uHTS identification of compounds inhibiting the binding between the RUNX1 Runt domain and CBFb via a fluorescence resonance energy transfer (FRET) assay.
The protein-protein interaction between the subunits of the heterodimeric transcription factor CBF, core binding factor b (CBFb) and Runx1 (CBFa), plays a critical role in hematopoiesis. Chromosomal rearrangements that target the core-binding factor genes are some of the most common mutations in leukemia. RUNX1 (AML1) is disrupted by the t(8;21)(q22;q22), t(12;21)(p13;q22), t(3;21)(q26;q22), more ..
BioActive Compounds: 993
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: X01 MH083230-01
Assay Provider: Dr. John Bushweller, University of Virginia Charlottesville, Charlottesville VA
The protein-protein interaction between the subunits of the heterodimeric transcription factor CBF, core binding factor b (CBFb) and Runx1 (CBFa), plays a critical role in hematopoiesis. Chromosomal rearrangements that target the core-binding factor genes are some of the most common mutations in leukemia. RUNX1 (AML1) is disrupted by the t(8;21)(q22;q22), t(12;21)(p13;q22), t(3;21)(q26;q22), t(16;21)(q24;q22), t(1;21)(p36;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22;q22), t(15;21)(q22;q22), and t(17;21)(q11.2;q22), all of which are associated with myeloid and lymphocytic leukemia. The gene coding for the CBFb subunit (CBFB) is also the target of a common chromosomal translocation, inv(16).
The role of CBFb binding for the (dys)function of AML1-ETO, product of the t(8;21) mutation, was recently examined. The introduction of point mutations into the Runt domain in AML1-ETO which abrogates CBFb binding 400-fold results in loss of the ability to immortalize lin- BM cells as well as a loss of leukemogenesis in a mouse model of AML1-ETO leukemia. These results validate this protein-protein interaction as an appropriate target for the development of a small molecule inhibitor which may have therapeutic usefulness for appropriate forms of leukemia.
The purpose of this assay to use HTS to identify inhibitors of the protein-protein interaction between the RUNX1 Runt domain and CBFb. This is accomplished by using a fluorescence resonance energy transfer (FRET) assay. When added together Cerulean-Runt domain and Venus-CBFb will bind and allow for energy transfer. If an inhibitor is present, binding will not occur and the FRET signal will be abated. This assay can be a counterscreen for AID:1434.
1) Cerulean-Runt domain, Venus-CBFb and unlabeled-CBFb was provided by Dr. John Bushweller (University of Virginia Charlottesville, Charlottesville VA).
1) The protein complex (PC); 100nM Cerulean-Runt domain, 100nM Venus-CBFb in 50mM Tris pH7.5, 100mM KCl, 10mM K2SO4, 2mM Mg2SO4, and 0.01% BSA is incubated in the dark at room temperature.
2) After 1 hour 4ul of PC is dispensed into columns 3 through 48 of a black 1536 well plate (Corning #3724)
3) 4ul of positive control (PC + 5uM unlabeled CBFb) is added to the wells in columns 1 and 2.
4) 40nl of 100% DMSO is added to wells in columns 1 through 4, while 40nl of 2mM compounds in 100% DMSO is added to wells in columns 5 through 48. Final compound concentration is 20uM, final DMSO concentration is 1%.
5) The plate is incubated for 1 hr at room temperature protected from light.
6) After 1 hour the plate is read on a BMG Pherastar 422ex, 530em and 480em
7) Raw values are the ratio of 530/480 signals.
Compounds with greater than 50% inhibition at 20 uM concentration are defined as actives of the primary screening.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
** Test Concentration.
Data Table (Concise)