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BioAssay: AID 435023

Fluorescence Polarization Cell-Free Homogeneous Dose Retest to Confirm Inhibitors of the LANA Histone H2A/H2B Interaction

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 Erythrocytes 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. ..more
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 Tested Compounds
 Tested Compounds
All(1430)
 
 
Active(83)
 
 
Inactive(1327)
 
 
Inconclusive(18)
 
 
Unspecified(2)
 
 
 Tested Substances
 Tested Substances
All(1430)
 
 
Active(83)
 
 
Inactive(1327)
 
 
Inconclusive(18)
 
 
Unspecified(2)
 
 
AID: 435023
Data Source: Broad Institute (2053-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-06-29
Modify Date: 2010-07-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 83
Related Experiments
AIDNameTypeComment
2629Fluorescence Polarization Cell-Free Homogeneous Primary HTS to Identify Inhibitors of the LANA Histone H2A/H2B InteractionScreeningdepositor-specified cross reference: Primary HTS
2659Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection ProjectSummarydepositor-specified cross reference: Project summary
463198Fluorescence Polarization Cell-Free Homogeneous Counter Screen to Identify Inhibitors of DNA IntercalationOthersame project related to Summary assay
463211Luminescent Cell-Based Counter Screen to Identify Non-Cytotoxic CompoundsConfirmatorysame project related to Summary assay
588510Fluorescence Polarization Biochemical Secondary LANA-Nucleosome Assay_2053_04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
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 Erythrocytes 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.
Protocol
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.
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. 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Qualifier'>', '=', or '<'String
2EC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_EC50_Mthe log of the EC50 (molar)Float
4Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_0.06uM (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.12uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.235uM (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.46uM (0.46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.9uM (0.9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.8uM (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_1.95uM (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_3.8uM (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_7.5uM (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R21 NS061738-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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