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Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies.

ABSTRACT: Backbone macrocyclic structures are often found in diverse bioactive peptides and contribute to greater conformational rigidity, peptidase resistance, and potential membrane permeability compared to their linear counterparts. Therefore, such peptide scaffolds are an attractive platform for drug-discovery endeavors. Recent advances in synthetic methods for backbone macrocyclic peptides have enabled the discovery of novel peptide drug candidates against diverse targets. Here, we overview recent technical advancements in the synthetic methods including 1) enzymatic synthesis, 2) chemical synthesis, 3) split-intein circular ligation of peptides and proteins (SICLOPPS), and 4) in vitro translation system combined with genetic code reprogramming. We also discuss screening methodologies compatible with those synthetic methodologies, such as one-beads one-compound (OBOC) screening compatible with the synthetic method 2, cell-based assay compatible with 3, limiting-dilution PCR and mRNA display compatible with 4.

SUBMITTER: Shinbara K

PROVIDER: S-EPMC7314982 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies.

Shinbara Koki K Liu Wenyu W van Neer Renier Herman Pieter RHP Katoh Takayuki T Suga Hiroaki H

Frontiers in chemistry 20200618

Backbone macrocyclic structures are often found in diverse bioactive peptides and contribute to greater conformational rigidity, peptidase resistance, and potential membrane permeability compared to their linear counterparts. Therefore, such peptide scaffolds are an attractive platform for drug-discovery endeavors. Recent advances in synthetic methods for backbone macrocyclic peptides have enabled the discovery of novel peptide drug candidates against diverse targets. Here, we overview recent te ...[more]

PMID: 32626683

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Project description:Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

Project description:RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise high throughput, cost efficient short-read RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward long-read sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5' and 3' UTRs is still in development.

Dataset Information

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies.

Publications

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies.

Similar Datasets

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