Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19 Public Deposited

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  • March 19, 2019
  • Perfetti, Michael
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Improving the 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 the regulation of DNA integrity and gene expression. Developing chemical tools for histone binding proteins that possess cellular activity will allow for further elucidation of the specific function of this class of histone regulating proteins. This research specifically targeted two different classes of Tudor domain containing histone binding proteins that are directly involved in the DNA damage response and modulation of gene transcription activities. The first methyl-lysine binding protein targeted was 53BP1, which is a DNA damage response protein. 53BP1 uses a tandem tudor domain (TTD) to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a post-translational modification (PTM) induced by double-strand DNA breaks. Through a cross-screening approach, two different small molecule ligands were identified for the 53BP1 TTD. Medicinal chemistry and structure-based design techniques were used, in addition to the development of a fragment-based screen, toward the goal of optimizing the potency and selectivity of the identified small molecule ligands. The later small molecule ligand, 61 (UNC2170), was further optimized as a micromolar inhibitor of 53BP1, which demonstrated a 17-fold selectivity for 53BP1 TTD as compared to other screened methyl-lysine (Kme) binding proteins. Structural studies revealed that the N-tert-butyl amine of 61 (UNC2170) anchors the compound in the Kme binding pocket of 53BP1 TTD, making it competitive with endogenous Kme substrates. X-ray crystallography demonstrated that 61 (UNC2170) bound at the interface between two tudor domains of a 53BP1 TTD dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process that requires a functional 53BP1 tudor domain. These results demonstrate that 61 (UNC2170) is a functionally active, fragment-like ligand for 53BP1 and the first selective small molecule ligand discovered for 53BP1 TTD. Additional research efforts were focused toward development of peptide-based ligands for the histone binding proteins PHF1 and PHF19. These proteins play an active role in the modulation of PRC2 activity that in turn controls various gene expression and repression outcomes. Structure-based design techniques were used to design peptide-based inhibitors for PHF1 and PHF19. Research efforts were focused on conducting an initial Structure Activity Relationship (SAR) study to develop a short, low molecular weight peptide via truncation of the endogenous histone 3 peptide. Structure based design techniques were used to develop a shorter 6- or 7-mer peptide that possessed various amino acid substitutions that specifically bound within the Tudor domain of PHF1 and PHF19 with emphasis on determining a quaternary amine mimic for the tri-methyl lysine 36 residue. These efforts proved fruitful and a 7-mer peptide (148) was developed that was equipotent to the endogenous histone peptide. Research efforts on both of these targeted proteins demonstrates that structure based design and medicinal chemistry techniques can successfully facilitate the development of small molecule and peptide based ligands for histone binding proteins.
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  • In Copyright
  • Strahl, Brian
  • Lawrence, David
  • Wang, Greg
  • Bowers, Albert
  • Frye, Stephen
  • Lee, Andrew
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2015
Place of publication
  • Chapel Hill, NC
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