TY - JOUR

T1 - Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy

AU - Huang, Yichao

AU - Nawatha, Mickal

AU - Livneh, Ido

AU - Rogers, Joseph M.

AU - Sun, Hao

AU - Singh, Sumeet K.

AU - Ciechanover, Aaron

AU - Brik, Ashraf

AU - Suga, Hiroaki

PY - 2020/6/26

Y1 - 2020/6/26

N2 - 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. Ideal macrocyclic peptides should have dissociation constants (KD) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3–1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides 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.

AB - 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. Ideal macrocyclic peptides should have dissociation constants (KD) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3–1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides 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.

KW - diubiquitin

KW - macrocyclic peptides

KW - messenger RNA display

KW - proteasome

KW - RaPID system

UR - http://www.scopus.com/inward/record.url?scp=85086170499&partnerID=8YFLogxK

U2 - 10.1002/chem.202000273

DO - 10.1002/chem.202000273

M3 - Journal article

C2 - 32105365

AN - SCOPUS:85086170499

VL - 26

SP - 8022

EP - 8027

JO - Chemistry: A European Journal

JF - Chemistry: A European Journal

SN - 0947-6539

IS - 36

ER -