Bookmark and Share
BioAssay: AID 743019

Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-03_Inhibitor_Dose_CherryPick_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
_
   
 Tested Compounds
 Tested Compounds
All(1026)
 
 
Active(23)
 
 
Inactive(1003)
 
 
 Tested Substances
 Tested Substances
All(1026)
 
 
Active(23)
 
 
Inactive(1003)
 
 
AID: 743019
Data Source: Broad Institute (7012-03_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-11-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 23
Related Experiments
Show more
AIDNameTypeComment
651666Broad Institute Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
651658Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
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
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.

The Compound Only Thermal Shift assay eliminates target protein from the protocol to identify compounds that give a thermal shift signal alone. This counter screen identifies compounds that should be eliminated from follow-up.

Expected Outcome:
Inactive compounds will not show a melting point by DSF.
Active compounds will show a melting point by DSF.
Protocol
Retest at Dose Protocol
45 mL MES buffer (pH6.0)
400uL DMSO
81 uL SYPro orange dye (5000x)
Dispense 5uL buffer/dye mixture into all wells of 384 well qpCR plate prepared with neutral control, and test compounds predelivered at 10mM (20nL) at 2x dilution
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; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.
NORMALIZATION:
No normalization was applied to the raw data signals.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 0.54
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC0.54_QualifierString
2AbsAC0.54_uM*FloatμM
3pAbsAC0.54_MString
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.285uM_(%) (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.3uM_(%) (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.35uM_(%) (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.56uM_(%) (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.6uM_(%) (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.68uM_(%) (0.68μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_1.1uM_(%) (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_1.2uM_(%) (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1.35uM_(%) (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_2.1uM_(%) (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_2.35uM_(%) (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_2.6uM_(%) (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_2.85uM_(%) (2.85μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_4.6uM_(%) (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_5uM_(%) (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_5.6uM_(%) (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_9uM_(%) (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_10uM_(%) (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_11uM_(%) (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_18uM_(%) (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_19.5uM_(%) (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_21uM_(%) (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_38uM_(%) (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_42uM_(%) (42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

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