Affinity Maturation of Macrocyclic Peptide Modulators of Lys48‐linked Diubiquitin by a Twofold Strategy

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. The desirable potencies of such macrocyclic peptides are to have dissociation constants (KD) as low as single-digit nM against a specific target of interest. Here, we describe a twofold strategy to discover optimized macrocyclic peptides within this affinity regime. First, we explored benzyl-thioether cyclized peptide libraries to identify tight binding hits. To obtain more insights into critical sequence information, we applied sequence alignment to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, we successfully obtained benzyl-thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2), displaying 0.3-1.2 nM KD values that represent two-orders of magnitude stronger binding than the macrocyclic peptide recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.