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BioAssay: AID 1442

E3 Ligase_Mutant_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 ..
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 Tested Compounds
 Tested Compounds
All(40)
 
 
Active(32)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(40)
 
 
Active(32)
 
 
Inactive(8)
 
 
 Related BioAssays
 Related BioAssays
AID: 1442
Data Source: PCMD (E3 Ligase_Mutant)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-12-02

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 32
Depositor Specified Assays
AIDNameTypeComment
1230E3 Ligase HTS_1536screeningE3 Ligase HTS_1536
1394E3 Ligase dose-response_384confirmatoryE3 Ligase_Dose response_384
1444E3 Ligase_WT_Dose Responseconfirmatory
Description:
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). To test selectivity of these compounds, these 40 hits were tested against a wild-type and a mutant strain. Hits obtained from this assay would be selctive MDM2 E3 ligase inhibitors. We report here the dose-response study on 40 compounds against a mutant strain of cells.

Reference:
1. Poyurovsky et. al., Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization. Mol Cell. 12:875-87 (2003)
Protocol
Materials

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.


Assay

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.

Dose-response protocol

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

Data analysis

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).




Comment
Activity scoring

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

Activity Outcome

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 >22 = Active
(2) EC50 >0 and Maximum percent enhancement <22 = Inactive

Hence,
Score >11 : Active compound
Score <11 : Inactive compound

Total number of compounds tested: 40
Number of active compounds: 32
Number of Inactive compounds: 8

Contributors

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.

Correspondence

Please direct all correspondence to Andrew Napper (napper@seas.upenn.edu)
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Mean Mutant EC50*FloatμM
2Mean Mutant maximum percent enhancementFloat%
3Mutant EC50#1 FloatμM
4Mutant EC50#1_Maximum percent enhancementFloat%
5Mutant EC50#1_Hill slopeFloat
6Mutant EC50#1_R-squaredFloat
7Mutant EC50#1_Luminescence signal at 100 microM (100μM**)Float
8Mutant EC50#1_Luminescence signal at 50 microM (50μM**)Float
9Mutant EC50#1_Luminescence signal at 25 microM (25μM**)Float
10Mutant EC50#1_Luminescence signal at 12.5 microM (12.5μM**)Float
11Mutant EC50#1_Luminescence signal at 6.25 microM (6.25μM**)Float
12Mutant EC50#1_Luminescence signal at 3.125 microM (3.125μM**)Float
13Mutant EC50#1_Luminescence signal at 1.56 microM (1.56μM**)Float
14Mutant EC50#1_Luminescence signal at 0.78 microM (0.78μM**)Float
15Mutant EC50#1_Luminescence signal at 0.39 microM (0.39μM**)Float
16Mutant EC50#1_Luminescence signal at 0.195 microM (0.195μM**)Float
17Mutant EC50#1_Luminescence signal at 0.097 microM (0.097μM**)Float
18Mutant EC50#1_Luminescence signal at 0.048 microM (0.048μM**)Float
19Mutant EC50#1_Luminescence signal at 0.024 microM (0.024μM**)Float
20Mutant EC50#1_Luminescence signal at 0.012 microM (0.012μM**)Float
21Mutant EC50#1_Luminescence signal at 0.006 microM (0.006μM**)Float
22Mutant EC50#1_Luminescence signal at 0.003 microM (0.003μM**)Float
23Mutant EC50#1_Control meanFloat
24Mutant EC50#1_Control standard deviationFloat
25Mutant EC50#1_Control percent CVFloat%
26Mutant EC50#2FloatμM
27Mutant EC50#2_Maximum percent enhancementFloat%
28Mutant EC50#2_Hill slopeFloat
29Mutant EC50#2_R squaredFloat
30Mutant EC50#2_Luminescence signal at 100 uM (100μM**)Float
31Mutant EC50#2_Luminescence signal at 50 uM (50μM**)Float
32Mutant EC50#2_Luminescence signal at 25 uM (25μM**)Float
33Mutant EC50#2_Luminescence signal at 12.5 uM (12.5μM**)Float
34Mutant EC50#2_Luminescence signal at 6.25 uM (6.25μM**)Float
35Mutant EC50#2_Luminescence signal at 3.125 uM (3.125μM**)Float
36Mutant EC50#2_Luminescence signal at 1.56 uM (1.56μM**)Float
37Mutant EC50#2_Luminescence signal at 0.78 uM (0.78μM**)Float
38Mutant EC50#2_Luminescence signal at 0.39 uM (0.39μM**)Float
39Mutant EC50#2_Luminescence signal at 0.19 uM (0.19μM**)Float
40Mutant EC50#2_Luminescence signal at 0.097 uM (0.097μM**)Float
41Mutant EC50#2_Luminescence signal at 0.048 uM (0.048μM**)Float
42Mutant EC50#2_Luminescence signal at 0.024 uM (0.024μM**)Float
43Mutant EC50#2_Luminescence signal at 0.012 uM (0.012μM**)Float
44Mutant EC50#2_Luminescence signal at 0.006 uM (0.006μM**)Float
45Mutant EC50#2_Luminescence signal at 0.003 uM (0.003μM**)Float
46Mutant EC50#2_Control MeanFloat
47Mutant EC50#2_Control standard deviationFloat
48Mutant EC50#2_Control percent CVFloat%
49Mutant EC50#3FloatμM
50Mutant EC50#3_Maximum percent enhancementFloat%
51Mutant EC50#3_Hill slopeFloat
52Mutant EC50#3_R squaredFloat
53Mutant EC50#3_Luminescence signal at 100 uM (100μM**)Float
54Mutant EC50#3_Luminescence signal at 50 uM (50μM**)Float
55Mutant EC50#3_Luminescence signal at 25 uM (25μM**)Float
56Mutant EC50#3_Luminescence signal at 12.5 uM (12.5μM**)Float
57Mutant EC50#3_Luminescence signal at 6.25 uM (6.25μM**)Float
58Mutant EC50#3_Luminescence signal at 3.125 uM (3.125μM**)Float
59Mutant EC50#3_Luminescence signal at 1.56 uM (1.56μM**)Float
60Mutant EC50#3_Luminescence signal at 0.78 uM (0.78μM**)Float
61Mutant EC50#3_Luminescence signal at 0.39 uM (0.39μM**)Float
62Mutant EC50#3_Luminescence signal at 0.19 uM (0.19μM**)Float
63Mutant EC50#3_Luminescence signal at 0.097 uM (0.097μM**)Float
64Mutant EC50#3_Luminescence signal at 0.048 uM (0.048μM**)Float
65Mutant EC50#3_Luminescence signal at 0.024 uM (0.024μM**)Float
66Mutant EC50#3_Luminescence signal at 0.012 uM (0.012μM**)Float
67Mutant EC50#3_Luminescence signal at 0.006 uM (0.006μM**)Float
68Mutant EC50#3_Luminescence signal at 0.003 uM (0.003μM**)Float
69Mutant EC50#3_Control MeanFloat
70Mutant EC50#3_Control standard deviationFloat
71Mutant EC50#3_Control percent CVFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03MH082369-01

Data Table (Concise)
Classification
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