Counter Screen for Luciferase-based Primary Stimulation Assays
This functional assay was developed for detection of compounds activating luciferase. These compounds would be observed as false positives of assays with increase-of-signal detection employing luciferase-based reactions. Potentially, the same compounds would act as false negatives in the decrease-of-signal luciferase-based assays. ..more
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 activating luciferase. These compounds would be observed as false positives of assays with increase-of-signal detection employing luciferase-based reactions. Potentially, the same compounds would act as false negatives in the decrease-of-signal luciferase-based assays.
Luciferase uHTS materials:
1) Enzyme working solution: ATPlite (PerkinElmer) containing luciferase and luciferin
2) Substrate working solution: 10.24 uM ATP in PBS buffer
3) Negative control wells: substrate working solution replaced with PBS
4) Positive control wells: contained enzyme and substrate in the absence of compounds
Luciferase uHTS 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 the substrate working solution 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
5) 1.5 uL enzyme working solution (PerkinElmer)
6) Luminescence is measured after 10 min at room temperature on a ViewLux plate reader (Perkin Elmer). The screening was performed on fully automated uHTS system (HRE).
7) Data analysis was performed using CBIS software
Luciferase activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC),
where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate.
Compounds with greater than or equal to 2-fold activation (AF >= 2) of Luciferase at 10 uM concentration are defined as actives of the primary screening.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the Luciferase assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. 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 the score is correlated with Luciferase activation factor demonstrated by a compound at 10 uM concentration:
a. If AF<1, then the assigned score is 0
b. For all other AF values,
Score = 40 - 40/AF
This formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
** Test Concentration.
Data Table (Concise)