title: Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1.
abstract: Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Perfetti
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Brandi M
authors.initials: BM
authors.lastname: Baughman
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Dickson
authors.affiliation: ‡Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, United States.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Mu
authors.affiliation: §Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Gaofeng
authors.initials: G
authors.lastname: Cui
authors.affiliation: ∥Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada, M5G 1L7.
authors.forename: Pavel
authors.initials: P
authors.lastname: Mader
authors.affiliation: ∥Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada, M5G 1L7.
authors.forename: Aiping
authors.initials: A
authors.lastname: Dong
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Jacqueline L
authors.initials: JL
authors.lastname: Norris
authors.affiliation: ⊥Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Rothbart
authors.affiliation: ⊥Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Strahl
authors.affiliation: ∥Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada, M5G 1L7.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Brown
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: William P
authors.initials: WP
authors.lastname: Janzen
authors.affiliation: ∥Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada, M5G 1L7.
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.affiliation: §Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Georges
authors.initials: G
authors.lastname: Mer
authors.affiliation: ‡Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, United States.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: McBride
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Lindsey I
authors.initials: LI
authors.lastname: James
authors.affiliation: †Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Frye
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism; D018360:Crystallography, X-Ray; D003959:Diamines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D057809:HEK293 Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008810:Mice, Inbred C57BL; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: C000607179:3-bromo-N-(3-(tert-butylamino)propyl)benzamide; D001549:Benzamides; D002843:Chromatin; D003959:Diamines; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D054852:Small Molecule Libraries; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D008239:Lysine