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

Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
Current opinion in chemical biology (Impact Factor: 6.81). 03/2012; 16(1-2):196-203. DOI: 10.1016/j.cbpa.2012.02.014
Source: PubMed


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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    • "In 2011, Suga's group published an important study in which highly diverse non-standard peptide libraries containing multiple different nonproteinogenic amino acids were constructed in an mRNA display format, and a novel non-standard peptide was selected from the libraries [5, 123]. Previously, the same group used the flexizyme system to examine the synthesis of various non-standard peptides in a ribosomal translation system [124] and reported the following findings: (1) an N-chloroacetyl-amino acid residue and cysteine residue on the same peptide were spontaneously reacted in situ using a reconstituted translation system to give a thioether-cyclized peptide without an intermolecular side reaction between the N-terminal chloroacetyl group on the peptide and the sulfhydryl group of the other translation component such as a cysteine monomer and DTT [3, 108, 112, 113, 115, 125]; (2) N-methyl amino acids with an aromatic side chain or noncharged and nonbulky side chains are efficiently incorporated into peptides by the ribosome, and multiple N-methyl amino acids can be incorporated simultaneously into a peptide to give various sequences of thioether-cyclized N-methyl-peptides [112]; and (3) the translation initiation apparatus accepts D-amino acids with hydrophobic side chain as relatively good initiators, and pre-N-acylation of D-aa-tRNA dramatically increases the efficiency of translation initiation [116]. "
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