The ubiquitin-proteasome pathway is present within all eukaryotic cells and plays roles in normal cellular functions and disease-related dysfunction. Proteins are tagged with a poly-ubiquitin chain that targets them for the proteasome, a multimeric protease, that degrades the protein into peptides and free ubiquitin. The proteasome has a key role in regulating cell cycle and growth and is known more ..
BioActive Compounds: 3
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
NCGC Assay Overview:
The ubiquitin-proteasome pathway is present within all eukaryotic cells and plays roles in normal cellular functions and disease-related dysfunction. Proteins are tagged with a poly-ubiquitin chain that targets them for the proteasome, a multimeric protease, that degrades the protein into peptides and free ubiquitin. The proteasome has a key role in regulating cell cycle and growth and is known to control the levels of cyclins and CDK inhibitors as well as tumor suppressor proteins. Additionally, the activation of NF- B is controlled by ubiquitin-labeling and degradation of the inhibitory protein I B . Inhibition of NF- B in cancer cell lines has been shown to make these cell lines more prone to apoptosis. With this in mind, proteasome inhibitors such as MG-132 and PS-341 have been used to inhibit tumor cell growth.
A cellular assay for ubiquitin-proteasome function was used to find chemical probes of this pathway. The assay used an ubiquitin-GFP construct expressed in U2OS cells (BioImage). Under basal conditions little GFP can be detected as it is degraded by the proteasome. However, treatment of the cells with a proteasome inhibitor such as MG-132 results in a rise in GFP levels that can be detected in as little as four hours. We used the Acumen-Explorer laser plate cytometer having a 488 nm laser as an excitation light source to detect the GFP. Compounds were screened in 1536-well plates using qHTS with a concentration-titration series range that varied from 46 uM to 2.9 nM.
NCGC Assay Protocol Summary:
Cells (U20S ps 2042) expressing ubiquitin (G76V-GFP) were maintained in DMEM with Glutamax-1 and high glucose, 10% FBS, 1% (v/v) penicillin-streptomycin, 0.5 mg/mL Geneticin (G418). The cells were seeded in 1536-well plates using a solenoid-based dispenser at 700 cells/5ul in DMEM medium with Glutamax-1 and high glucose containing 0.5% FBS, w/o phenol red, 25 mM HEPES. The plates were incubated at 37 #C/5% CO2 overnight. Then 23 nl of MG-132 in DMSO was added to the control wells at a final concentration of 10 uM. Next, 23 nL of library compounds in DMSO were added to the sample wells. The plates were incubated for 4 hrs at 37 #C/5% CO2. Following this incubation the plates were read on the Acumen using a cell object definition of 15 to 100 um in width and depth and a window of Total Intensity of between 77,200 and 2,720,000 FLU to enumerate GFP-expressing cells. See Table.
The %Activity was determined from the basal and MG-132 treated control wells. %Activity was determined by normalizing to the difference in signal between basal cells (0% Activity) and cells incubated with 10 uM of the proteasome inhibitor MG-132 (100% Activity). Concentration-response curves were fitted to the signals arising from the GFP fluorescence.
Assay Protocol Summary Table
Sequence Parameter Value Description
1. Reagent 5 uL 700 U2OS ps 2040 cells/well
2. Time O/N 37C incubation
3. Reagent 23 nL MG132
4. Compound 23 nL Libraries (46 uM to 2.9 nM)
5. Time 4 hr 37C incubation
6. Detector Acumen Total Intensity, GFP fluorescence (488 ex/515 em)
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, BioImage, proteasome, ubiquitin-GFP, qHTS, NCGC
1. Compounds are first classified as being probable activators (compounds causing an increase in GFP flourescence, presumably through inhibition of proteasome activity), inactive, or inconclusive based on flourescence readout in the cells.
2. Within the activators, compounds were ranked by efficacy and potency.
Data Table (Concise)