Chemical Probes of Kaposi's Sarcoma Herpes Virus Latent Infection
Kaposi's sarcoma associated herpesvirus (KSHV) is associated with cancer in immunosuppressed hosts. KSHV latently infects cells and the viral genome persists as a circular episome (plasmid). KSHV LANA tethers the viral genome to mitotic chromosomes to segregate episomes to daughter nuclei. N-terminal LANA binds histones H2A/H2B to attach to chromosomes. Here, we report a screen for inhibitors of more ..
BioActive Compounds: 150
Kaposi's sarcoma associated herpesvirus (KSHV) is associated with cancer in immunosuppressed hosts. KSHV latently infects cells and the viral genome persists as a circular episome (plasmid). KSHV LANA tethers the viral genome to mitotic chromosomes to segregate episomes to daughter nuclei. N-terminal LANA binds histones H2A/H2B to attach to chromosomes. Here, we report a screen for inhibitors of N-terminal LANA peptide binding to nucleosomes. A fluorescence polarization assay was used to detect N-LANA binding to nucleosomes. Hits were obtained in the primary screen, but none of the hits survived all counterscreens, which included screens for DNA intercalators and a confirmatory non fluorescent (ELISA) assay to detect LANA binding to nucleosomes.
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.
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.
Data Table (Concise)