E3 Ligase_WT_Dose Response
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 substrate 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: 40
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 substrate 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: Poyurovsky 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.
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.
We have earlier screened 218,724 compounds of the MLSCN library (AID 1230) and identified 220 actives. Of the 200 compounds tested, 40 showed a good dose-response and were reported as hits against E3 ligase (AID 1394). These 40 compounds were retested for dose-response in triplicate, which is reported here.
1. Poyurovsky 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 384 well, white, tissue-culture treated plates from Greiner (Cat#781080). Breathe-easy membranes (Cat#Z380059) were from Sigma.
Cells (wild-type or mutant) were plated and allowed to settle at 37C overnight. The cells were then incubated with different concentrations of compound (final DMSO conc:0.25%) at 37C for 2 hrs. SteadyLite luciferase reagent was added, plates incubated for 30 mins and then read on Envision reader.
1.Fill 384 well plate with 24 uL of cells (wild-type or mutant) in DMEM (7500 cells per well) using Wellmate (all columns except 1 and 23)
2.Add 24 uL DMEM to columns 1 and 23 using Wellmate
3. Seal plates with Breathe-easy membranes and incubate at 37C overnight
4.Dilute 1 ul of compound from dose-response plate with 79 ul DMEM to get a diluted dose-response plate (top concentration 125 uM)
5. Add 6 ul of diluted compound to the overnight-grown cells, seal with Breathe-easy membrane and incubate at 37C for 2 hrs
6.Add 30 ul SteadyLite HTS reagent and incubate at room temperature for 30 min
7.Read luminescence on Envision reader
The data was analyzed in IDBS ActivityBase. Each dose-response plate contained compounds in columns 3-22, controls (wild-type or mutant cells, no compound) in columns 2 and 24, and blanks (no cells) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 25 uM to 0.75 nM. Percent enhancement of signal was calculated for each well from the signal in luminescence units (LU) 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)))
Dose response curves of percent enhancement were fit using XLfit equation 205 (four parameter logistic model).
IC50 scores were calculated based on the Maximum percent enhancement and the EC50 value as follows:
(1) For EC50 >0, Score = Maximum percent enhancement/2
(2) For EC50 =0 (bad curve fit), Score = 0
EC50 values were determined as described in protocol above. Maximum percent enhancement shown by each compound is reported and is used to determine potency of the compound along with the EC50 value.
Activity outcome is reported as follows:
(1) EC50 >0 and Maximum percent enhancement >30 = Active
(2) EC50 >0 and Maximum percent enhancement <30 = Inactive
Score >15 : Active compound
Score <15 : Inactive compound
Total number of compounds tested: 40
Number of active compounds: 40
Number of Inactive compounds: 0
This assay was submitted to the PCMD by Brent Stockwell from Columbia University. Dose-response studies were carried out, and data was submitted by Nuzhat Motlekar of the University of Pennsylvania.
Please direct all correspondence to Andrew Napper (email@example.com)
* Activity Concentration. ** Test Concentration.
Data Table (Concise)