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 ..
BioActive Compounds: 110
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.
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 buffer: 50 mM TRIS-HCl pH 7.5, 50 mM NaCl, 1 mM DTT. Add 0.05% BSA prior to reaction setup.
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.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)