Ribosomal production and in vitro selection of natural product-like peptidomimetics: The FIT and RaPID systems
ABSTRACT 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.
- SourceAvailable from: Hiroshi Murakami[Show abstract] [Hide abstract]
ABSTRACT: The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.Journal of nucleic acids 10/2012; 2012:713510. DOI:10.1155/2012/713510
Article: Polycyclic Peptide Therapeutics[Show abstract] [Hide abstract]
ABSTRACT: Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases.ChemMedChem 03/2013; 8(3). DOI:10.1002/cmdc.201200513 · 3.05 Impact Factor
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ABSTRACT: In the past decade, in vitro transcription/translation technologies have emerged as discovery tools for screening large protein expression libraries, for the selection of engineered polypeptide libraries, and as alternatives to conventional heterologous expression for protein production. Therapeutic proteins and peptides discovered using ribosome-based display methods that link genetic information to the encoded polypeptide generated by cell-free extracts, or purified translation components, are beginning to move forward into human clinical trials. This review details the significant progress in in vitro translation for novel protein and non-natural amino acid containing peptide discovery platforms, as well as advances in the clinical-scale production of therapeutic proteins using cell-free transcription/translation.Current opinion in chemical biology 03/2013; 17(3). DOI:10.1016/j.cbpa.2013.02.014 · 7.65 Impact Factor