Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR - 7012-04_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
BioActive Compounds: 24
Depositor Specified Assays
fibroblast growth factor (FGF), FGF22, FGF7, label-free Thermal Shift
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.
Counter screen replaces FGF22 with FGF7 to identify binders to FGF7 for elimination from FGF22 hits.
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.
Inactive compounds (non-FGF7 binders) will not change the melting point of FGF7.
Active compounds (binders to FGF7) will show a shift in the melting point of FGF7 by changing the temperature at which fluorescent signal increases.
Retest at Dose Protocol
120mL batch FGF7/dye mixture (44ug/mL FGF7/9x dye)
90 mL MES buffer (pH 6.0)
216 uL SYPro orange dye (5000x)
5.25 mg FGF22 @ 0.177 mg/mL
Dispense 5uL FGF7/dye mixture into all wells of 384 well qpCR plate prepared with positive control, 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.
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.
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 0.38999998569488525
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)
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.
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)