Systematic Variation of Both the Aromatic Cage and Dialkyllysine via GCE-SAR Reveal Mechanistic Insights in CBX5 Reader Protein Binding

Abstract

Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein–methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure–activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation−π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends.

Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function

“…52 However, binding to an aromatic cage without a cation-p interaction, as proposed for OC9 valine insertion, is precedented by the all-carbon, neutral, non-natural tert-butyl norleucine (t-BuNle) substituted H3K4 derived peptides which retain affinity against Kme3-binding PHD-ngers. 52,53 In such cases, dispersion forces and hydrophobic effects are proposed to drive the aromatic cage engagement, as exemplied for CBX5 chromodomain, where affinity increases with increasing size of alkyl substituent on lysine 3-N. 54 Further characterisation of OC9 binding was carried out from partial 1 H chemical shi assignments derived for bound-OC9 obtained from isotope ltered 2D experiments (Fig. S24B †).…”

Virtual Special Issue: Epigenetics 2022

“…There are also reports of ligand development for non-BET bromodomains including ATAD2, BPTF, and CREBBP . While there has been significant progress in the development of ligands for bromodomains, ligands for methyl-lysine readers have been more challenging, so it is encouraging to see papers focusing on the development of peptido­mimetic ligands for the plant homeo­domain (PHD) of plant homeodomain finger protein 1 (PHF) and for the CBX5 chromodomain …”

“…59 While there has been significant progress in the development of ligands for bromodomains, ligands for methyl-lysine readers have been more challenging, so it is encouraging to see papers focusing on the development of peptidomimetic ligands for the plant homeodomain (PHD) of plant homeodomain finger protein 1 (PHF) 60 and for the CBX5 chromodomain. 61 The lysine/histone deacetylases (K/HDAC) are an established epigenetic therapeutic target. In this collection there are reports of HDAC inhibitors being investigated for the treatment of T-cell prolymphocytic leukemia 62 and the repurposing of human HDAC6 inhibitors to treat multi-drugresistant malaria-causing parasites.…”

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

“…Structurally, they are well suited for capturing extensive protein-protein interactions that lack binding pockets and cover a large surface area. Encouraged by reports of the successful development of peptide-derived inhibitors for CBX proteins, [22][23][24][25][26] we set to target the PHD3 domain using peptide-based modalities.…”