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BioAssay: AID 652254

qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Clonogenic Assay

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 ..
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AID: 652254
Data Source: NCGC (USP2605)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-02
Hold-until Date: 2014-01-16
Modify Date: 2014-01-21

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
2281qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a: SummarySummarydepositor-specified cross reference: Summary AID
927qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2aConfirmatorysame project related to Summary assay
1455Confirmation Concentration-response Assay for Inhibitors of Ubiquitin-specific Protease USP2aConfirmatorysame project related to Summary assay
463106qHTS Validation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the ReporterConfirmatorysame project related to Summary assay
463254qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the ReporterConfirmatorysame project related to Summary assay
493168Confirmation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the ReporterConfirmatorysame project related to Summary assay
493169CHOP2 Reporter Counterscreen Assay for Inhibitors of Ubiquitin-specific Protease USP2aConfirmatorysame project related to Summary assay
493170Ub-rhodamine 110 based assay for inhibitors Ubiquitin-specific Protease USP2aConfirmatorysame project related to Summary assay
652168qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: K633 CounterscreenConfirmatorysame project related to Summary assay
652170qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cytotox AssayConfirmatorysame project related to Summary assay
652172Confirmation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: K633Confirmatorysame project related to Summary assay
652173qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: K484 SubstrateConfirmatorysame project related to Summary assay
652174Confirmation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: SARConfirmatorysame project related to Summary assay
652251qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cyclin D1 Western BlotOthersame project related to Summary assay
652264qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cell CycleOthersame project related to Summary assay
652277qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Caspase 6 AssayConfirmatorysame project related to Summary assay
Description:
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
Protocol
2 mL 300 cells/well were dispensed into a 6-well plate in DMEM with 10% FBS and 1% Pen-Strep and then incubated for 48 hours at 37 deg C, 5% CO2. Then 3 uL of compound in DMSO was added to each well, which was followed by a 10 day incubation at 37 deg C, 5 %CO2. All medium is then aspirated with a pipet, the cells are washed with PBS, and then 1 mL of crystal violet in 10% methanol is added and kept for 30 min at RT. The reagent is then aspirated with a pipet before the plate is repeatedly submeged in water for washing puproses. The plate is dried overnight and then read on a GE Typhoon FLA 9500 in end-point mode (Ex 635, Em Long-Pass Red Filter)
Comment
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50 (uM)FloatμM
2Compound QCString
Additional Information
Grant Number: MH079852

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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