uHTS identification of compounds inhibiting the binding between the RUNX1 Runt domain and CBFb-SMMHC via a fluorescence resonance energy transfer (FRET) assay.
The protein-protein interaction between the subunits of the heterodimeric transcription factor CBF, core binding factor beta (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: 1621
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 beta (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-SMMHC, a potential therapeutic approach for inv(16) related leukemia. This is accomplished by using a fluorescence resonance energy transfer (FRET) assay. When added together Cerulean-Runt domain and Venus-CBFb-SMMHC will bind and allow for energy transfer. If an inhibitor is present, binding will not occur and the FRET signal will be abated.
1) Cerulean-Runt domain and Venus-CBFb-SMMHC was provided by Dr. John Bushweller (University of Virginia Charlottesville, Charlottesville VA).
2) Fluorescent CBFa/b complex working solution: 50 nM Cerulean-Runt (CBFa) domain, 50 nM Venus-CBFb-SMMHC in 62.5 mM Tris-HCl pH 7.4, containing 125 mM KCl, 12.5 mM K2SO4, 2.5 mM Mg2SO4, and 0.0125% BSA is incubated in the dark at room temperature for 1h.
3) Positive control (PC) solution contained untagged CBFb-SMMHC added at 1.25 uM concentration to the CBFa/b working solution
1) 4ul of CBFa/b working solution is dispensed into columns 3 through 48 of a black 1536-well plate (Corning #3724)
2) 4ul of positive control is added to the wells in columns 1 and 2.
3) 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%.
4) The plate is incubated for 1 hr at room temperature protected from light.
5) After 1 hour the plate is read on a BMG Pherastar 422ex, 530em and 480em
6) The FRET signal is determined as the ratio of 530/480 signals.
Dose-response confirmation protocol:
1) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were 2-fold serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration.
2) 4 uL compounds in 10% DMSO were transferred into columns 3-22. Columns 1-2 and 23-24 contained 4 uL of 10% DMSO.
3) 16 ul of CBFa/b working solution is dispensed into columns 3 through 24 of a black 384-well plate (Greiner #784076)
4) 16 ul of positive control is added to columns 1 and 2.
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) The FRET signal is determined as the ratio of 530/480 signals.
Compounds with greater than 50% inhibition at 20 uM concentration are defined as actives of the primary screening. The primary screening actives are then cherry picked and retested in duplicated at 20 uM in the primary assay. Compounds with an average of >= 50% inhibition in the reconfirmation assay are considered active. These compounds are then tested in an alternative assay that employs TR-FRET as the readout. Compounds that are active in the TR-FRET assay then proceed to the dose-response confirmation stage in both assays.
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
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound#s potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the RunX assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
A score of 44 is given to active compounds selected from plates:
a) That do not have a Hill coefficient associated with them and have a qualifier of < or >.
b) The value of + 6*(pEC50/IC50-3)*QC, is < 0.500
Active compounds will have a score >= 44.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)