External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: HMS791

Protocol: Prior to screening, FITC LANA1-23 was stored lyophilized at -80 degC and freshly purified chicken nucleosomes were stored at 4 degC in 20 mM Tris pH 7.5, 600 mM NaCl, 0.2 mM EDTA, 0.5 mM B-Mercaptoethanol.

On the day of screening, FITC LANA1-23 was resuspended at 50 uM in TEN-BT buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 2.5 mM NaCl, 5 mM Beta-mercaptoethanol, 0.01% Triton-X-100) and diluted to a final concentration of 50 nM in TEN-BT buffer plus 240 nM of purified nucleosomes (480 nM LANA peptide binding sites). 30 uL per well were dispensed in column 1-22 in Corning #3575 black 384 well plates.

Wells in column 23 contained 30 uL of the same mixture (for pilot screen) or with the addition of 10 uM monensin (for HTS) as a negative control. In column 24, 1250 nM unlabeled WT LANA1-23 peptide (for pilot screen) or 10 uM mitoxantrone (for HTS) was added to the wells as a positive control. Compounds were transferred into wells via stainless steel pin array (100 nL) and the reaction was incubated at room temperature for 10 to 45 minutes (stable for up to 2 hours). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Following a room temperature incubation of 10 to 45 minutes, the assay is read on a EnVision plate reader using a 480 nM excitation filter, 535 nM S and P emission filters and D505fp/D535 dichoric mirror. mP value for FP measurement = 1000*(S-G*P)/(S+G*P) where S= , P=, G= G-factor. The G Factor = 1.

Comment: LANA 1-23 peptide containing the first 23 amino acids of the LANA protein from Kaposi's sarcoma herpesvirus (KSHV) was synthesized with an N-terminal FITC via a beta alanine linker and HPLC purified (peptide sequence: [FITC]-Beta alanine-MAPPGMRLRSGRSTGAPLTRGSC-[NH2]).

Data analysis: Z-scores were calculated for each replicate well using the mean and standard deviation of plate experimental well FP values. Compounds were considered active if the Z-score for both replicates < -2. Wells with high total fluorescence intensity (high S and P channel values) were excluded from further consideration. Activity scores were calculated based on replicate average FP Z-scores. For wells with average Z-score >= 0, the activity score was set to 0. For wells with replicate average Z-score < 0, replicate Z-score was divided by 4 and multiplied by -100. The replicate average was then used to determine the well activity score. Values > 100 (replicate average Z-score < -4) were set to 100.