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
BioActive Compounds: 2976
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.
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
Compounds with >50% inhibition at 10 uM concentration are defines 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. 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.
** Test Concentration.
Data Table (Concise)