TES1 - eGFP vs TES2 -dsRED Pathway Differentiation Measured in Cell-Based System Using Imaging - 2122-04_Inhibitor_Dose_CherryPick_Activity
NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, cytotoxicity, EBV-transformed TES 1 - eGFP and TES2 - dsRED ..more
BioActive Compounds: 113
NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, cytotoxicity, EBV-transformed TES 1 - eGFP and TES2 - dsRED
Epstein-Barr Virus is a ubiquitous Herpesvirus that is an important cause of Hodgkin's Disease, other Lymphoproliferative Diseases, and Nasopharyngeal Carcinoma. EBV infection mimics NF-kB hyperactivation states present in many malignancies. The EBV oncoprotein LMP1 constitutively activates both canonical and noncanonical NF-kB pathways in a ligand-independent fashion. LMP1 is expressed in most EBV-associated lymphoproliferative and epithelial malignancies. LMP1 activates NF-kB via two cytoplasmic signaling domains. The membrane proximal "TES1" domain activates a non-canonical NF-kappaB pathway, while the membrane distal "TES2" domain activates canonical NF-kappaB. This assay uses a dual color reporter system in HEK293T cells stably transfected with a contruct containing EGFP driven by a TES-1 responsive promoter and a dsRED reporter driven by a Tet-On tetracycline responsive element.
Compounds that are inhibiting the eGFP are inhibiting the TES pathway, while compounds inhibiting the TET-ON dsRed signal are not specific for NF-kB. Compounds that are of interest will selectively inhibit the eGFP signal and not the dsRED production and will be carried forward. A window of at least 2-fold is desired. Partial inhibition of eGFP in this assay are of interest, as they could be acting through just one of the canonical/non-canonical pathways.
LMP1 - Secondary Screen 2 Secondary Screen Protocol
(eGFP/dsRED Assay )
Day 0, cell grown in t175 TC flask to 95% confluence to yield 30 Million (TrypLE phenol free) and resuspended to dispensing at 125,000 cells / mL of phenol free DMEM
Day 1, plate cells 5000 per well in 40 uL media (phenol red free DMEM/10% Tet Free FBS/Pen/Strep/L-Glutamine); incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 24 hours.
Day 2, add 10 uL per well of stimulant (3ug/mL doxycycline and 3uM 4-hydroxytamxoifen in phenol free DMEM medium) with a Combi multidrop (Thermo);
add 100 nL 3.75 mM compound library into 50 uL assay volume in Black Clear bottom Corning 8816BC barcoded 384 well plates using a pin tool (CyBio). Final compound library concentration was 9.4 uM. MG132 (Calbiochem 474790), a proteasome inhibitor that blocks degradation of NF-kappaB inhibitor Ikappa-Balpha, was added to positive control wells to a final concentration of 12.5 uM.
Incubate 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 4, remove plate from incubator ; Read on Acumen eX3 with 488 excitation laser and detect with green and red filters, gated at 2SD and 562 voltage. <
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 in the eGFP channel only, with a 2-fold less potent response in the dsRED channel
The compounds were measured in multiple channels; each instance is called a 'TEST'. TEST_1 corresponds to the dsRED channel, and TEST_2 corresponds to the eGFP channel. For each channel, 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 (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: The plate pattern correction algorithm 'Assay Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
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.
Once the data for each channel was processed, the channel name was appended to all column headers in that channel's data set. The individual channel results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when AC50 of the dsRED channel (TEST1) is greater than 2 * AC50 of the eGFP channel (TEST2), or dsRED (TEST1) was inactive and eGFP (TEST2) < AC_limit;
b. '1' (inactive) when all channel outcomes were '1' (inactive), or when AC50 of the dsRED channel (TEST1) is less than 2 * AC50 of the eGFP channel (TEST2).
c. '3' (inconclusive) when one or more channel outcomes were inconclusive.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the value of the eGFP channel (TEST2);
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE for active compounds is equal to the activity score of the eGFP channel (TEST2).
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)