Bookmark and Share
BioAssay: AID 588364

C-LANA Counter Screen: DNA intercalators Measured in Biochemical System Using Plate Reader - 2117-02_Inhibitor_Dose_DryPowder_Activity

We wish to exclude "hits" which non-specifically inhibit C-LANA DNA binding by intercalating into DNA. Compound libraries contain many flat molecules which may intercalate into DNA. We modified and miniaturized an assay to counterscreen for DNA intercalators. Acridine orange fluoresces and intercalates into DNA, and this binding can be measured by fluorescence polarization (FP). DNA more ..
_
   
 Tested Compounds
 Tested Compounds
All(20)
 
 
Active(4)
 
 
Inactive(16)
 
 
 Tested Substances
 Tested Substances
All(20)
 
 
Active(4)
 
 
Inactive(16)
 
 
 Related BioAssays
 Related BioAssays
AID: 588364
Data Source: Broad Institute (2117-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-09-16
Hold-until Date: 2012-09-15
Modify Date: 2012-09-18

Data Table ( Complete ):           Active    All
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
504431Broad Institute Inhibitors of Kaposi's Sarcoma Herpes virus LANA-C Inhibitor Probe ProjectsummarySummary assay
Description:
Keywords: DNA, intercalation, acridine orange


Assay Overview:
We wish to exclude "hits" which non-specifically inhibit C-LANA DNA binding by intercalating into DNA. Compound libraries contain many flat molecules which may intercalate into DNA. We modified and miniaturized an assay to counterscreen for DNA intercalators. Acridine orange fluoresces and intercalates into DNA, and this binding can be measured by fluorescence polarization (FP). DNA intercalators compete for acridine orange binding. Titration of DNA into a solution of 50 nM acridine orange results in a steep increase in FP signal followed by a plateau. This assay is specific since excess mitoxantrone, a DNA intercalator, strongly inhibits FP signal.

Expected Outcome: Since the primary assay involves DNA binding, the lead compounds will consist of DNA intercalators and other binders. The goal is to find compounds that are specific to the LANA DNA binding site and not general DNA intercalating compounds. Compounds that have an IC50 of greater than 30 uM will be of interest while any active compounds in this assay will be eliminated from contention due to their ability to bind DNA in a non-specific manner.
Protocol

Protocol:
Compounds will be tested for their ability to bind DNA. Acridine orange is a DNA intercalator that binds independent of a specific DNA target sequence. 50 nM acridine orange (Invitrogen) and 6 ug/mL salmon sperm DNA (Invitrogen) are incubated with compounds for 30 minutes. Mitoxantrone, a known DNA intercalator, is used as the positive control at 10 uM. 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 the reaction volume is 30 uL per well. Compounds are examined over a range of doses.


HEN assay Buffer: 10 mM HEPES, pH 7.5; 1 mM EDTA pH 7.5; 100 mM NaCl

500mL: 10 mL 1M HEPES, 20 mL 2.5M NaCl, 2 mL 0.5 M EDTA 468 mL water

Acridine orange: make 1 mM stock by dissolving 3 mg/mL in 10 mL water (Milli Q)

Salmon sperm DNA: stock is 10 mg/mL

ASSAY DEVELOPMENT SETUP:

6 ug/mL DNA, 50 nM Acridine orange (final concentrations in HEN buffer)


Dispense 30 uL per well to Corning non-binding 3575 black opaque 384 well plates, immediately add compound in 100 nL volume with a CyBio pinning apparatus and incubate at room temperature (in dark) for 20 minutes.

Read on the Perkin Elmer Envision plate reader using the 480 nM excitation filter, 535 S and P emission filters and D505fp/D535 dichroic mirror. Apply polarization calculation to determine mP values.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.005uM_(%) (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.015uM_(%) (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.046uM_(%) (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.42uM_(%) (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_1.2uM_(%) (1.2μM**)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_11uM_(%) (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21 NS067638-01A1

Data Table (Concise)
PageFrom: