title: Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1.
abstract: The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602, USA. yzheng@uga.edu.
authors.forename: Yepeng
authors.initials: Y
authors.lastname: Luan
authors.forename: Levi L
authors.initials: LL
authors.lastname: Blazer
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.forename: Taraneh
authors.initials: T
authors.lastname: Hajian
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Scott
authors.initials: S
authors.lastname: Houliston
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.forename: Masoud
authors.initials: M
authors.lastname: Vedadi
authors.forename: Yujun George
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005453:Fluorescence; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D051788:Myeloid-Lymphoid Leukemia Protein / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012436:S-Adenosylmethionine / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; C497422:KMT2A protein, human; D008024:Ligands; D054852:Small Molecule Libraries; D051788:Myeloid-Lymphoid Leukemia Protein; D012436:S-Adenosylmethionine; D011495:Histone-Lysine N-Methyltransferase