Dose Response 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 ..
BioActive Compounds: 1000
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 . 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.
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.
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)
Procedure for dose-response confirmation assay
1) Using Labcyte Echo550, dispense 40 nL of serially diluted compounds to columns 5-48 and DMSO to columns 1-4.
2) Using Thermo Scientific MultiDrop Combi, dispense 2.5 uL of Assay Buffer to each well into columns 1-2.
3) Using Thermo Scientific MultiDrop Combi, 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, 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, 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 Thermo Scientific MultiDrop Combi, dispense 1 uL of Stabiliser Mix to each well into columns 1-48.
11) Incubate for 30 minutes at room temp.
12) Read plates on Perkin Elmer Viewlux using at 630 nM in absorbance mode
Compounds with an IC50 < 80 uM 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 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 and is not applicable in this assay.
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 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 this assay
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)