RaPID Selection of α-Hydrazino Acid-Containing Macrocyclic Peptides

Incorporation of α-hydrazino acid (α-Hza) into peptide chain can stabilize the secondary structures, such as helices and turns, and give rise to rigidification of peptide conformation. Consequently, such peptides potentially acquire enhanced binding affinity to target proteins. Moreover, the hydrazidic bond in α-Hza-containing peptides contributes to proteolytic resistance. Despite such favorable characteristics for therapeutic peptides, there is no example of success in screening de novo α-Hza-containing peptides against target proteins of interest. Here we report the construction of diverse, mRNA-encoded α-Hza-containing macrocyclic peptide libraries and their application to the Random nonstandard Peptides Integrated Discovery (RaPID) selection against two enzyme targets, Janus kinase 2 (JAK2) and human factor XIIa (FXIIa). The affinity-based enrichment of ligands from these libraries yielded potent binders with low-to-sub-nM dissociation constants, also exhibiting potent inhibitory activity, target specificity, proteolytic stability, and membrane permeability. Mutational studies of active macrocycles underscored critical roles of α-Hza residues in functional potencies. This study establishes a platform for de novo discovery of bioactive α-Hza-containing macrocyclic peptides against proteins of choice, thereby expanding the accessible chemical space for the RaPID system.