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BioAssay: AID 2014

uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (PABP)

Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is more ..
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 Tested Compounds
 Tested Compounds
All(290921)
 
 
Active(2982)
 
 
Inactive(285717)
 
 
Inconclusive(2226)
 
 
 Tested Substances
 Tested Substances
All(291075)
 
 
Active(2985)
 
 
Inactive(285864)
 
 
Inconclusive(2226)
 
 
AID: 2014
Data Source: Burnham Center for Chemical Genomics (BCCG-A235-PABP-FP-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-23
Modify Date: 2011-01-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2982
Related Experiments
AIDNameTypeComment
2012uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (eIF4H)Confirmatorydepositor-specified cross reference
2030Summary assay for the identification of translation initiation inhibitors (PABP)Summarydepositor-specified cross reference
435011SAR analysis for the identification of translation initiation inhibitors (eIF4H)Confirmatorydepositor-specified cross reference
435018SAR analysis for the identification of translation initiation inhibitors (PABP)Confirmatorydepositor-specified cross reference
449744SAR analysis for the identification of translation initiation inhibitors (eIF4H) via a crosslinking assayOtherdepositor-specified cross reference
449751SAR analysis for the identification of translation initiation inhibitors (PABP) via an Electrophoretic Mobility Shift Dose Response AssayConfirmatorydepositor-specified cross reference
449752SAR analysis for the identification of translation initiation inhibitors (PABP) via an Electrophoretic Mobility Shift AssayScreeningdepositor-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: 1R03MH084835-01
Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada

Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppressor genes directly influence the translation process and recently, chemoresistance in vivo has been linked to deregulated translation initiation. In a transformed setting, where translation can be inhibited by a small molecule modulator (e.g. rapamycin), decreased translation rates are associated with reversal of chemoresistance, possibly by inhibition of pro-survival pathways or resetting of pro-apoptotic program. These results validate translation initiation as a potential chemotherapeutic target.

A fluorescence polarization HTS assay has been developed to identify small molecules that block translation initiation by targeting an important components of this pathway, poly(A) binding protein (PABP). This protein requires RNA binding to mediate its effects on translation initiation and the screen is designed to find inhibitors that block this process
Protocol
HTS Assay
Assay materials:
1) GST-PABP RRM1/2 (Obtained from Assay Provider)
2) 5'-fluorescein-labeled poly(A)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20
Procedure:
1. Dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
2. Add 2 ul of PABP protein, 15 nM final concentration into columns 3-48
3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 5 nM final concentration into columns 1-48
4. Using a HighRes biosolutions pintool dispense 70 nl of 2 mM compounds in DMSO to columns 5-48
5. Using a HighRes biosolutions pintool dispense 70 nl of DMSO to columns 1-4.
6. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode
i. Positioning delay = 0.0
ii. Flashes/well = 10
7. Data analysis was performed using CBIS software (ChemInnovations, Inc).
8. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.
Dose Response Protocol
Assay materials:
1) GST-PABP RRM1/2 (Obtained from Assay Provider)
2) 5#-fluorescein-labeled poly(A)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20
Procedure:
1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 (Control wells), while varying volumes of test compounds are transferred to columns 5-48 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. After compounds have been added, dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
3. Add 2 ul of PABP protein, 15 nM final concentration into columns 3-48.
4. Add 2 ul of 5#-fluorescein-labeled poly(A)10 5 nM final concentration into columns 1-48.
5. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
6. Data analysis was performed using CBIS software (ChemInnovations, Inc).
7. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.
Comment
Compounds that demonstrated % activity of >= 50 % and 0.4 =< F_ratio <= 1.5 at 35 uM concentration are defined as actives of the primary screen in this assay. Compounds with activity >50% that demonstrate F_ratios outside these boundaries are optically compounds interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".
Selected compounds from the primary screen were retested and those that confirmed in duplicate at 20uM concentration were considered "active". If the results of the duplicates were not consistent they are marked as "inconclusive."
Compounds were tested in a dose response assay and those with an IC50 < 100 uM are considered to be "active."
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 for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with % displacement in the assay demonstrated by a compound at 35 uM concentration:
a. If primary % displacement is less than 0%, then the assigned score is 0
b. If primary % displacement is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary % displacement is between 0% and 100%, then the calculated score is (% Displacement)*0.4/(1+(F_ratio-1)^2)
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's 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- or pathway-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 eIF4H 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 all 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 resynthesized true positives and their analogues and is not applicable in to this assay
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
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
5Ave %Inhibition at 20uM (20μM**)Average of duplicate samplesFloat%
6Std.Err(Repeats) (20μM**)Standard error of the duplicate samplesFloat%
7%inhibition at 35uM (35μM**)% inhibition in primary screeningFloat%
8F_RatioFluorescence intensity normalized to the average fluorescence intensity value of the plate negative contirolsFloat
9Mean HighMean Fluorescence Polarization of negative controls in the corresponding plateFloatmP
10STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatmP
11Mean LowMean Fluorescence Polarization of positive controls in the corresponding plateFloatmP
12STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatmP

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084835-01

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