Nrf2 PathHunter Nuclear Translocation assay in U2OS cells Measured in Cell-Based System Using Plate Reader - 2119-04_Inhibitor_Dose_DryPowder_Activity
Keap1-Nrf2 interaction, nuclear translocation, Enzyme Fragment Complementation, beta-galactosidase, U2OS cells ..more
BioActive Compounds: 12
Keap1-Nrf2 interaction, nuclear translocation, Enzyme Fragment Complementation, beta-galactosidase, U2OS cells
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 induce a nuclear translocation of Nrf2 is evaluated using PathHunter U2OS Keap1-Nrf2 functional assay (DiscoveryRx, Fremont CA).
This assay is using Enzyme Fragment Complementation (EFC) technology in which the beta-galactosidase (beta-gal) enzyme has been split into two inactive fragments. A small 4 kDa complementing fragment of beta-gal , termed PK (i.e., the ProLinktrade mark tag) has been fused recombinantly to the NRF2 protein. The larger portion of beta-gal, termed EA for 'enzyme acceptor', is anchored in the nucleus. The inhibition of Keap1-NRF2 interaction results in Nrf2 nuclear translocation. The nuclear accumulation of Nrf2 results in PK-EA complementation and formation of a functional enzyme. Enzyme activity is then quantitatively detected using the chemiluminescent substrate in the PathHunter(R) Detection Kit.
The compounds that inhibit Keap1-Nrf2 interaction show an increase of chemluminescence.
1. U2OS cells co-expressing Keap1-Nrf2 and beta-Gal in different cellular compartments (DiscoverX, Product code 93-0821C3) are cultured in 225mm TC flasks with 40mls of growth media, MEM (Gibco), 10% Heat Inactivated FBS (Hyclone), 1X penn/strep/glutamine (Gibco)) supplemented with 500ug/ml Geneticin (Gibco) and 250ug/ml Hygromycin (Invitrogen) in a 37C incubator (5%CO2). Do let cells go around 95% confluency. Split cells 2X106 cells for subsequent passage every three days.
2. Day 1, cells on T225 mm cell culture flasks are washed once with 1XPBS (Gibco), and digested with 3ml 1X Versen (Gibco #15040) for 2-3 minutes.
3. Add 15-20mL assay media (MEM (Gibco), 10% Heat Inactivated FBS (Hyclone), 1X penn/strep/glutamine (Gibco)) to the plate, mix cells, then transfer the cells to a 50ml centrifuge tube. Centrifuge cells at 1000 rpm for 5 minutes.
4. Aspirate off the supernatant, and resuspend the cells in assay media and count the cell numbers.
5. Add 20uL/well assay media in white 384 well plates (Corning 8867BC) with Combi (Thermo),
5. Plate cells in white 384 well plates (Corning 8867BC), 10000 cells/25uL/well with Combi (Thermo) while gently stirring the media. Incubate cell plates 20 hours in a 37 degree Celsius incubator (10% CO2).
6. Day2. Remove the media from wells by gentle tipping plate to paper towels,
7. Add 25uL of HBSS (+Ca++, Mg++, Invitrogen) buffer to each well with Combi (Thermo)
8. Pin transfer 100 nL/well of compound and PosCon to the cells.
9. Incubate at 37C in 5%CO2 incubator for 3 hours.
10. Remove the plate from the incubator, add 7uL/well of beta-Gal reagent (ABI) with Combi multidrop (Thermo), incubate at room temperature for 1h 20min.
11.#Measure luminescence in Envision (Perkin Elmer)
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.
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.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)