Sequence: Kaposi's Sarcoma Herpesvirus (KSHV) Latency associated nuclear antigen (LANA)

Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
504431	Broad Institute Inhibitors of Kaposi's Sarcoma Herpes virus LANA-C Inhibitor Probe Project	summary	Summary assay

Keywords: CellTiterGlo, HeLa, cytotoxicity, ATP

Assay Overview:
At this stage in the project, interesting compounds have been studied in biochemical reactions. This assay examines if any potential probes are cytotoxic to cells. A standard human cell line, HeLa cells, are exposed to compounds for 24 hours with various concentrations of the compounds. Promega's Cell Titer Glo, which measures cellular ATP levels, is used to examine cellular viability in a luminescence-based assay.

Expected Outcome:
A small percentage of compounds identified as hits will be toxic to cells at less than 10 uM. This will lead to a reduction in cellular ATP levels which correlates with a decreased luminescence signal and increased cytotoxicity. Compounds that exhibit no cytotoxicity at <30 uM will be prioritized for follow-up studies.

Protocol:
HeLa cells are seeded at 3,000 cells per well in 30 uL phenol-red free DMEM/10% fetal bovine serum to opaque, white Corning 8867BC 384 well plates. The following day, 100 nL of compound is added per well with the CyBio Vario pinning apparatus. DMSO is added to neutral control wells and mitoxantrone is used as a positive control due to its cytotoxic capacity. HeLa cells are incubated in the Liconic incubators for 24 hours at 37 degrees C. 30 uL of Cell Titer Glo (Promega) is added per well, shaken for 1 minute, incubated at room temperature for 10 minutes and then read on the Perkin Elmer Envision plate reader with standard luminescence parameters. Compounds which exhibit no cytotoxicity at 10 uM and/or below will be prioritized for follow-up. Compounds will only be considered valid for subsequent studies if they do not kill mammalian cells.

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 order for a compound to meet probe attributes, it must not have activity in this assay (IC50>30 uM).

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: 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)

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: R21 NS067638-01A1