL of compounds or control solutions added to 384-well plate:
a) positive control (EDTA, 12.5 mM final concentration) to columns 1-2
b) negative control (10% DMSO) to columns 23-24
c) serially diluted compounds to columns 3-22
2) 8 uL ATP substrate solution (6.4 uM final concentration) and 8 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 an EnVision plate reader (Perkin Elmer).
4) Data analysis was performed using sigmoidal dose-response equation through non-linear regression
Compounds with EC50 values < 100 uM are defined as 'actives' in the outcome column. Compounds that show <50% inhibition at all the concentration values are defined as 'inactive' in the outcome field. Compounds with EC50 below 195 nM, the lowest tested concentration, were assigned IC50 value of < 0.2 uM and defined as 'active'.
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 and therefore is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds are assigned a score value equal 41.
b. For compounds with EC50<100 uM, the score is linearly correlated with a compound#s potency using the following equation
Score = 44 + 6*(pEC50 - 3)*2.6*(exp(-0.5*nH^2)-exp(-1.5*nH^2)),
where pEC50 is negative log(10) of the EC50 value expressed in mole/L concentration units and nH is Hill coefficient value.
c. Compounds with EC50 below the lowest tested concentration are assigned a score value equal 80.
3) Third tier (81-100 range) is reserved for resynthesized confirmed positives and their analogues and is not applicable in this assay.
* Activity Concentration.
Data Table (Concise)