Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_Dose_CherryPick_Activity
Keywords: NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2 ..more
BioActive Compounds: 645
Keywords: NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2
Assay Overview: 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.
The primary screen uses a stably transfected HEK293 cell line with a doxycycline & 4-hydroxytamoxifen inducible LMP1 TES2 construct and a NF-kappaB luciferase reporter. Induction of the system will activate the NF-kappaB pathway and result in expression of the luciferase reporter gene. Inhibitors of the the pathway will block expression of the reporter construct.
Expected Outcome: Inhibitors of the LMP1 pathway will show a reduction in luminescence measured by a commercial luciferase kit (SteadyGlo, Promega.) Later assays will determine whether this loss of signal is due to inhibition of the pathway of interest or due to off-target or general toxicity effects.
LMP-1 Screening Protocol
(Luciferase reporter assay)
Day 0, cell grown in HyperFlask (Corning) to 95% confluence to yield 273 Million (TrypLE phenol free) and resuspended to dispensing at 150,000 cells / mL of phenol free DMEM
Day 1, plate cells 3000 per well in 20 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 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). Final compound library concentration was 12.5 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 16.7 uM.
Incubate 16 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 3, remove plate from incubator to cool for 15 minutes to room temperature; add 20 uL 50% Promega Steady glo (diluted 1:1 with PBS pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.1 sec per well.
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 (10)(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)
Note: Partially inhibited curves (Sinf > -100) are also of interest for this particular assay system.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)