Tyrosinase-Catalyzed Peptide Macrocyclization for mRNA Display

Abstract

mRNA display of macrocyclic peptides has proven itself to be a powerful technique to discover high-affinity ligands for a protein target. However, only a limited number of cyclization chemistries are known to be compatible with mRNA display. Tyrosinase is a copper-dependent oxidase that oxidizes tyrosine phenol to an electrophilic o-quinone, which is readily attacked by cysteine thiol. Here we show that peptides containing tyrosine and cysteine are rapidly cyclized upon tyrosinase treatment. Characterization of the cyclization reveals it to be widely applicable to multiple macrocycle sizes and scaffolds. We combine tyrosinase-mediated cyclization with mRNA display to discover new macrocyclic ligands targeting melanoma-associated antigen A4 (MAGE-A4). These macrocycles potently inhibit the MAGE-A4 binding axis with nanomolar IC 50 values. Importantly, macrocyclic ligands show clear advantage over noncyclized analogues with ∼40-fold or greater decrease in IC 50 values.

Late-stage reshaping of phage-displayed libraries to macrocyclic and bicyclic landscapes using multipurpose linchpin

“…Cyclization of peptides also can minimize proteolytic liability, 3,4 and decrease conformational flexibility leading to stronger association with cognate targets. 5 Genetically-encoded (GE) libraries displayed on phage 6,7 , mRNA 8,9 , bacteria [10][11][12] and yeast 13 as well as DNA-encoded libraries (DEL) of macrocyclic topology 14,15 offer uniquely advantages in discovery of macrocyclic peptides because they associate the unique DNA message with structure of each individual macrocycle. The genotype-phenotype connection allows amplification from a single copy number, repetitive rounds of selection or maturation, and makes it possible to start discovery campaigns from libraries of 10 6 -10 12 scale.…”