qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cytotox Assay
Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible more ..
BioActive Compounds: 48
Depositor Specified Assays
Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible and dynamic. Deubiquitination, the reverse process, is catalyzed through the action of enzymes referred to as isopeptidases or deubiquitinating enzymes (DUBs). This group of enzymes is collectively responsible for maintaining adequate pools of free ubiquitin and regulating the ubiquitination status of cellular proteins. The class of DUBs referred to as the ubiquitin-specific proteases (USP) family functions endoproteolytically to cleave Ub chains from a wide range of protein substrates. USP2a deubiquitinates fatty acid synthase (FASN) which has recently been identified as an emerging oncology target. To identify inhibitors of USP2a a cell-free qHTS assay was employed.
Confirmed qHTS active compounds were tested for cytoxicity in HCT116 cells (human colon cancer cells) using the Cell Titer Glo luciferase-coupled ATP detection system from Promega. This assays monitors cell proliferation by quantitating the amount of ATP present by luciferase, which in the presence of ATP converts pro-luciferin into the luminescent luciferin product. A compound that inhibits cell proliferation would result in a decrease in luminescence. The data were normalized to control columns representing maximum signal (cells with DMSO) and background (no cells with DMSO). The cells were cultured in DMEM with 10% FBS and plated in white solid-bottom tissue-culture treated plates.
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
5 uL of HCT116 (600 cells/well) were plated into a 1536 well plate and then incubated for 6 hr at 37 deg C, 5% CO2. Compounds are added through pinning before the plate is incubated for 72 hr at 37 deg C, 5% CO2. Then, 3 uL of Cell Titer Glo is added, the plate is left at RT for 10 min, before luminescence is detected using the ViewLux.
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 inhibitors are ranked higher than compounds that showed apparent activation.
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)