Targeting of the bromodomain and extra-terminal domain (BET) subfamily of acetyllysine-binding bromodomains shows considerable therapeutic promise in the treatment of a range of diseases, including several cancers. Despite extensive medicinal chemistry efforts, identification of selective inhibitors of these reader proteins has proved elusive owing to the close structural conservation both within the BET family and amongst the wider family of bromodomain-containing proteins. With their larger size and functionality, we hypothesised that cyclic peptides could provide an alternative strategy to selectively inhibit these proteins.
DNA-encoded library technologies provide ideal platforms for identifying such peptides. Here, we employed the RaPID (Random non-standard Peptide Integrated Discovery) system, to identify cyclic peptide binders, using a tailored library allowing incorporation of an acetyllysine warhead. We identified a range of high-affinity BET binders with improved potency and selectivity over previous compounds. Structural studies by NMR and X-ray crystallography, reveal that whilst the peptides bind in the classical acetyl-lysine binding pocket, they also contact a significantly larger surface than previously identified inhibitors, likely contributing to this selectivity and allowing scope for further improvements in specificity. Solution structures demonstrate that some peptides adopt a high degree of structural preorganisation, likely contributing to the high affinities observed. The structures also reveal significant structural diversity amongst the peptide binding motifs, highlighting the wide range of structures present in the initial libraries. Overall we envisage that these peptides will provide a starting point for the development of next-generation BET bromodomain inhibitors.