qHTS screen for small molecules that induce genotoxicity in human embryonic kidney (HEK293T) cells expressing luciferase-tagged ELG1
Cancer cells divide rapidly compared to normal cells in the body. During cell division, cancer cells need to duplicate their genome by DNA replication. The failure of genome duplication leads to cancer cells' death. Therefore, inhibitors of DNA replication could specifically kill cancer cells. Based on this concept, many chemotherapeutic agents were developed and have been used to treat cancer more ..
BioActive Compounds: 4174
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH092164-01
Assay Provider: Kyungjae Myung, NHGRI, NIH
Keywords: MLPCN, qHTS, NCGC, genotoxicity, cancer
Cancer cells divide rapidly compared to normal cells in the body. During cell division, cancer cells need to duplicate their genome by DNA replication. The failure of genome duplication leads to cancer cells' death. Therefore, inhibitors of DNA replication could specifically kill cancer cells. Based on this concept, many chemotherapeutic agents were developed and have been used to treat cancer patients. However, there are a limited number of agents available in the clinical setting, and some of those that do exist have low efficacy and severe side effects. Therefore, if we can find the compounds that effectively block DNA replication either by directly damaging DNA or by inhibiting other cellular mechanisms, the options for cancer treatment will be broadened. With this in mind, we developed a quantitative high-throughput ELG1-luciferase reporter gene assay. Since the level of human ELG1 protein is enhanced in response to genetic stresses, increases in luciferase activity can be used to identify compounds that cause genetic stress.
Assay Protocol Summary:
For genotoxicity screening, 2,000 cells in 5 uL/well were dispensed into white, solid-bottom 1536-well plates using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, the plates were incubated 16 hr at 37oC and 5% CO2. 5 ul ONE-Glo (Promega) was then added to each plate and following a 30 min incubation at room temperature, luciferase activity was quantified on a ViewLux CCD-based plate reader.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)