Sequence: LANA [Human herpesvirus 8]

Gene:HHV8GK18_GP81     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
2659	Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection Project	summary
435023	Fluorescence Polarization Cell-Free Homogeneous Dose Retest to Confirm Inhibitors of the LANA Histone H2A/H2B Interaction	confirmatory
463211	Luminescent Cell-Based Counter Screen to Identify Non-Cytotoxic Compounds	confirmatory

Primary Collaborators:
Kenneth Kaye,Brigham & Womens,Boston MA,kkaye@rics.bwh.harvard.edu,617-525-4256
Chantal Beauchemin,Brigham & Womens,Boston MA,cbeauchemin@rics.bwh.harvard.edu,617-525-4256

Keywords: KSHV, LANA, fluorescence polarization, nucleosomes, viral persistence

Assay Overview:
Screening for inhibitors that reduce or prevent the binding of LANA to the acidic region of the H2A/H2B histone dimer interface. Synthetic LANA peptide is labeled with an amino terminal FITC fluorophore linked via a beta alanine residue. The peptide contains the first 23 amino acids of the LANA protein that is essential for binding to the H2A/H2B interface. Nucleosomes were purifed from chicken eryhrocytes as a source of intact H2A/H2B dimers. Fluorescence is measured in the S and P planes with a Perkin Elmer Viewlux after 1 hour incubation.

Expected Outcome:
Compounds will be identified that inhibit the interaction of LANA 1-23 peptide with purified nucleosomes (containing H2A/H2B histone dimers). Inhibitors will have a reduced mP value.

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. Purified chicken erythrocyte nucleosomes are provided by the assay provider and purified from chicken red blood cells. They are stored at -80C at 30 uM.

Assay buffer (TEN-BT) is 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

LANA peptide is diluted to a final concentration of 50 nM in TEN-BT buffer with 100 nM of purified nucleosomes in black 1536 well plates with 10 uL per well. 7.5 nL of compounds are pinned per well. The reaction is incubated at room temperature for 60 minutes (but is stable for up to 2 hours). The assay is read on a Viewlux plate reader using a 480 nM excitation filter, 535nM 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.

HTS Data Analysis

Negative control wells (DMSO) were included on every plate.
Active compounds result in decreased readout signal.

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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.

The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata applied to the normalized plate data.

The replicate activity scores were multiplied by -1 to convert Genedata negative percent inhibition values to Pubchem positive percent activity values.
The final PUBCHEM_ACTIVITY_SCORE was set as equal to the most active replicate.

The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of 25%:

Activity_Outcome = 1 (inactive)
None of the replicates fell outside the threshold.

Activity_Outcome = 2 (active)
At least one of the replicates fell outside the threshold.

Activity_Outcome = 3 (inconclusive)
None.

Grant Number: 1 R21 NS061738-01