DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related Bromodomain and ExtraTerminal domain
(BET) family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide
complexes reveal diverse structures and binding modes, which nevertheless display several
conserved features. Some peptides demonstrate significant paralogue-level specificity, though
the physicochemical explanations for this specificity are often not clear. Our data demonstrate
the power of cyclic peptides to discriminate between very similar proteins with high potency
and hint that differences in conformational dynamics might modulate the affinity of these
domains for particular ligands.