AID	Name	Type	Comment
504586	Broad Institute Epstein-Barr LMP-1 Inhibitor Probe Project	summary	Summary assay

Keywords:
NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, cytotoxicity, EBV-transformed lymphoblastoid cells (LCLs)

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 Secondary Screen use EBV-transformed lymphoblastoid cells (LCLs) to confirm the cytoxicity in relevant target cells of the dose retested compounds from the primary screen. EBV-transformed lymphoblastoid cells (LCLs) require perpetual LMP1/NF-kB activity for growth and survival. shRNAs that block LMP1 expression provoke LCL apoptosis. We are therefore particularly interested in discovering small molecule probes that selectively block LMP1/NF-kB.

Expected Outcome:
Compounds that are cytotoxic will inhibit LCL viability, either due to LMP-1 specific action or off target toxicity. Compounds that are of interested will diminish cell growth at the IC50 threshold and lower, and will be carried forward. Off-target toxicity will be determined in a separate counterscreen. Partial inhibition & cytostatic effects 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
(LCL Cytotoxicity 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 5000 per well in 50 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 100 nL 3.75 mM compound library into 50 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). 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 to cool for 15 minutes to room temperature; add 20 uL 50% Promega CellTiterGlo (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).

NORMALIZATION:
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)

PUBCHEM_ACTIVITY_OUTCOME:
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.

PUBCHEM_ACTIVITY_SCORE:
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.

Note:
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.

Grant Number: 2 R01 CA085180-06A1