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BioAssay: AID 1984

Fluorescence for the identification of compounds that decrease p/CIP protein stability

Genetic studies have demonstrated that loss of the transcriptional co-activator p/CIP leads to resistance to obesity and diabetes, especially in extreme mouse obesity models. The goal of this research is to find small molecules that decrease p/CIP protein stability, thus providing new understanding and novel approaches to cure obesity and diabetes. In an in vitro white fat differentiation system, more ..
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 Tested Compounds
 Tested Compounds
All(292329)
 
 
Active(36)
 
 
Inactive(292293)
 
 
 Tested Substances
 Tested Substances
All(292483)
 
 
Active(36)
 
 
Inactive(292447)
 
 
AID: 1984
Data Source: Burnham Center for Chemical Genomics (BCCG-A215-p/CIP-Inhibitors)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-14
Modify Date: 2010-12-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 36
Related Experiments
AIDNameTypeComment
1995Summary assay for compounds that decrease p/CIP protein stabilitySummarydepositor-specified cross reference
2005uHTS fluorescence for the identification of compounds that decrease EGFP protein stabilityConfirmatorydepositor-specified cross reference
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084852-01
Assay Provider: Zhiyong Wang PhD, The Salk Institute for biological studies, San Diego CA

Genetic studies have demonstrated that loss of the transcriptional co-activator p/CIP leads to resistance to obesity and diabetes, especially in extreme mouse obesity models. The goal of this research is to find small molecules that decrease p/CIP protein stability, thus providing new understanding and novel approaches to cure obesity and diabetes. In an in vitro white fat differentiation system, p/CIP is important for fat cell formation. A fluorescence-based primary assay employing this in vitro cellular model has been developed to screen for small molecules that destabilize a p/CIP-EGFP fusion protein. Individual compounds will be incubated with the stably transfected cell line containing this GFP (green fluorescence) fusion protein. Small molecules will be identified that only diminish the GFP signal from the p/CIP-EGFP fusion protein cells.

The assay described below is a cell-based HTS assay that utilizes an NIH3T3 derived subline F442A, a fibroblast line capable of differentiation into adipocytes in vitro, transfected with a p/CIP-EGFP fusion protein driven by a CMV promoter. Drugs interfering with p/CIP expression levels should directly affect the level of EGFP expression. The assay measures total fluorescence intensity of EGFP.
Protocol
Assay materials:

1) F442A-pCIP-EGFP cell line obtained from the assay provider's laboratory.

2) Assay media: F12 supplemented with 5% Bovine Calf Serum, 4.5g/L glucose, 2mM L-glutamine, 1mM Na-pyruvate, 0.5 mg/mL G418, & 50 IU/mL penn/strep.

Primary Screen and Single-concentration confirmation

Day 1 Procedure

1) Harvest F442A-pCIP-EGFP at 100% confluency

2) Dispense with Multidrop 5 uL (5000 cells)/well to columns 5-48 of a 1536 TC-treated black/clear-bottom plate (Corning # 3896).

3) Dispense with Multidrop 5 uL assay media to columns 1-4 of a 1536 TC-treated black/clear-bottom plate (Corning # 3896).

4) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.

5) Using a HighRes biosolution pintool equipped with V&P Scientific pins, stamp 25nl of 2mM cmpds in DMSO (col 5-48) and 25nl DMSO controls (col 1-4) to plates. 10uM final assay concentration

6) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O

7) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).

8) Lid Plates. Incubate overnight (16 hours) in 37 degrees C 10% CO2 incubator

Day 2 Procedure

1) Equilibrate plates to room temp for 10 min.

2) Wash plates with 5uL 1X PBS using Biotek EL406 plate washer. (Aspirate 3uL, dispense 5uL PBS, aspirate 6uL.)

3) Read FITC fluorescence intensity (bottom read) on Perkin Elmer Envision.

p/CIP Dose Response assay


Assay materials:

1) F442A-pCIP-EGFP cell line obtained from the assay provider's laboratory.

2) Assay media: F12 supplemented with 5% Bovine Calf Serum, 4.5g/L glucose, 2mM L-glutamine, 1mM Na-pyruvate, 0.5 mg/mL G418, & 50 IU/mL penn/strep

Day 1 Procedure

1) Harvest MEF p/CIP-EGFP at 100% confluency

2) Dispense with Multidrop 5 uL (5000 cells)/well to columns 5-48 of a 1536 TC-treated black/clear-bottom plate (Corning # 3896).

3) Dispense with Multidrop 5 uL assay media to columns 1-4 of a 1536 TC-treated black/clear-bottom plate (Corning # 3896).

4) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.

5) Serial compound dilutions: dispense 50nl 100% DMSO (columns 1-4, 47-48) or compounds (columns 5-46) using with Labcyte Echo 550 into plates from step 2

6) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O

7) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).

8) Lid Plates. Incubate overnight (16 hours) in 37 degrees C 10% CO2 incubator

Day 2 Procedure

1) Equilibrate plates to room temp for 10 min.

2) Wash plates with 5uL 1X PBS using Biotek EL406 plate washer. (Aspirate 3uL, dispense 5uL PBS, aspirate 6uL.)

3) Read FITC fluorescence intensity (bottom read) on Perkin Elmer Envision.
Comment
ZScore is calculated as (wellValue - Mean) / StdDev. Where the mean is the mean of all of the valid wells on the plate and the STDDev is the standard deviation of all of the wells on the plate

Compounds with a Z score of less than or equal to -3 OR >= 30% efficacy at 10 uM were defined as actives in the primary screen.

Compounds determined to be actives in the primary screen were retested in duplicate at 10 uM and 20 uM. Compounds with a Z score of less than or equal to -3 OR >= 30% efficacy at 20 uM are considered to be active.

Confirmed actives were subsequently run in dose response mode. Compounds with an IC50 < 40 uM were "actives" in the dose response screen.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization is described below.


1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 10 uM concentration:
a. If primary %Efficacy is less than 0%, then the assigned score is 0
b. If primary %Efficacy is greater than 100%, then the assigned score is 40
c. If primary %Efficacy is between 0% and 100%, then the calculated score is (%Efficacy)*0.4
d. If primary %Efficacy < 30% and the ZScore is < -3, then the assigned score is 30

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for re-synthesized true positives and their analogues and is not applicable in this assay
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%Efficacy at 10 uM (10μM**)% Efficacy in primary screeningFloat
6ZScore (10μM**)ZScore in the primary screenFloat
7Ave %Efficacy of Repeats at 10uM (10μM**)Average %Efficacy of the repeats at 10 uMFloat%
8Std.Err(Repeats) at 10uM (10μM**)Standart Error of the repeats at 10 uMFloat%
9ZScore at 10uM (10μM**)ZScore of the repeats at 10uMFloat
10Ave %Efficacy of Repeats at 20uM (20μM**)Average %Efficacy of the repeats at 20 uMFloat%
11Std.Err(Repeats) at 20uM (20μM**)Standart Error of the repeats at 20 uMFloat%
12ZScore at 20uM (20μM**)ZScore of the repeats at 20uMFloat
13Mean High (10μM**)Mean luminescence signal of positive controls in the corresponding plateFloatCPS
14STD Deviation High (10μM**)Standard deviation (n=64) of positive controls in the corresponding plateFloatCPS
15Mean Low (10μM**)Mean luminescence signal of negative controls in the corresponding plateFloatCPS
16STD Deviation Low (10μM**)Standard deviation (n=64) of negative controls in the corresponding plateFloatCPS

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084852-01

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