title: Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology.
abstract: Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z'-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats.
authors.affiliation: High-Throughput Screening and Drug Discovery Lab, Nemours Center for Childhood Cancer Research, A.I. duPont Hospital for Children, Wilmington, Delaware, USA.
authors.forename: Venita Gresham
authors.initials: VG
authors.lastname: Watson
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Drake
authors.forename: Yu
authors.initials: Y
authors.lastname: Peng
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Napper
mesh_terms: D004268:DNA-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors*; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D051788:Myeloid-Lymphoid Leukemia Protein / Q000032:analysis / Q000037:antagonists & inhibitors*; D009687:Nuclear Proteins / Q000032:analysis / Q000037:antagonists & inhibitors*; D015514:Oncogene Proteins, Fusion / Q000032:analysis / Q000037:antagonists & inhibitors*; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping / Q000379:methods*; D035602:Transcriptional Elongation Factors
chemical_list: D004268:DNA-Binding Proteins; C474558:MLL-AF9 fusion protein, human; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D035602:Transcriptional Elongation Factors; D051788:Myeloid-Lymphoid Leukemia Protein; C081449:AFF1 protein, human