Supplementary Figure S1 from Ribosomal incorporation of negatively charged d-α- and <i>N</i>-methyl-l-α-amino acids enhanced by EF-Sep

Tricine SDS-PAGE analysis of model peptides P1 and P2

Supplementary Table S1 from Ribosomal incorporation of negatively charged d-α- and <i>N</i>-methyl-l-α-amino acids enhanced by EF-Sep

List of RNAs and the corresponding primers

Ribosomal Synthesis of an Amphotericin‑B Inspired Macrocycle

Here we report in vitro ribosomal synthesis of a natural product-like macrocyclic peptide, inspired by the structure of amphotericin B (AmB), an amphiphilic and membrane-interacting antifungal natural product. This AmB-inspired macrocyclic peptide (AmP), one side of which is composed of hydrophobic terpene, and the other side comprises a peptidic chain, was synthesized utilizing flexizyme-assisted in vitro translation via an unusual but successful initiation with a d-cysteine derivative. The established method for the synthesis of AmPs is applicable to the generation of a diverse AmP library coupled with an in vitro display format, with the potential to lead to the discovery of artificial bioactive amphiphilic macrocycles

<i>In Vitro</i> Selection of Anti-Akt2 Thioether-Macrocyclic Peptides Leading to Isoform-Selective Inhibitors

The Akt kinase family, consisting of three isoforms in humans, is a well-validated class of drug target. Through various screening campaigns in academics and pharmaceutical industries, several promising inhibitors have been developed to date. However, due to the mechanistic and structural similarities of Akt kinases, it is yet a challenging task to discover selective inhibitors against a specific Akt isoform. We here report Akt-selective and also Akt2 isoform-selective inhibitors based on a thioether-macrocyclic peptide scaffold. Several anti-Akt2 peptides have been selected from a library by means of an <i>in vitro</i> display system, referred to as the RaPID (Random nonstandard Peptide Integrated Discovery) system. Remarkably, the majority of these “binding-active” anti-Akt2 peptides turned out to be “inhibitory active”, exhibiting IC₅₀ values of approximately 100 nM. Moreover, these peptides are not only selective to the Akt kinase family but also isoform-selective to Akt2. Particularly, one referred to as Pakti-L1 is able to discriminate Akt2 250- and 40-fold over Akt1 and Akt3, respectively. This proof-of-concept case study suggests that the RaPID system has a tremendous potential for the discovery of unique inhibitors with high family- and isoform-selectivity

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

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

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

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

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

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

Reevaluation of the d‑Amino Acid Compatibility with the Elongation Event in Translation

The compatibility of d-amino acids with peptide elongation during translation has been examined in several studies. However, some of the studies have reported that d-amino acids are incompatible with translation, whereas others have reported that d-amino acids are incorporated into polypeptides. Here, we have reevaluated the incorporation of a series of d-amino acids into the nascent chain of short peptides with a reprogrammed genetic code by using the flexible in vitro translation (FIT) system. The FIT system enables the compatibility of each d-amino acid with elongation to be assessed quantitatively in the absence of potential competitors. The incorporation efficiencies were determined by Tricine-SDS-PAGE and the full-length peptide was detected by MALDI-TOF-MS. The d-amino acids were categorized into three groups based on their incorporation efficiencies relative to the corresponding l-amino acid. The d-isomers in group I showed efficiencies of 40% or higher (Ala, Ser, Cys, Met, Thr, His, Phe, and Tyr), and those in group II showed efficiencies of 10–40% (Asn, Gln, Val, and Leu). The d-amino acids in group III produced truncated peptides or no detectable full-length peptides (Arg, Lys, Asp, Glu, Ile, Trp, and Pro). When group I d-amino acids were used consecutively or were alternated with l-amino acids, this completely inhibited their elongation. However, when two or three l-amino acids were inserted between the d-amino acids, the double-incorporation efficiency was restored. Our results quantitatively reveal the compatibility of d-amino acids with peptide elongation and raise new questions about the mechanism of d-amino acid selection and incorporation by the ribosome

Ribosomal Synthesis of Peptides with Multiple β‑Amino Acids

The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple β-amino acids. The experiments of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, l-βhAla, l-βhGln, l-βhPhg, l-βhMet, and d-βhPhg) showed high incorporation efficiencies, and seven (l-βhLeu, l-βhIle, l-βhAsn, l-βhPhe, l-βhLys, d-βhAla, and d-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (l-βhPro, l-βhTrp, and l-βhGlu). Subsequent double-incorporation experiments using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of β-amino acids at multiple positions

Nonstandard Peptide Expression under the Genetic Code Consisting of Reprogrammed Dual Sense Codons

We here demonstrate a translation system that is governed by a reprogrammed genetic code consisting of “dual sense codons.” A dual sense codon assigns two distinct amino acids for initiation and elongation. Because multiple dual sense codons independently function without cross-readings, this system enables the expansion of the repertoire of initiators as well as elongators that can be used simultaneously