A range of isoxazole-containing amino acids has been synthesized, which displace acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues <i>in situ</i>. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of human histone H3. The complementary approaches that we report, which enable incorporation of effective and stable KAc mimics in a site-selective manner, will allow precise activation of KAc-mediated protein-protein interactions, facilitating study of their downstream effects in an unprecedented manner. We envisage that this approach could be of high value in developing peptide ligands for bromodomains with no known binding partners, underpinning epigenetic probe- and drug discovery.

Isoxazole-derived amino acids are bromodomain-binding acetyl-lysine mimics when incorporated in histone H4 peptides and histone H3

252nd National Meeting (2016)

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