title: The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8.
abstract: SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA strelow_john_mark@lilly.com.
authors.forename: John M
authors.initials: JM
authors.lastname: Strelow
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Xiao
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA.
authors.forename: Rachel N
authors.initials: RN
authors.lastname: Cavitt
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Fite
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Margolis
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA.
authors.forename: Kyu-Jin
authors.initials: KJ
authors.lastname: Park
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism; D000074463:PR-SET Domains / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D006657:Histones; D009707:Nucleosomes; D010455:Peptides; D011495:Histone-Lysine N-Methyltransferase; C470730:KMT5A protein, human; D008239:Lysine; D000241:Adenosine; C006235:sinefungin
keywords: SAM; SETD8; methyltransferase; nucleosomes