In Vitro Selection of Macrocyclic α/β³-Peptides against Human EGFR

Abstract

Here, we report ribosomal construction of thioether-macrocyclic α/β3-peptide libraries in which β-homoglycine, β-homoalanine, β-homophenylglycine, and β-homoglutamine are introduced by genetic code reprogramming. The libraries were applied to the RaPID (Random nonstandard Peptides Integrated Discovery) selection against human EGFR to obtain PPI (protein–protein interaction) inhibitors. The resulting peptides contained up to five β3-amino acid (β3AA) residues and exhibited outstanding binding affinity, PPI inhibitory activity, and proteolytic stability, which were attributed to the β3AAs included in the peptides. This showcase work has demonstrated that the use of such β3AAs enhances the drug-like properties of peptides, providing a unique platform for the discovery of de novo macrocycles against a protein of interest.

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

“…However, tRNA AsnE2 and tRNA GluE2 may be required in the few circumstances that EF-P decreases translation efficiency. Regardless, tRNA Pro1E2 has been used for the incorporation of β 3 -AAs, ,, β 2 -AAs, ,, β 2,3 -amino acids, and cyclic β 2,3 -AAs (Figure ). ,,, …”

“…Wakabayashi and co-workers constructed a RaPID library using tRNA Pro1E2 with β 3 -Phg, β 3 -Ala, β 3 -Gly, and β 3 -Gln at the codons AUU, ACU, GCU, and CAU, respectively . This library was screened against hEGFR with discovered teMPs containing up to 35% β 3 -AAs per sequence.…”

“…24,295,583,606 Wakabayashi and co-workers constructed a RaPID library using tRNA Pro1E2 with β 3 -Phg, β 3 -Ala, β 3 -Gly, and β 3 -Gln at the codons AUU, ACU, GCU, and CAU, respectively. 604 This library was screened against hEGFR with discovered teMPs containing up to 35% β 3 -AAs per sequence. L2β contained three β 3 -Phg residues and bound to hEGFR with a K D of 47.0 nM and IC 50 of 66.8 nM, while D3β had one of each of β 3 -Phg, β 3 -Ala, and β 3 -Gln and possessed a K D of 34.1 nM and an IC 50 of 20.3 nM.…”

In Vitro Selection of Macrocyclic l-α/d-α/β/γ-Hybrid Peptides Targeting IFN-γ/IFNGR1 Protein–Protein Interaction

“…33,34 The FIT system allows the construction of random peptide libraries, comprising over 10 12 unique members, applicable to an in vitro selection methodology, referred to as the Random nonstandard Peptides Integrated Discovery (RaPID) system, based on the mRNA display method. 30,35 We constructed macrocyclic peptide libraries containing combinations of L-α-, D-α-, β-, and γ-amino acids, such as [L-αand D-α-amino acids], 10 [L-α-and β-amino acids], 36 and [L-α-, D-α-, and β-amino acids], 24,36−38 which were then applied to the RaPID system against various targets. The selection results revealed the contribution of D-α-and βamino acids to the proteolytic stability of peptide inhibitors and the crucial role of cβAA residues in inhibitory activity by folding peptides into rigid β-sheets.…”

“…Taking advantage of the remarkable properties of these nonproteinogenic amino acids, high-throughput screening of nonstandard peptides from ribosomally synthesized libraries, utilizing genetic code reprogramming, has been conducted for discovering novel bioactive peptides. − To ribosomally synthesize nonstandard peptides, we have developed the flexible in vitro translation (FIT) system which comprises the reconstitutedEscherichia colitranslation system with precharged nonproteinogenic aminoacyl-tRNA prepared by the flexizymes. , The FIT system allows the construction of random peptide libraries, comprising over 10 12 unique members, applicable to an in vitro selection methodology, referred to as the Random nonstandard Peptides Integrated Discovery (RaPID) system, based on the mRNA display method. , We constructed macrocyclic peptide libraries containing combinations of l -α-, d -α-, β-, and γ-amino acids, such as [ l -α- and d -α-amino acids], [ l -α- and β-amino acids], and [ l -α-, d -α-, and β-amino acids], ,− which were then applied to the RaPID system against various targets. The selection results revealed the contribution of d -α- and β-amino acids to the proteolytic stability of peptide inhibitors and the crucial role of cβAA residues in inhibitory activity by folding peptides into rigid β-sheets.…”