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BioAssay: AID 2662

qHTS Fluorescence Polarization Assay for Inhibitors of MLL CXXC domain - DNA interaction

The MLL (Mixed Lineage Luekemia) gene is involved in chromosomal translocation that results in either acute lymphoid leukemia (ALL) or acute myeloid leukemia (AML). Chromasomal translocation of MLL gene is responsible for the fusion of N-terminal MLL to more than 60 different partner genes in frame. Many of the MLL partner genes contain transcription activation domains or function as more ..
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 Tested Compounds
 Tested Compounds
All(294067)
 
 
Active(110)
 
 
Inactive(285261)
 
 
Inconclusive(8738)
 
 
 Tested Substances
 Tested Substances
All(294978)
 
 
Active(110)
 
 
Inactive(286120)
 
 
Inconclusive(8748)
 
 
AID: 2662
Data Source: NCGC (MLLD723)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-24
Modify Date: 2010-09-28

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 110
Depositor Specified Assays
AIDNameTypeComment
2698qHTS Fluorescence Polarization Assay for Inhibitors of MLL CXXC domain - DNA interaction: SummarysummarySummary assay.
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: DA027717-01A1
Assay Submitter (PI): John Bushweller

NCGC Assay Overview:

The MLL (Mixed Lineage Luekemia) gene is involved in chromosomal translocation that results in either acute lymphoid leukemia (ALL) or acute myeloid leukemia (AML). Chromasomal translocation of MLL gene is responsible for the fusion of N-terminal MLL to more than 60 different partner genes in frame. Many of the MLL partner genes contain transcription activation domains or function as transcriptional activators. Despite the heterogeneity of fusion partners, the N-terminus of MLL fusion proteins always consists of the N-terminal menin binding motif and the CXXC domain. The MLL-CXXC domain specifically recognizes unmethylated CpG sequences of the HOX-A gene. Disruption of the MLL-CXXC and HOX-A gene interaction leads to down regulation of HOX-A gene expression and may potentially reverse leukemogenesis. We have miniaturized and optimized an in-vitro assay to screen for inhibitors of the MLL-CXXC domain and HOX-A DNA interaction, which could have utility in MLL fusion leukemias.
Protocol
The affinity purified MLL-CXXC domain, unlabeled HOX-A9 DNA strand, fluorescein (FLSN) labeled HOX-A9 DNA strand were kindly supplied by the laboratory of Dr. John Bushweller, University of Virginia. All reagents were stored in single use aliquots at -80C.

Assay Reagents:
Assay buffer: 50 mM TRIS-HCl pH 7.5, 50 mM NaCl, 1 mM DTT. Add 0.05% BSA prior to reaction setup.

Assay Procedure:
1.Dispense 1 ul of a 7 uM MLL-CXXC solution prepared in assay buffer into column 1-48.
2.Transfer 23 nl of compounds and unlabeled HOX-A9 per well.
3.Incubate for 5 minutes at ambient.
4.Dispense 1ul of a 50 nM FLSN-HOX-A9 solution prepared in assay buffer into columns 1-48
5.Centrifuge for 30 seconds at 1000 RPM.
6.Incubate for 30 minutes at ambient.
7.Read using the Envision reader fluorescent polarization protocol.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Fluorescence_Polarization-Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Total_Fluorescence-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Total_Fluorescence-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Total_Fluorescence-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Total_Fluorescence-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Total_Fluorescence-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Total_Fluorescence-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Total_Fluorescence-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Total_Fluorescence-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Total_Fluorescence-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Total_Fluorescence-Activity at 0.00736 uM (0.00736μM**)% Activity at given concentration.Float%
15Total_Fluorescence-Activity at 0.037 uM (0.0368μM**)% Activity at given concentration.Float%
16Total_Fluorescence-Activity at 0.184 uM (0.184μM**)% Activity at given concentration.Float%
17Total_Fluorescence-Activity at 0.920 uM (0.92μM**)% Activity at given concentration.Float%
18Total_Fluorescence-Activity at 4.600 uM (4.6μM**)% Activity at given concentration.Float%
19Total_Fluorescence-Activity at 10.30 uM (10.3μM**)% Activity at given concentration.Float%
20Total_Fluorescence-Activity at 23.00 uM (23μM**)% Activity at given concentration.Float%
21Total_Fluorescence-Activity at 51.40 uM (51.4μM**)% Activity at given concentration.Float%
22Total_Fluorescence-Activity at 115.0 uM (115μM**)% Activity at given concentration.Float%
23Fluorescence_Polarization-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
24Fluorescence_Polarization-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
25Fluorescence_Polarization-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
26Fluorescence_Polarization-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
27Fluorescence_Polarization-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
28Fluorescence_Polarization-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
29Fluorescence_Polarization-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
30Fluorescence_Polarization-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
31Fluorescence_Polarization-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
32Fluorescence_Polarization-Activity at 0.00736 uM (0.00736μM**)% Activity at given concentration.Float%
33Fluorescence_Polarization-Activity at 0.037 uM (0.0368μM**)% Activity at given concentration.Float%
34Fluorescence_Polarization-Activity at 0.184 uM (0.184μM**)% Activity at given concentration.Float%
35Fluorescence_Polarization-Activity at 0.920 uM (0.92μM**)% Activity at given concentration.Float%
36Fluorescence_Polarization-Activity at 4.600 uM (4.6μM**)% Activity at given concentration.Float%
37Fluorescence_Polarization-Activity at 10.30 uM (10.3μM**)% Activity at given concentration.Float%
38Fluorescence_Polarization-Activity at 23.00 uM (23μM**)% Activity at given concentration.Float%
39Fluorescence_Polarization-Activity at 51.40 uM (51.4μM**)% Activity at given concentration.Float%
40Fluorescence_Polarization-Activity at 115.0 uM (115μM**)% Activity at given concentration.Float%
41Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA027717-01A1

Data Table (Concise)
Classification
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