qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cell Cycle
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.
Selected active compounds with corresponding inactive controls were tested for their effect on the cell cycle in HCT116 cells. The USP2 substrate cyclin D1 is important for progression for G1 to S in the cell cycle. An inhibitor of USP2 would lead to a decrease in the amount of cyclin D1 and would arrest the cells in G1. Because the cells are predominantly in G1 in the absence of compound treatment, nocodazole (a known G2 blocker) was used to trap cells that made it through G1. Cell cycle was visualized using PI staining. Cells in G2 have twice the fluorescence as cells in G1, and the Acumen allows the cells to be binned by their individual fluorescence properties. Therefore, in the presence of an active USP2 inhibitor the fluorescence of a cell would be lower since it would not progress from G1 to S and then G2. The cells were cultured in DMEM with 10% FBS and 1% Pen-Strep and plated in black clear bottom collagen-coated 96-well plates
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
HCT116 cells at 600 cells/well were plated in 96-well plates and allowed to adhere overnight (37C, 50% CO2). The following day media containing a fixed concentration of nocodazole (final concentration of 0.6nM) and serial dilutions of the test compounds (highest concentration at 39 uM) was added to the cells. The cells were incubated for 16 hours. The cells were fixed with 8% paraformaldehyde for 1 hour at room temperature and washed 3x with 100 uL PBS/0.02% Triton solution. The cells were permeabilized with PBS/0.5% Triton-X for 10 min. Twenty five microliter of Propidium iodide / RNase reagent was added and the plate was incubated at 37C for 1 hour. Intensity of the DNA stain per cell was quantified using the Acumen plate reader. A cell cycle histogram report was generated to assess the effects of the compound on cell cycle.
If 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.
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Data Table (Concise)