Poster Presentation International Peptide Symposium 2023

Ribosomal synthesis of macrocyclic D/L-α/β/γ-hybrid peptide libraries for discovery of IFNGR1 inhibitors (#312)

Takashi Miura 1 , Takayuki Katoh 1 , Hiroaki Suga 1
  1. The University of Tokyo, Tokyo, Japan

An increasing number of bioactive macrocyclic peptides containing nonproteinogenic amino acids, such as D-α-amino acids (DAA), β-amino acids (βAA), and γ-amino acids (γAA), have been reported[1]. DAAs and γAAs are known to increase proteolytic stability of peptides, while βAAs can induce rigid turn/helix structure of peptide and thereby improve binding affinity and specificity to target molecules[2]. We have previously screened bioactive peptides that contain DAAs, βAAs, or γAAs by means of the Random non-standard Peptide Integrated Discovery (RaPID) system[3,4]. Hit peptides containing combinations of these amino acids contributed to the proteolytic stability, binding affinity, and target specificity; however, we have not yet obtained any D/L-α/β/γ-hybrid peptides which contain all of DAAs, βAAs, γAAs, and proteinogenic L-α-amino acids. If every amino acids of them were incorporated into one piece peptide sequence, we can expect to obtain a bioactive peptide with improved proteolytic stability as well as potency. Here we report, for the first time, in vitro selection of D/L-α/β/γ-hybrid peptides against a therapeutic target, interferon gamma receptor 1 (IFNGR1).

We first constructed Library A with a randomized region containing two βAAs, two γAAs, and eleven LAAs which was flanked by N-chloroacetyl-D-tyrosine and D-cysteine for macrocyclization of peptides. Library A was applied to the RaPID selection against IFNGR1, where D/L-α/β/γ-hybrid peptides were selected with IC50 values of up to 67 nM. The selected inhibitors shared an N-terminal conserved motif containing a βAA, which was essential for their activity. To obtain stronger inhibitors, we repeated the selection using Library B with the conserved motif fixed at the N-terminus, resulting in new peptides with a 5.6-fold increase in potency. Mutational scanning revealed that the βAA in the motif was required for the inhibitory activity, while the γAAs and DAAs contributed to the serum stability. Thus, D/L-α/β/γ-hybrid peptides exhibited high inhibitory activity and proteolytic stability.

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  4. T. Miura et al. In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease. Nat. Chem. (2023). https://doi.org/10.1038/s41557-023-01205-1