qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: K484 Substrate
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: 228
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) [1, 2]. 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 in orthogonal USP2 assays utilizing a di-ubiquitin substrate labeled with a quencher on one ubiquitin and a fluorescent compound on the other, i.e. the substrate has internally quenched fluorescence or IQF (Lifesensors). In the presence of active USP2, the diubiquitin (DiUb) is cleaved and the fluorescence at 598 nm increases. This assay is run in kinetic mode. Upon inhibition of USP2, the fluorescence signal from cleaved substrate increases less over time than in the absence of an inhibitor. This assay was also used during SAR. The buffer consisted of 20 mM Tris pH 8, 2 mM beta-mercaptoethanol and 0.05% Chaps, and the assay was run in black solid-bottom Greiner plates.
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
3 uL of 5 nM USP2 (or no enzyme) was dispensed in assay buffer was dispensed into a 1536-well plate and then compounds were added through pinning. 1 uL of 100 nM DiUb K48-4IQF subtsrate was added before fluoresence on the ViewLux in kinetic moder (Ex 525/Em 598).
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)