Flexizymes-facilitated Genetic Code Reprogramming Leading to the Discovery of Drug-like Peptides

Abstract

Flexizymes are a family of aminoacylation ribozymes devised by in vitro selection in our research group. They charge a wide variety of nonproteinogenic amino acids onto virtually any kind of tRNAs, enabling us to reprogram the genetic code in a custom-made cell-free translation system. This genetic code reprogramming method was integrated with a mRNA display method, which not only expresses nonstandard peptides such as macrocyclic peptides but also selects ligands specifically binding to target proteins. This system is referred to as random nonstandard peptide integrated discovery (RaPID) system, which has yielded inhibitors against disease-related target proteins. In this review, we summarize the evolutionary history and recent applications of the flexizymes and RaPID system.

Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

“…As discussed above, the methodologies of ncAA incorporations into polypeptides have given various applications in chemical biology. Non-standard macrocyclic peptides are useful scaffolds for developing drug leads and co-crystallization molecules against protein targets [ 81 , 97 , 98 ]. The mRNA-based selection system integrated with the methodology of genetic code reprogramming, such as the RaPID system [ 99 ], is an ideal platform technology to achieve such goals.…”

“…In addition, this methodology can expand the repertoire of initiators and structural diversity of peptides simultaneously synthesized in one translation mixture. Thus, such an expression system can be applied for the discovery of bioactive non-standard peptides using mRNA-encoded non-standard peptide libraries [ 81 ]. Indeed, a random cyclic peptide library containing d Y and K tf designated by a dual sense AUG codon was constructed by a FIT system, and cyclic peptides armed with a mechanism-based warhead (K tf ) that selectively inhibit NAD (nicotinamide adenine dinuclotide)-dependent deacetylase sirtuin-2 were successfully developed [ 82 ].…”

Ribosome-mediated synthesis of natural product-like peptides via cell-free translation

“…In this work, a custom-made reconstituted cell-free translation system called Flexible In Vitro Translation (FIT) system [3] was used. Central to the FIT system is a ribozyme-mediated aminoacylation of tRNAs (called flexizyme technology) [20,21] and genetic code reprogramming [22,23] for the incorporation of non-canonical amino acids [24].…”

A Post-translational Cyclodehydratase, PatD, Tolerates Sequence Variation in the C-terminal Region of Substrate Peptides

“…We previously devised an in vitro reconstituted biosynthetic system for artificial Az-peptides by combining a custom-made cell-free translation (Flexible In-vitro Translation; FIT) system 8,9 with a post-translational cyclodehydratase PatD. 10 In this system, referred to as the FIT-PatD system, 11 precursor peptides composed of an N-terminal leader peptide (LP) region, upstream recognition sequence, (uRS), core sequence (CS), and downstream recognition sequence (dRS) 1214 are ribosomally synthesized from the corresponding synthetic DNA templates.…”