qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Caspase 6 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 proteaosome. However, ubiquitination is highly reversible more ..
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 proteaosome. However, ubiquitination is highly reversible and dynamic. Deubiquitination, the reverse process, is catalyzed through the action of enzymes referred to as isopeptidases for 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 deubiquinates fatty acid synthase (FASN) which has recently been identified as an emerging oncology target.
To ascertain whether the USP2 inhibitors also inhibit other cysteine proteases, a caspase 6 (cysteine-aspartic acid protease) assay coupled to luciferase was developed using the Caspase 6 Glo kit (Promega, Madison, WI). This assay monitors caspase 6 activity with a luminogenic caspase 6 substrate. Upon cleavage of the substrate by caspase 6, the product can be acted upon by luciferase to lead to luminescence that is proportional to the caspase 6 activity. A compound that inhibits caspase 6 would result in a decrease in luminescence. The assay is run at 5 uM substrate concentration, which is the Km reported by Promega of their substrate. Therefore, this assay is sensitive to identify non-, un- and competitive inhibitors of caspase 6. This assay is run in kinetic mode. Inactivity is the desired outcome for this assay.
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
Two and a half uL of 0.5 U/mL caspase 6 in buffer was dispensed in a 1536 well plate. The assay plate was then pinned with 23 nL compound using the Kalypsis pintool. After 30 minutes at room temperature, 25 uL of Caspase 6 Glo detection reagent was added and luminescence signal was read using the PerkinElmer ViewLux plate reader with a 1 second exposure.
The data were normalized to control columns representing maximum signal (all components) and background (no enzyme). The buffer consisted of 10 mM Hepes pH 7.2, 2 mM DTT, 10% glycerol and 0.05% CHAPS, and the assay was run in white solid-bottom Greiner plates.
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)