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
BioActive Compounds: 5128
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.
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.
45mL batch FGF22/dye mixture (1uM FGF22/9x dye)
39.1 mL MES buffer (pH 6.0)
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)
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.
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.
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.
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
** Test Concentration.
Data Table (Concise)