Single concentration confirmation of uHTS hits for Peg3 Promoter Inhibitors via a luciferase reporter assay
Cancer is a progressive disease culminating in the acquisition of metastatic potential by a subset of evolving tumor cells. Although extensively investigated, the precise molecular events underlying tumor development and cancer progression remain unclear. Progression elevated gene-3 (PEG-3), originally identified in rodents, displays elevated expression as cancers become more aggressive. The more ..
BioActive Compounds: 1063
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBIMR, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 2R01 GM068385-06
Assay Provider: Paul B. Fisher, M.Ph., Ph.D., Virginia Commonwealth University
Cancer is a progressive disease culminating in the acquisition of metastatic potential by a subset of evolving tumor cells. Although extensively investigated, the precise molecular events underlying tumor development and cancer progression remain unclear. Progression elevated gene-3 (PEG-3), originally identified in rodents, displays elevated expression as cancers become more aggressive. The minimal promoter region controlling PEG-3 expression, PEG-Prom, has been isolated and shown to display elevated expression in a wide range of both human and rodent tumors, with minimal expression in normal cells. The PEG-Prom is transcriptionally activated following transformation by diverse acting oncogenes or as a consequence of unidentified genetic factors mediating cellular transformation. We have determined the mechanism for this selectivity and it involves transcription (gene regulatory) factors, AP-1 and PEA-3, which are expressed at elevated levels in virtually all rodent and human cancers. Moreover, in cases of cancer reversion or blocking expression of specific transforming oncogenes, PEG-Prom activity is decreased. In these contexts, the PEG-Prom represents a valuable predictive tool and readout to identify molecules with potential antitumor activity, without a priori identification of the genetic changes causative of the transformed/tumorigenic phenotype. The primary goal of this screen is to identify chemical inhibitors of the PEG-Promoter generated from high-throughput screening methods. These small molecule chemical probes might have potential anticancer properties and could provide a foundation for the development of novel antitumor drugs.
The primary goal of this project is to identify chemical inhibitors of the PEG-Promoter generated from high-throughput screening methods using HeLa cells stably expressing the PEG-Promoter upstream of firefly luciferase. These small molecule chemical probes might have potential anticancer properties and could provide a foundation for the development of novel antitumor drugs.
The goal of this assay is to confirm hits from "uHTS identification of Peg3 Promoter Inhibitors in a luminescence assay", AID 588405.
1. Su ZZ, Goldstein NI, Jiang H, Wang MN, Duigou GJ, Young CS, Fisher PB. 1999. PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis. Proc Natl Acad Sci U S A 96(26):15115-15120.
2. Su ZZ, Emdad L, Sarkar D, Randolph A, Valerie K, Yacoub A, Dent P, Fisher PB. 2005. Potential molecular mechanism for rodent tumorigenesis: mutational generation of progression elevated gene-3 (PEG-3). Oncogene 24(13):2247-2255.
3. Su ZZ, Sarkar D, Emdad L, Duigou GJ, Young CS, Ware J, Randolph A, Valerie K. Fisher PB. 2005. Targeting gene expression selectively in cancer cells using the progression-elevated gene-3 promoter. Proc Natl Acad Sci U S A 102(4):1059-1064.
PEG-prom-Luc HeLa cells
DMEM 4.5g/l glu w/glu w/o phenol red (Cellgro cat#17-205-CV)
Fetal Bovine Serum (Hyclone Cat# SH30396.03)
L-glutamine (100X ) (Invitrogen Cat#25030081 )
Hygromycin B (CellGro Cat#30-240-CR)
Steady GLo (Promega Cat#E2550)
Assay plate: Aurora 1536 white Solid Bottom Plate
I. Cell Suspension
1- Dispense 4 uL/well of assay media to columns 1-2 using combi dispenser.
2- Dispense 4 uL/well of cells at 3.75X10;5 cells/mL to columns 3-48 using combi Dispenser
3- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.
II. Compound Addition:
4- Using LabCyte Echo, transfer 8 nL from 10 mM DPI reordered compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 0.2% DMSO), and 8 nL of DMSO to control wells in Columns 1-4.
5- Spin down plates on Vspin at 1000 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.
III. Reagent Addition
7- Retrieve plates from incubator, remove lids and allow to plates to cool at room temp for 10 minutes
8- Add 3uL/well of Steady-Glo reagent to all wells using Biotek dispenser.
9- Spin down plates without lids on Vspin at 2000 rpm for 2 min
10- Put lid on, and then incubate plates at room temp for 10 minutes.
IV. Reading plates:
11- Read the plate on PerkinElmer-EnVision plate reader luminescence protocol
Compounds that demonstrated average % activity of >=40 for four replicates at 20 uM concentration are defined as actives in this assay.
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.
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 assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)