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BioAssay: AID 652163

S100A4: HTS Measured in Biochemical System Using Plate Reader - 7045-01_Inhibitor_SinglePoint_HTS_Activity

Assay Overview: Fluorescence polarization HTS to identify inhibitors of S100A4/TAMRA-Myosin IIA heavy chain interaction. The primary assay consists of a TAMRA-labeled peptide derived from the S100A4 binding site on the myosin-IIA heavy chain (TAMRA-Ahx-TETADAMNREVSSLKNKLRRGDLP-amide) and untagged human S100A4. The S100A4/myosin-IIA interaction is detected by fluorescence polarization with a BMG PHERAstar FS multimode microplate reader using excitation and emission wavelengths of 540 +/- 10 and 590 +/- 10 nm, respectively. ..more
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 Tested Compounds
 Tested Compounds
All(352231)
 
 
Active(501)
 
 
Inactive(351267)
 
 
Inconclusive(491)
 
 
 Tested Substances
 Tested Substances
All(356325)
 
 
Active(502)
 
 
Inactive(355332)
 
 
Inconclusive(491)
 
 
 Related BioAssays
 Related BioAssays
AID: 652163
Data Source: Broad Institute (7045-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 501
Related Experiments
AIDNameTypeComment
652158Broad Institute Old Drugs and New Strategies for Tumor Metastasis Inhibitor Probe ProjectSummarydepositor-specified cross reference
Description:
Keywords: S100A4, metastasis, breast cancer, S100 calcium-binding protein A4


Assay Overview: Fluorescence polarization HTS to identify inhibitors of S100A4/TAMRA-Myosin IIA heavy chain interaction. The primary assay consists of a TAMRA-labeled peptide derived from the S100A4 binding site on the myosin-IIA heavy chain (TAMRA-Ahx-TETADAMNREVSSLKNKLRRGDLP-amide) and untagged human S100A4. The S100A4/myosin-IIA interaction is detected by fluorescence polarization with a BMG PHERAstar FS multimode microplate reader using excitation and emission wavelengths of 540 +/- 10 and 590 +/- 10 nm, respectively.


Expected Outcome: Decrease of fluorescence polarization calculated value indicates inhibition of binding.
Protocol
Assay Mix
1 mM S100A4 monomer
5 nM labeled MyoIIA1904-1927
50 mM HEPES, pH 7.2
15 mM NaCl
100 mM KCl
10 mM CaCl2
0.10% Triton X-100
2 mM DTT
Step 1. Add 5 ul of the Assay Mix to all wells of a 1536-Well Assay Plate, Black, square well, in which the test compounds (or DMSO for neutral control wells) have previously been dispensed. Add 0.5 uL of TFP control to positive control wells.
Step 2. Wait 5 minutes and use Envision microplate reader to top-read the fluorescence polarization of TAMRA-MIIA1908-1923 (lex=530 nm, lex=590 nm). FP signal is stable between 5 - 60 min.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -70.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_10.7uM_(%) (10.7μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_10.7uM_(%) (10.7μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: R01CA129598-01A1

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