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BioAssay: AID 651658

Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-01_Inhibitor_SinglePoint_HTS_Activity

A fluorescence-based thermal shift assay will be used to identify small molecule binders to FGF22. This label-free approach is based on the principal of ligand-induced thermodynamic stabilization of proteins. Changes in protein thermal stability can be induced by ligand binding, and these changes can be measured using an environmentally-sensitive fluorescent dye. ..more
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 Tested Compounds
 Tested Compounds
All(339647)
 
 
Active(5128)
 
 
Inactive(334541)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(343089)
 
 
Active(5128)
 
 
Inactive(337955)
 
 
Inconclusive(6)
 
 
AID: 651658
Data Source: Broad Institute (7012-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-19
Modify Date: 2013-02-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5128
Related Experiments
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AIDNameTypeComment
651666Broad Institute Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Inhibitor Probe ProjectSummarydepositor-specified cross reference
687022Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
720646Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-04_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743017Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743019Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-03_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743039Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743116Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743182HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
743185HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
Description:
Keywords:
fibroblast growth factor (FGF), FGF22, FGF7, label-free Thermal Shift


Assay Overview:
A fluorescence-based thermal shift assay will be used to identify small molecule binders to FGF22. This label-free approach is based on the principal of ligand-induced thermodynamic stabilization of proteins. Changes in protein thermal stability can be induced by ligand binding, and these changes can be measured using an environmentally-sensitive fluorescent dye.
Recombinant FGF22 is reconstituted in MES buffer and added to the dye SYPRO orange. 5nL of either positive control molecule (Sucrose octasulfate), test compounds, or DMSO (neutral control), are added to each well of a 384-well qPCR plate at a concentration of 10mM. 5uL of the reconstituted FGF22/dye solution is added to each well for a final compound concentration of 10uM. Protein melting curves are aquired for each protein-ligand mixture using a Roche Light cycler 480 qPRC instrument with heating at a rate of 3.6 deg. C/minute from 25c to 85c and analyzed using Roche protein melting analysis software to determine the melting point (Tm) of FGF22. When the protein melts, or begins to unfold, the hydrophoblic reqions of the protein become exposed and cause the dye to begin to fluoresce.


Expected Outcome:
Inactive compounds (non-FGF22 binders) will not change the melting point of FGF22.
Active compounds (binders to FGF22) will show a shift in the melting point of FGF22 by changing the temperature at which fluorescent signal increases.
Protocol
45mL batch FGF22/dye mixture (1uM FGF22/9x dye)
39.1 mL MES buffer (pH 6.0)
400uL DMSO
81 uL SYPro orange dye (5000x)
5.5 mL FGF22 @ 0.129 mg/mL
Dispense 5uL FGF22/dye mixture into all wells of 384 well qpCR plate prepared with positive control, neutral control, and test compounds predelivered at 10mM (5nL).
Incubate the plate for 30 minutes at room temperature in the dark.
Place plate in Roche Light cycle 480 and run protein melt protocol (melting from 25c-85c, 3.6 deg C / minute ramping, 10 acquisitions per deg C, Ex 465nm and Em 580 nm)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
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.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized 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 44.28.
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 1 (inactive) :
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_9.99uM_(%) (9.99μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1R03MH094173-01A1

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