Secondary Assay for Inhibitors of Ubiquitin-specific Protease USP2a: PLA2 Counterscreen
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 Compound: 1
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN 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 [3-6]. To identify inhibitors of USP2a a cell-free assay was employed.
This is assay is a counterscreen used for the for the USP2a-PLA2 coupled assay. In this assay the murine PLA2 reporter enzyme is screened alone using NBD C6-HPC (Invitrogen) as a fluorogenic substrate.
NCGC Assay Protocol Summary:
The assay buffer, prepared fresh at the day of the assay, contains 20mM Tris-HCl pH 8.0, 2mM CaCl2, 2mM beta-mercaptoethanol, 0.1% DMSO. 3ul of 10nM (5nM final) free PLA2, which is stored on ice, is dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Aurora BioRaptr. The assay plate is then pinned with 23nL compound with the Kalypsys pintool. The controls were pinned as follows: row 31 2M NEM and row 32 DMSO. Subsequently, 3uL 40uM (20uM final) NBD C6-HPC (stored on ice and protected from light) were dispensed into the plate using the BioRaptr dispenser. Plates were read immediately on an Envision (Perkin Elmer) plate reader using wavelengths of Ex 460nm /Em 535nm. Then, the assay plates were incubated for 2.5 hours and read again on the Envision using the same settings.
Data were normalized to the to AC100 inhibition (NEM). 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.
Keywords: ubiquitination, deubiquitination, proteases, profluorescent, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
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".
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)