Differential Scanning Fluorimetry (Thermal Shift) Binding Assay for validation of Inhibitors of Scp-1 phosphatase
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: 378
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.
The goal of this differential scanning fluorimetric (aka Thermal Shift) assay is to eliminate promiscuous hits in "HTS Colorimetric assay for identification of inhibitors of Scp-1" (AID 493091), that inhibit by denaturing the protein nonspecifically. The delta Tm of the protein in the presence of the test compounds is determined to flag the compounds that reduce the Tm of the protein.
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.
4. Pantoliano, M.W., Petrella, E.C., Kwasnoski, J.D., Lobanov, V.S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B.A., Lane, P., Salemme, F.R. High-density miniaturized thermal shift assays as a general strategy for drug discovery, J Biomol Screen, 2001. 6: p. 429-440.
5. Niesen, F.H., H. Berglund, and M. Vedadi, The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc, 2007. 2(9): p. 2212-21.
1) SCP-1 protein phosphatase (catalytic domain 76-261aa) was obtained from the Dr. Yan Jessie Zhang laboratory.
2) Sypro Orange 5000x from Invitrogen (Cat # S6651)
3) Sodium orthovanadate was purchased from Sigma Aldrich (Cat # S6508-10G)
4) Assay Buffer: 50mM Tris Acetate pH 5.5, 10 mM MgCl2, 0.005% Tween-20
5) 384 white Lightcycler MultiwellPlate (Roche Cat# 04 729 749 001)
Single dose Thermal Shift procedure
1) Using Labcyte Echo555, dispense 40 nl of 10 mM compound into columns 3 through 22, while 100 nL of 100% DMSO into columns 23 and 24.
2) Using Labcyte Echo555, dispense 100 nL of 100% DMSO into columns 23 and 24.
3) Using Viaflow 12.5 ul electronic pipettor dispense 1 uL of 100 mM Sodium Orthovanadate into columns 1 and 2
4) Using Thermo Scientific MultiDrop Combi, dispense 5 ul of 10 uM Scp-1 protein in Assay Buffer into columns 1 through 24.
5) Using Thermo Scientific MultiDrop Combi, add 5 ul of 10x Sypro Orange in Assay Buffer to all wells.
6) Centrifuge plates for 1 min at 1000 rpm
7) Using Roche LightCycler480(R) qPCR instrument perform the temperature gradient was performed in the range of 20-95 degrees C with a ramp rate of 0.03 degrees C /s. The detection of protein unfolding was performed at an excitation wavelength of 465 nm and an emission of
8) Tm Calling Analysis was performed using LightCycler(R) 480 Software, to determine the Tm values in presence of the test compound.
%Activity in this assay is:
(Temp of the compound - Temp of the negative control)/( Temp of the positive control - Temp of the negative control)
Compounds that demonstrated an %Activity_Mean >= 5.3% at 100 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 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
** Test Concentration.
Data Table (Concise)