CHOP2 Reporter Counterscreen Assay for Inhibitors of Ubiquitin-specific Protease USP2a
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: 130
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: XO1-MH079852-01
PI Name: Nicholson, Ben. Progenra Inc, Malvern, PA
NCGC Assay Overview:
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 assay was employed.
This assay is a counterscreen against the reporter enzyme used to measure USP2a activity in the primary qHTS assay. The reporter enzyme is a protease (enterokinase, EK; EC 188.8.131.52), which is activated when a Ub sequence is cleaved by USP2a. The counterscreen assay is performed in the same buffer and conditions as the primary USP2a coupled assay but uses fully activated EK alone. The assay gives a fluorescent read-out at excitation/emission wavelengths of 485/531. The assay was provided by Progenra, Inc. and has been licensed for sale by LifeSensors, Inc.
NCGC Assay Protocol:
The assay buffer, prepared fresh at the day of the assay, contains 20mM Tris-HCl pH 8.0, 2mM CaCl2, 2mM beta-Mercaptoethanol, 0.05% CHAPS. 3uL of 100 pM EK (50 pM final) was dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Kalypsys dispenser. The assay plate was then pinned with 23nL compounds in DMSO with the Kalypsys pintool in columns 5-48 (0.3% final DMSO concentration). The negative control was buffer/DMSO only in column 3. The assay plate is incubated at RT for 30min. Subsequently, 3uL of reporter substrate (25nM final) were dispensed into the plate using the Kalypsys dispenser. Plates were read after 10 min at RT on a ViewLux (Perkin Elmer) plate reader using the following wavelengths: Ex 485nm /Em 531nm.
Data were normalized to the AC100 inhibition (buffer alone). Concentration-response curves were fitted to the normalized data and the concentration-response curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. The time zero reading was used to flag fluorescence artifacts.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)