Ribosomal synthesis of macrocyclic D/L-α/β/γ-hybrid peptide libraries for discovery of IFNGR1 inhibitors — ASN Events
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  3. Ribosomal synthesis of macrocyclic D/L-α/β/γ-hybrid peptide libraries for discovery of IFNGR1 inhibitors

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.

  1. A. Vinogradov, Y. Yin, and H. Suga, Macrocyclic Peptides as Drug Candidates: Recent Progress and Remaining Challenges. J. Am. Chem. Soc. 141, 4167 (2019).
  2. K. Tsuchiya, T. Kurohara, K. Fukuhara, T. Misawa, and Y. Demizu, Helical Foldamers and Stapled Peptides as New Modalities in Drug Discovery: Modulators of Protein-Protein Interactions. Processes 10, 924 (2022).
  3. Y. Goto and H. Suga, The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides. Acc. Chem. Res. 54, 3604 (2021).
  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
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