E3 Ligase HTS_1536
The E3 ligases are involved in regulating other proteins by covalent ligation to the 76 amino acid protein ubiquitin. This post-translational modification can result in altered conformation, altered activity, or degradation of the sustrate protein. Thus, E3 ligases are effectors of a major means of post-translational modification of proteins in many species, including mammals. The dipeptide more ..
BioActive Compounds: 220
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Brent Stockwell, Columbia University
MLSCN Grant: R03MH082369-01
The E3 ligases are involved in regulating other proteins by covalent ligation to the 76 amino acid protein ubiquitin. This post-translational modification can result in altered conformation, altered activity, or degradation of the sustrate protein. Thus, E3 ligases are effectors of a major means of post-translational modification of proteins in many species, including mammals. The dipeptide boronic acid bortezomib is a potent proteasome inhibitor, has selective anticancer activity in tumor cells and in mice and was recently approved for clinical use in multiple myeloma. MDM2 E3 ligase is involved in numerous types of human cancer. Selective E3 ligase inhibitors would be preferable as they would be more selective and less toxic.
Inhibitors of the MDM2-UBCH5 interaction should disrupt the E3 ligase activity of MDM2 and therefore its oncogenic activity. Such inhibitors could be developed into novel therapeutic agents for sarcomas involving MDM2 overexpression or amplification, irrespective of their p53 status. Patients with sarcomas are likely to
benefit from this approach because of their high frequency of MDM2 protein overexpression and MDM2 gene amplification.
Cellular autoubiquitination assay: Prives et al. reported that an MDM2-GFP fusion protein autoubiquitinates and autodegrades in vivo. This approach was adapted to luciferase, because luciferase is easier to detect in high-throughput assays. A cellular assay for autoubiquitination in 384-well format was designed. An MDM2 E3-luciferase fusion protein was stably transfected into mammalian cells. The fusion protein autoubiquitinates, targeting itself for degradation. Luminescence is used to monitor protein abundance. A negative control of inactive RING domains fused to
luciferase, known to have no ubiquitination activity, has been developed as a counterscreen. The assay has been successfully miniaturized and validated in 1536-well format.
We have used a cell-based MDM2 autoubiquitination screen to identify small molecules that inhibit MDM2 E3 ligase activity. The cell based assay uses full-length MDM2 fused to luciferase. This construct autoubiquitinates itself and autodegrades. The screen will identify compounds that prevent this autodegradation and stabilize the MDM2-luciferase construct by blocking MDM2 E3 ligase activity. Thus inhibitors will show an enhanced luminescence signal.
HTS was performed using 218,724 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 3 ul 1536 well assay plates (final concentration of compound in assay plate:5 uM).
1. Prives et. al., Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization. Mol Cell. 12:875-87 (2003)
DMEM (Cat#11995-081), Fetal Bovine serum (Cat#26140-079), Streptomycin (Cat#15140-155), Trypsin-EDTA (Cat#25200-106) and Zeocin (Cat#R250-01) were all purchased from Invitrogen. SteadyLite HTS gene assay reporter system (Cat#6016989) was purchased from Perkin Elmer. The Luminescence assay was carried out in 1536-well white plates from Corning (Cat#3727). Breathe-easy membranes (Cat#Z380059) were from Sigma.
Cells were plated and allowed to settle at 37C for 4 hrs. The cells were then incubated with 5 uM compound (final DMSO conc:0.2%) at 37C for 2 hrs. SteadyLite luciferase reagent was added, plates incubated for 15 mins and then read on Envision reader.
1.Fill 1536 well plate with 3 uL of cells in DMEM (2000 cells per well) using Aquamax DW4
2.Add 3 uL DMEM to columns 1, 2, 45, and 46 using Aquamax DW4
3. Seal plates with Breathe-easy membranes and incubate at 37C for 4 hours
4.Add 6 nL of compound (0.25 mM in DMSO) using Evolution 1536 pintool
5. Seal plates with Breathe-easy membranes and incubate at 37C for 2 hrs
6.Add 3 uL SteadyLite HTS reagent and incubate at room temperature for 15 min
7.Read luminescence on Envision reader
The data was analyzed in IDBS ActivityBase. Each HTS plate had a single test compound (5 uM in 0.2% DMSO) in columns 5-44, controls (cells, no compound) in columns 3, 4, 47, and 48, and blanks (DMEM) in columns 1, 2, 45, and 46. Percent enhancement of signal was calculated for each compound from the signal in luminescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% enhancement = 100*(((signal-blank mean)-( control mean-blank mean ))/(control mean-blank mean)))
Activity scores were calculated as follows:
For positive percent enhancement, score = 0.4 x Percent inhibition
For negative percent enhancement, score = 0
Activity outcome is reported as follows:
(1) Percent enhancement >= 30 = active
(2) Percent enhancement < 30 = inactive
Analysis of screening results
A hit cut-off of 30% enhancement was selected. Based on this cutoff, a hit rate of 0.078% was observed.
This assay was submitted to the PCMD by Brent Stockwell from Columbia University. Assay development was done by Nuzhat Motlekar, HTS was carried out by Nuzhat Motlekar and Subramaniam Ramanathan, and data was submitted by Nuzhat Motlekar all of the University of Pennsylvania.
Our Thanks go to Olga Lozynska for carrying out the cell culture work related to this project.
Please direct all correspondence to Andrew Napper (email@example.com)
** Test Concentration.
Data Table (Concise)