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_Dose_CherryPick_Activity
Keywords: Keap1- Nrf2 complex, Kelch domain, Inhibitor screening, biochemical fluorescence polarization assay ..more
BioActive Compounds: 76
Keywords: Keap1- Nrf2 complex, Kelch domain, Inhibitor screening, biochemical fluorescence polarization assay
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 the purified, recombinant Keap1 Kelch domain to a FITC-labeled peptide from the highly conserved Keap1 binding region of Nrf2 (FITC-9mer-Nrf2-NH2). When the probe binds to the 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
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
Final concentration for FP assay: 10nM FITC-9mer-Nrf2-NH2, 100nM Kelch, 2uM Ac-9mer-Nrf2 (positive control)
FP buffer: 10mM HEPES, pH7.4, 3.4mM EDTA, 150mM NaCl, 0.005% Tween-20
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing 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 (2)(Max_Concentration).
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.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
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 >= -70% but <= -30%, 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)