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BioAssay: AID 504378

qHTS Assay for Compounds Blocking the Interaction Between CBF-beta and RUNX1 for the Treatment of Acute Myeloid Leukemia: SAR for Probe with Orthogonal Assay

Core Binding Factor (CBF) abnormalities are associated with 20-25% of all acute myeloid leukemias (AML), of which 5-10% are further sub classified as acute myelomonocytic leukemia with eosinophilia, also known as M4Eo in the FAB classification scheme. This subtype of leukemia is usually associated with chromosome 16 inversion (p13:q22). Chromosome 16 inversion results in formation of the more ..
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 Tested Compounds
 Tested Compounds
All(29)
 
 
Inactive(25)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(32)
 
 
Inactive(26)
 
 
Inconclusive(6)
 
 
AID: 504378
Data Source: NCGC (CBFB008)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-28
Hold-until Date: 2011-08-25
Modify Date: 2011-10-03

Data Table ( Complete ):           All
Targets
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
1477qHTS Assay for Compounds Blocking the Interaction Between CBF-beta and RUNX1 for the Treatment of Acute Myeloid LeukemiaconfirmatoryPrimary screen.
1484Quantitative High-Throughput Screen for Compounds Blocking the Interaction Between CBF-beta and RUNX1 for the Treatment of Acute Myeloid Leukemia: SummarysummarySummary assay for project.
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: X01MH083259-01
Assay Submitter (PI): Paul Liu

NCGC Assay Overview:

Core Binding Factor (CBF) abnormalities are associated with 20-25% of all acute myeloid leukemias (AML), of which 5-10% are further sub classified as acute myelomonocytic leukemia with eosinophilia, also known as M4Eo in the FAB classification scheme. This subtype of leukemia is usually associated with chromosome 16 inversion (p13:q22). Chromosome 16 inversion results in formation of the fusion oncogene CBFB-MYH11, which encodes the fusion protein CBF-beta-SMMHC. This fusion protein binds to RUNX1 (AML1) with high affinity and dominantly inhibits RUNX1 function preventing definitive hematopoiesis.

It has been hypothesized that the interference of CBF-beta and RUNX1 binding could have therapeutic implications for patients with CBF mediated leukemias. We developed a CBF-beta and RUNX1 binding assay in AlphaScreenTM format and optimized it for high throughput screening to search for inhibitors. The goal of this project is to screen MLPCN's compound collection (MLSMR) for identifying the inhibitors of the interaction between CBF-beta and RUNX1 proteins as research probes. If these probes work well in the animal models, the ultimate goal of this project is to development a new drug treatment for this disease.

This assay can not be used to assess how apparent inhibitors interfere with protein binding, and whether inhibitors specifically bind to either protein. Disruption of this protein-protein interaction, through any mechanism, is the intended target activity.

The present data set was run to confirm the activity of hits from the primary screen using an orthogonal assay technology for measuring protein-protein interaction disruption.
Protocol
NCGC Assay protocol:

ME-1 suspension cells were grown in T225 flasks to 80% density under a standard cell culture condition (ATCC) using Dulbecco's Modified Eagle Medium (DMEM) + 10% fetal bovine serum, harvested in OPTI-MEM (phenol free) + 10% FBS. Seeded at 500 cells/well in 5uL medium into 1536-well white solid-bottom plates using Multidrop Combi dispenser, and incubated the plates at 37 C with 5% CO2 and 95% humidity for 2 hours prior to pinning compound. Subsequently, 23 nL/well compound solutions or DMSO controls were dispensed to the assay plates via a pintool workstation. Plates were then transferred to 37 C with 5% CO2 and 95% humidity for 48 hours, and then ATP content was measured following the addition of 4uL/well of ATPlite reagent and a 20 min incubation on a ViewLux plate reader.

Keywords: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, Core binding factor, CBF, CBF-beta, Acute myeloid leukemia
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 "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
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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
5Curve_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 signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00109 uM (0.00109μM**)% Activity at given concentration.Float%
15Activity at 0.00217 uM (0.00217μM**)% Activity at given concentration.Float%
16Activity at 0.00439 uM (0.00439μM**)% Activity at given concentration.Float%
17Activity at 0.00941 uM (0.00941μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.018μM**)% Activity at given concentration.Float%
19Activity at 0.035 uM (0.035μM**)% Activity at given concentration.Float%
20Activity at 0.076 uM (0.076μM**)% Activity at given concentration.Float%
21Activity at 0.156 uM (0.156μM**)% Activity at given concentration.Float%
22Activity at 0.301 uM (0.301μM**)% Activity at given concentration.Float%
23Activity at 0.629 uM (0.629μM**)% Activity at given concentration.Float%
24Activity at 1.265 uM (1.265μM**)% Activity at given concentration.Float%
25Activity at 2.582 uM (2.582μM**)% Activity at given concentration.Float%
26Activity at 5.419 uM (5.419μM**)% Activity at given concentration.Float%
27Activity at 10.86 uM (10.86μM**)% Activity at given concentration.Float%
28Activity at 22.10 uM (22.1μM**)% Activity at given concentration.Float%
29Activity at 41.05 uM (41.05μM**)% Activity at given concentration.Float%
30Activity at 82.37 uM (82.37μM**)% Activity at given concentration.Float%
31Activity at 230.4 uM (230.4μM**)% Activity at given concentration.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01MH083259-01

Data Table (Concise)
Classification
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