Display Selection of Exotic Macrocyclic Peptides Expressed under a Radically Reprogrammed 23 Amino Acid Genetic Code

Abstract

Bioactive naturally occurring macrocyclic peptides often exhibit a strong bias for hydrophobic residues. Recent advances in in vitro display technologies have made possible the identification of potent macrocyclic peptide ligands to protein targets of interest. However, such approaches have so far been restricted to using libraries composed of peptides containing mixtures of hydrophobic and hydrophilic/charged amino acids encoded by the standard genetic code. In the present study, we have demonstrated ribosomal expression of exotic macrocyclic peptides under a radically reprogrammed, relatively hydrophobic, genetic code, comprising 12 proteinogenic and 11 nonproteinogenic amino acids. Screening of this library for affinity to the interleukin-6 receptor (IL6R) as a case study successfully identified exotic macrocyclic peptide ligands with high affinity, validating the feasibility of this approach for the discovery of relatively hydrophobic exotic macrocyclic peptide ligands.

In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides

“…30 We withdrew the natural initiator amino acid Met and its cognate aaRS during the construction of a custom reconstituted in vitro translation system, 31 and supplemented this with initiator tRNA loaded with ClAc-D-Phe, 22 using an artificial ribozyme flexizyme. 32 We then further reprogrammed the genetic code to remove the charged amino acids Glu, Asp, and Arg, and add the following non-canonical amino acids: 22,29,33 three N-methylated: MeGly, MeAla and MePhe; two D-stereochemistry: D-Phe and D-Ala, and the linear hydrophobic side-chain Aoc (Fig. 1A).…”

“…A potential route to such molecules is to screen cyclic peptide libraries which include from the beginning non-canonical amino acids known to confer desirable physical properties. 22,29 However, it is not yet known if cyclic peptides from such a library can succeed at a challenging molecular recognition task like specific Ub chain binding. Here, we constructed and screened a highly nonproteinogenic peptide library to discover de novo cyclic peptides capable of modulating specific Lys48-linked ubiquitin chains.…”

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies

“…Owing to the broad tolerance of flexizyme for tRNAs and amino acids, including NAAs, various NAA-tRNAs can be prepared. The combination of a reconstituted E. coli translation system and NAA-tRNAs prepared by flexizyme is referred to as the flexible in vitro translation (FIT) system, which enables the synthesis of various peptides containing NAAs by means of genetic code reprogramming (Yamagishi et al, 2011;Katoh et al, 2017;Passioura et al, 2018a;Katoh and Suga, 2019).…”

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

“…A lysine derivative with biotin group ( 88 ) was used as an affinity handle. The ncAAs with varying hydrophobicity and properties ( 89-91 ) (Passioura et al, 2018 ) were used to build libraries with expanded diversity for peptide selection (Kawakami et al, 2013b ). A warhead ncAA, ε-N-trifluoroacetyl-Lysine ( 92 ), in peptide sequence was used to achieve selective inhibition of the de-acylation activity of Human Deacetylase SIRT2 (Morimoto et al, 2012 ).…”