Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

Cyclic peptide display screening techniques can identify drug leads and biological probes with exceptional affinity and specificity. To date, however, the structural and functional diversity encoded in such peptide libraries remains unexplored. We have used the Random nonstandard Peptide Integrated Discovery (RaPID) system to develop cyclic peptide inhibitors of several acetyllysine-binding bromodomains from the Bromodomain and Extra-Terminal domain (BET) family of epigenetic regulators. These peptides have very high affinities for their targets and exhibit extraordinary selectivity (up to 106-fold), making them the highest-affinity and most specific BET-binding molecules discovered to date. Crystal structures of 13 distinct peptide-bromodomain complexes, which all target the acetyllysine-binding pocket, reveal remarkable diversity in both peptide structure and binding mode, and include both -helical and {beta}-sheet type structures. The peptides can exhibit a high degree of structural pre-organization and bivalent binding of two BDs by one peptide was common, flagging the potential for a new direction in inhibitor design that could bring stronger discrimination between BET-family paralogues. Our data demonstrate for the first time the enormous potential held in these libraries to provide a wide array of modes against a single target, maximizing the opportunity to attain high potency and specificity ligands to a wide variety of proteins.