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BioAssay: AID 540281

Single concentration confirmation of uHTS hits for Scp-1 phosphatase using a colorimetric assay

Human Scp phosphatases are regulators involved in neuronal gene silencing. This family of phosphatases exhibits specificity for phosphoryl-Ser5 in the heptad repeats of the C-terminal domain of RNA polymerase II (RNA Pol II) and thus inhibit initiation of transcription [1, 2]. Scps strongly associate with the REST/NRSF neuronal silencing complex that functions to inhibit transcription of neuronal more ..
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 Tested Compounds
 Tested Compounds
All(2438)
 
 
Active(1487)
 
 
Inactive(951)
 
 
 Tested Substances
 Tested Substances
All(2452)
 
 
Active(1487)
 
 
Inactive(965)
 
 
AID: 540281
Data Source: Burnham Center for Chemical Genomics (SBCCG-A670-SCP1-Inhibitor-CP-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1487
Related Experiments
AIDNameTypeComment
493091uHTS Colorimetric assay for identification of inhibitors of Scp-1Screeningdepositor-specified cross reference
493120Summary assay for small molecule Inhibitors of SCP-1Summarydepositor-specified cross reference: Summary AID.
540297Dose Response confirmation of uHTS hits for Scp-1 phosphatase using a colorimetric assayConfirmatorysame project related to Summary assay
540313Single concentration colorimetric interference counterscreen of uHTS hits for Scp-1 phosphataseScreeningsame project related to Summary assay
540329Differential Scanning Fluorimetry (Thermal Shift) Binding Assay for validation of Inhibitors of Scp-1 phosphataseOthersame project related to Summary assay
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 Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 DA030556-01A1
Assay Provider: Dr. Yan Jessie Zhang, University of Texas, Austin, TX

Human Scp phosphatases are regulators involved in neuronal gene silencing. This family of phosphatases exhibits specificity for phosphoryl-Ser5 in the heptad repeats of the C-terminal domain of RNA polymerase II (RNA Pol II) and thus inhibit initiation of transcription [1, 2]. Scps strongly associate with the REST/NRSF neuronal silencing complex that functions to inhibit transcription of neuronal genes in neuronal stem cells and in nonneuronal tissues [1]. Inhibition of Scps by dominant negative forms of the enzyme or by premature expression of the nervous system-specific microRNA miR-124, results in differentiation of neuronal stem cells into mature neurons [1, 3]. Thus small molecule inhibitors of Scps will be valuable reagents to facilitate study and understanding of nervous system development.

This biochemical assay employs a colorimetric readout based on the ability of the SCP-1 phosphatase to release an ortho phosphate from the phosphorylated 17-mer peptide substrate.

The goal of this assay is to confirm hits in "uHTS Colorimetric assay for identification of inhibitors of Scp-1", AID 493091.

References:
1. Yeo, M., et al., Small CTD phosphatases function in silencing neuronal gene expression. Science, 2005. 307(5709): p. 596-600.
2. Yeo, M., et al., A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5. J Biol Chem, 2003. 278(28): p. 26078-85.
3. Visvanathan, J., et al., The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development. Genes Dev, 2007. 21(7): p. 744-9.
Protocol
Assay materials:
1) SCP-1 protein phosphatase (catalytic domain 76-261aa) was obtained from the Dr. Yan Jessie Zhang laboratory.
2) 17-mer phopsphorylated peptide SPSYSPTSPSYSPT-pS-PS was custom synthesized by AnaSpec, Inc.
2) Assay Buffer: 50mM Tris Acetate pH 5.5, 10 mM MgCl2, 0.005% Tween-20
3) PiColorlock Gold Reagent Kit was purchased from Novus Biologicals (Cat # 303-0125)
4) Corning 1536 well black clear bottom plate (Cat # 3891)
uHTS Procedure
1) Using LabCyte Echo, transfer 50 nL of test compounds from a 2 mM compound source plate into assay plate Cols. 5-48 (final concentration of test compounds is 20 uM, 1.0 % DMSO). Transfer 50 nL of 100% DMSO into assay plate Col. 1-4.
2) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
Prepare 1X Assay Buffer, and then prepare four master mixes: Scp1 Mix, BIEB-1 Substrate Mix, Colorlock Gold/Accelerator Mix, and Stabiliser Mix as described in sections E. Recipe and H. Calculation.
3) Using Thermo Scientific MultiDrop Combi, dispense 2.5 uL of Assay Buffer to each well into columns 1-2.
Using Thermo Scientific MultiDrop Combi nL, dispense 2.5 uL of 3 nM Scp1 solution in Assay Buffer to each well into columns 3-48.
4) Using Thermo Scientific MultiDrop Combi nL, dispense 2.5 uL of 120 uM Peptide Substrate solution in Assay Buffer to each well into columns 1-48.
5) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
6) Incubate for 45 minutes at room temp.
7) Using Thermo Scientific MultiDrop Combi nL, dispense 1.5 uL of Colorlock Gold/Accelerator Mix to each well into columns 1-48.
8) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
9) Incubate for 5 minutes at room temp.
10) Using Combi nL, dispense 1 uL of Stabiliser Mix to each well into columns 1-48.
11) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
12) Incubate for 30 minutes at room temp.
13) Read plates on Perkin Elmer Viewlux using at 630 nM in absorbance mode
Comment
Compounds that demonstrated an inhibition of >= 30% at 20 uM concentration are defined as actives in this assay.
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.
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 % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 20 uM (20μM**)inhibition in primary screeningFloat%
2Value (20μM**)Value of the sampleFloatOD
3Mean LowMean Absorbance of positive controls in the corresponding plateFloatOD
4Std Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatOD
5Mean HighMean Absorbance of negative controls in the corresponding plateFloatOD
6Std Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatOD

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA030556-01A1

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