Ribosomal production and in vitro selection of natural product-like peptidomimetics: The FIT and RaPID systems

Bioactive natural product peptides have diverse architectures such as non-standard sidechains and a macrocyclic backbone bearing modifications. In vitro translation of peptides bearing these features would provide the research community with a diverse collection of natural product peptide-like molecules with a potential for drug development. The ordinary in vitro translation system, however, is not amenable to the incorporation of non-proteinogenic amino acids or genetic encoding of macrocyclic backbones. To circumvent this problem, flexible tRNA-acylation ribozymes (flexizymes) were combined with a custom-made reconstituted translation system to produce the flexible in vitro translation (FIT) system. The FIT system was integrated with mRNA display to devise an in vitro selection technique, referred to as the random non-standard peptide integrated discovery (RaPID) system. It has recently yielded an N-methylated macrocyclic peptide having high affinity (Kd = 0.60 nM) for its target protein, E6AP.

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► Flexizymes are used to produce tRNA charged with non-standard amino acids.
► Flexizymes were integrated with genetic code reprogramming to create the FIT system.
► The FIT system was integrated with mRNA display to create the RaPID system.
► E6AP-binding non-standard cyclic peptides were selected from a random library.
► Selected peptides possess a d-amino acid and an N-methylated cyclic backbone.