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	Project summary

Keywords: Acridine orange, DNA intercalation, Fluorescence polarization

Assay Overview:
The overall goal of the project is to identify compounds that inhibit the binding of the LANA peptide of Kaposi's Sarcoma Herpes Virus (KSHV) to the H2A/H2B histone dimer interface. Compounds that disrupt the interaction were identified in a previous assay. However, DNA intercalators that disrupt this interaction are not of interest.
Acridine orange is a known DNA intercalator. A fluorescence polarization assay that measures the binding of acridine orange to salmon sperm DNA has been used to identify intercalators. The assay determines if the compound can prevent or compete with acridine orange for binding to DNA.

Expected Outcome:
Some compounds will be DNA intercalators and reduce binding of acridine orange to DNA. Compounds that are inactive in this assay will be candidates for further development.

Compounds will be tested for their ability to bind DNA. Acridine orange is a DNA intercalator. 50 nM acridine orange and 6 ug/mL salmon sperm DNA are incubated with compounds for 20 minutes. Fluorescence polarization is measured using a 480 nM excitation filter and 535 nM S and P emission filters with a D505 FP/D535 dichroic mirror. The S and P values are processed with the standard FP calculation formula (mP=1000*(S-G*P)/(S+G*P) where G is the G-factor and is approximately 1). The assay is formatted for 384 well plates and reaction volume is 30 uL per well.

Data Analysis

Negative control wells (NC) and positive control wells (PC) 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.

No plate pattern correction algorithm in Genedata was applied to the normalized plate data.

AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE

Inactive compounds = 0
Active compounds = -10*Log(AC50)

PUBCHEM_ACTIVITY_OUTCOME

Activity_Outcome = 1 (inactive) when:
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when:
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive) when:
Curve fitting strategy resulted in a constant fit with an activity score >30% but <70%
or
The fit was not valid due to poor fit quality.

Activity_Outcome = 4 (unspecified) when:
No usable data was obtained from the tested compound

Grant Number: 1 R21 NS061738-01