Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_SinglePoint_HTS_Activity
Keywords: Keap1- Nrf2 complex, Kelch domain, Inhibitor screening, biochemical fluorescence polarization assay ..more
BioActive Compounds: 489
Keywords: Keap1- Nrf2 complex, Kelch domain, Inhibitor screening, biochemical fluorescence polarization assay
The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and subsequent proteosomal degradation. Under oxidative stress conditions, Keap1 liberates Nrf2 from its repression, resulting in an increased oxidative stress response. This response may play a role in several diseases, including cancer, inflammation, and neurodegeneration.
The assay uses fluorescence polarization (FP) to measure the binding of purified recombinant Keap1 Kelch domain protein to a probe, FITC-labeled peptide from highly conserved Keap1 binding region of Nrf2 (FITC-9mer-Nrf2-NH2). When the probe binds to Kelch domain, the binding results in a high FP signal. Any compound that decreases the high FP signal may indicate the disruption of the interaction between Keap1 and Nrf2. Autofluorescent or quenching compounds will be removed by using total fluorescence filter and subsequent counterscreens.
Compounds of interest will inhibit the Keap1-Nrf2 interaction, indicated by a decrease of fluorescence polarization signal generated by the binding between Keap1 protein and Nrf2 peptide. Total fluorescence should not be affected, as a change may indicate small molecule interference through quenching or autofluorescence.
Human Keap1 Kelch domain protein is cloned into expression vector pET15b and transformed into E.coli BL21 (DE3) strain
The protein is purified through Ni-NTA affinity column due to 6x his tag.
Purified protein is stored in the buffer containing 20 mM HEPES (pH7.4), 5mM DTT at -80 degrees C.
Solution preparation (For a run of 135 plates):
1) Prepare 1100mL 20nM FITC-9mer-Nrf2-NH2 in FP buffer
Take 2.689mL of 8.18uM FITC-9mer-Nrf2-NH2 in 100% DMSO in 1100ml FP buffer
2) Prepare 60mL 8uM Ac-9mer-Nrf2 in FP buffer
Take 0.48mL of 1mM Ac-9mer-Nrf2 in 100% DMSO in 60mL FP buffer
3) Prepare 500mL 400nM Kelch domain in FP buffer
Take 9mL 0.75mg/mL Kelch-xtal in 500mL FP buffer
Final concentration for FP assay: 10nM FITC-9mer-Nrf2-NH2, 100nM Kelch
Final concentration for PosCon: 2uM Ac-9mer-Nrf2
Setup reagents on automation instrument
1) Add the solutions of Ac-9mer-Nrf2, Kelch domain and FP buffer to the bottles of BioRaptr (Beckman) based on the dispense table
2) Add FITC-9mer-Nrf2-NH2 solution to the bottle of combi NL (Thermo Scientific)
3) Add 1536-well assay ready plates (ARPs) to incubator
Run automation protocol
1) Dispense 4uL/well of the solutions from BioRaptr based on the dispense table to 1536-well ARPs (Aurora, Cat#: 00019180BX)
(Positive control wells receive 2uL/well of Ac-9mer-Nrf2 and 2uL/well of Kelch solution; all the other wells receive 2uL/well of FP buffer and 2uL/well of Kelch solution)
2) Incubate the plates for 10mins at 25 degrees C
3) Dispense 4ul/well of 20nM FITC-9mer-Nrf2-NH2 solution to the plates by Combi NL
4) Incubate the plates for 60mins at 25 degrees C
5) Read the plates on ViewLux (PerkinElmer) with excitation wavelength of 480nm and emission wavelength of 535 nm with dichroic mirror of D505fp/D535
FP buffer: 10mM HEPES, pH7.4, 3.4mM EDTA, 150mM NaCl, 0.005% Tween-20
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.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' 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 -1 times 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 12, which was 3 times the standard deviation of the neutral control wells.
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 50.
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 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)