The inhibition of Keap1-Nrf2 interaction in cells by beta-lactamase reporter assay Measured in Cell-Based System Using Plate Reader - 2119-03_Inhibitor_Dose_DryPowder_Activity_Set4
The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and proteosomal degradation. Under oxidative stress condition, Nrf2 dissociates from Keap1 and translocates into the nucleus. In the nucleus, Nrf2 dimerizes with a small protein Maf and binds to an antioxidant response element (ARE) in the promoter to increase the transcription of cytoprotective genes. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration. ..more
BioActive Compounds: 21
Keywords: Inhibition, Keap1-Nrf2 interaction, beta-lactamase reporter assay, ARE-bla HepG2
The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and proteosomal degradation. Under oxidative stress condition, Nrf2 dissociates from Keap1 and translocates into the nucleus. In the nucleus, Nrf2 dimerizes with a small protein Maf and binds to an antioxidant response element (ARE) in the promoter to increase the transcription of cytoprotective genes. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration.
We conducted a high throughput screen to identify the inhibitors to disrupt the interaction between Keap1 and Nrf2 by a biochemical fluorescence polarization assay. The ability of the hits to disrupt this interaction at cellular level is evaluated by a cell-base beta-lactamase reporter assay.
The CellSensortrade mark ARE-bla HepG2 cell line (Invitrogen, Cat# K1208) is a stable cell line expressing beta-lactamase reporter gene regulated by ARE in the promoter region. Therefore, the inhibition of Keap1-Nrf2 interaction upon compound treatment is directly correlated with an increase of the beta-lactamase activity.
The beta-lactamase activity is measured by LiveBLAzertrade mark-FRET BG Loading Kit (Invitrogen, Cat#1089). The substrate of beta-lactamase is in its membrane-permeant ester forms and labeled with two fluorophores (Coumarin -emission 460nm and Fluorescein-emission 530nm). This lipophilic ester readily enters the cell and is cleaved by endogeneous cytoplasmic esterases rapidly. The resulted negatively charged forms, thereby is trapped in the cytosol. In the absence of beta-lactamase activity or without inhibitor, the intact molecule of the substrate in the cells results in high FRET signal or low 460nm/530nm ratio. In the presence of beta-lactamase activity caused by inhibitor, the substrate is cleaved and the separation of the two fluorophores spatially. This cleavage results a low FRET signal or a high 460nm / 530nm ratio.
The compounds that inhibit Keap1-Nrf2 interaction will increase beta-lactamase activity, resulting an increase of 460nm/530nm ratio in ARE-bla HepG2 cells.
Cell line: ARE-bla Hep G2 (Invitrogen, Cat# K1208)
Growth Media (500 mL)
50 mL Dialyzed FBS (Invitrogen, Cat# 26400-044)
5 mL Nonessential amino acids (Invitrogen, Cat# 11140-050)
12.5 mL HEPES (pH 7.3) (Invitrogen, Cat# 15630-080)
5 mL Penicillin/Streptomycin (Invitrogen, Cat# 15070-063)
0.25 mL Blasticidin (Invitrogen, Cat# A11139-02)
430 mL DMEM with GlutaMAXtrade mark (Invitrogen, Cat# 10569-010)
Assay Media has the same ingredients as the growth media, but does not contain Blasticidin.
1.Thaw 2x10;6 cells/mL and culture them in T75 tissue culture flask with 15mL Growth Medium without Blasticidin in a humidified 37 degrees C/5% carbon dioxide incubator.
2.At first passage, switch to Growth Medium with Blasticidin.
3.To passage cells, aspirate medium, rinse once in PBS, add Trypsin/EDTA and swirl to coat the cells evenly. Cells usually detach after 2-5 minutes exposure to Trypsin/EDTA. Add an equal volume of Growth Medium to inactivate Trypsin.
4.Verify under a microscope that cells have detached and clumps have completely dispersed.
5.Spin down cells and resuspend in Growth Medium.
6.Pass or feed cells at least twice a week and maintain them between 10% and 90% confluency. Do not allow cells to reach confluence.
7.It is recommend by Invitrogen that passing cells for three passages after thawing before using them in the beta-lactamase assay
1.The Cells for the assay should be grown to reach 60 to 75% confluency. Lower confluent cells will give lower response.
2.Change medium with Assay Medium on the day before the plating
3.Harvest cells and resuspend in Assay Medium at a density of 4.1x105 cells/mL.
4.Dispense 30 uL/well of the cell suspension to Black-wall, clear-bottom, 384-well assay plates (Corning, 3712)
5.Incubate the plates in a humidified 37 degrees C/5% carbon dioxide incubator for 5 hours.
1.Take the assay plates out from incubator and equilibrate them at room temperature for 30 mins
2.Prepare 2% DMSO in Assay Medium
3.Dispense 10uL/well of 2% DMSO solution to assay plates
4.Pin 200 nL/well compounds in dose and 100nL/well positive control tBHQ at 50mM (PubChem SID: 137276090) to assay plates
Final compound concentration: 0.26-68uM
5.Incubate the assay plates in a humidified 37 degrees C/5% carbon dioxide incubator for 15 hours.
Beta-lactamase activity detection (LiveBLAzertrade mark FRET - B/G Loading Kit, Cat#1089)
1.Prepare 6x loading solution:
a.Prepare Solution A
Add 182 iL of the provided DMSO to 200 ug of CCF4-AM and mix well.
b.Add 6 iL of Solution A to 60 iL of Solution B and vortex.
c.Add 934 iL of Solution C to the combined Solutions A and B and vortex.
Note: Under typical laboratory conditions, 6X CCF4-AM Substrate Loading Solution is stable for up to 12 hours.
2.Remove the assay plates from the humidified 37 degrees C/5% carbon dioxide incubator and equilibrate the plates for 30 mins at RT
3.Add 8 uL/well of 6X Substrate Loading Solution to the assay plates to 1X final concentration
4.Cover the assay plates to protect it from light and evaporation.
5.Incubate the assay plates at room temperature for 2 hours
6.Read the assay plates on EnVison in bottom-read mode (General dual mirror bs50/bs50) with excitation of the Coumarin (410 nm) and detection of the blue Coumarin emission (460 nm) and green Fluorescein emission (530 nm).
7.The data is expressed as a ratio of 460nm/530nm
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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.
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.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 40.0
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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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)