qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cyclin D1 Western Blot
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
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.
To further visualize and quantitate the effect of compounds on cell proliferation, a colony forming or clonogenic assay was performed in 6-well plates using HCT116 cells. The cells were allowed to adhere to the plates for 2 days and then compound was added. The cells were allowed to grow for 10 additional days at which point the cells were fixed and labeled with crystal violet. The colonies were visualized on the Typhoon imager (GE) using Cy5 detection, and quantitated with the associated software. An inhibitor of USP2 would be expected to lead to a decrease in the size and perhaps the number of colonies. The cells were cultured in DMEM with 10% FBS and 1% Pen-Strep and plated in 6-well clear 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
. HCT116 cells were grown in 6-well tissue-culture plates to ~75% confluence and treated with compound. To obtain total lysate, the cells were resuspended in lysis buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1X protease inhibitor cocktail (Cell Signaling)] and lysed on ice for 30 min. Proteins were separated by SDS-PAGE using a Novex 4-12% Tris-glycine gel (Life Technologies) and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Life Technologies). The membrane was blocked with 5% nonfat dry milk in PBS/0.1% Tween 20 for 1 h at room temperature, incubated with mouse anti-cyclin D1 (Cell Signaling, 1:1,000 dilution in PBS/0.1% Tween 20) overnight at 4 degrees C, and then incubated with HRP-conjugated anti-mouse IgG (Cell Signaling, 1:5,000 dilution in in PBS/0.1% Tween 20) for 1 h at room temperature. After each incubation, the membrane was washed with PBS/0.1% Tween 20. Proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher) and the Chemidoc XRS Imaging System (Bio-Rad). The membrane was then stripped using Restore Western Blot Stripping Buffer (Thermo Fisher) and re-probed with rabbit anti-GAPDH (Abcam,1:5,000 dilution in PBS/0.1% Tween 20) followed by HRP-conjugated anti-rabbit IgG (MBL International Corporation,1:5,000 dilution in PBS/0.1% Tween 20). Protein levels were quantified using ImageJ.
f a compound has an IC50 <=5 uM, the compound is considered "active" and a score of 90 is assigned; if a compound has an IC50 > 5 uM and < 10 uM then the compound is considered "inconclusive" and a score of 50 is assigned; if a compound has an IC50 >= 10 uM it is considered "inactive" and a score of 10 is assigned.
Data Table (Concise)