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 ..
Depositor Specified Assays
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.
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
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)