A Simple Two-Step System to Produce a Cyclic Peptide Library for Cell-Based Assays

The pharmaceutical market consists mainly of chemical and biological drugs. These drugs act on different types of targets and have distinct pharmacological properties. Generally, chemical drugs bind to the active site of target enzymes and easily penetrate the cell membrane owing to their small size; however, biological drugs can bind to the protein–protein interaction site but are less stable due to their protein properties. Cyclic peptides possess the pharmacological merits of both chemical and biological drugs, such as the ability to bind to the protein–protein interaction site and penetrate cell membranes. In this study, we developed a simple two-step system to generate a cyclic peptide library using the split intein of Npu DnaE and Gateway cloning. The first step is the PCR of Ready-to-use(R) template DNA having the coding sequences of random cyclic peptides between two split intein elements NpuC and NpuN and the recombination recognition site of Gateway cloning. The second step is the transformation of the PCR products via Gateway cloning to produce colonies with expression vectors to produce cyclic peptides comprising random amino acid sequences. The expression vectors in randomly chosen transformed colonies were confirmed to have random cyclic peptide sequences and all the clones, except ones having a stop codon in the cyclic peptide coding region, showed the expected protein splicing result. This simple two-step system for bacterial expression systems may be modified to suit various expression systems for cell-based assays.

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Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03311-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5500-5509 ◽

Cited By ~ 15

Author(s):

Shaima A. El-Mowafi ◽

John N. Alumasa ◽

Sarah E. Ades ◽

Kenneth C. Keiler

Keyword(s):

Gene Expression ◽

Cyclic Peptides ◽

Fluorescent Protein ◽

Cyclic Peptide ◽

Yellow Fluorescent Protein ◽

Mobility Shift ◽

Content Type ◽

Antibiotic Development ◽

Split Intein ◽

Gel Mobility Shift

ABSTRACTNoncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced inEscherichia coliusing split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similarKivalues. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

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Biosynthetic Strategies for Macrocyclic Peptides

Molecules ◽

10.3390/molecules26113338 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3338

Author(s):

Wei Wang ◽

S. Cyrus Khojasteh ◽

Dian Su

Keyword(s):

Cyclic Peptide ◽

Chemical Space ◽

Combinatorial Libraries ◽

Building Blocks ◽

Peptide Library ◽

In Vitro Translation ◽

Split Intein ◽

Macrocyclic Peptides

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

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A Target-Based Method for Designing Heterochiral Cyclic Peptide Binders: De Novo Inhibitors of the PD-1/PD-L1 Interaction

10.26434/chemrxiv.11663337.v2 ◽

2020 ◽

Author(s):

Salvador Guardiola ◽

Monica Varese ◽

Xavier Roig ◽

Jesús Garcia ◽

Ernest Giralt

Keyword(s):

Protein Interactions ◽

Cyclic Peptides ◽

General Framework ◽

Large Scale ◽

Cyclic Peptide ◽

De Novo ◽

Inhibitory Effect ◽

Original Text ◽

Retraction Notice ◽

Pharmaceutical Properties

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above. ------------------------------------------------------------------------ Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

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Faculty Opinions recommendation of NMR-Based Determination of the 3D Structure of the Ligand-Protein Interaction Site without Protein Resonance Assignment.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726190110.793515339 ◽

2016 ◽

Author(s):

Gottfried Otting

Keyword(s):

Resonance Assignment ◽

Protein Interaction ◽

3D Structure ◽

Interaction Site ◽

Protein Resonance Assignment

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Research Progress on Plant Expression Vectors for Gateway Cloning Technology

Plant Science Journal ◽

10.3724/sp.j.1142.2012.50528 ◽

2012 ◽

Vol 30 (5) ◽

pp. 528

Author(s):

Su-Qin XIAO ◽

Zhen SUN ◽

Xiu-Xia XUAN ◽

Li-Mei CHEN

Keyword(s):

Research Progress ◽

Expression Vectors ◽

Gateway Cloning ◽

Plant Expression ◽

Cloning Technology

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metaPIS: A Sequence-based Meta-server for Protein Interaction Site Prediction

Protein and Peptide Letters ◽

10.2174/0929866511320020015 ◽

2012 ◽

Vol 20 (2) ◽

pp. 218-230

Author(s):

Junfeng Huang ◽

Riqiang Deng ◽

Jinwen Wang ◽

Hongkai Wu ◽

Yuanyan Xiong ◽

...

Keyword(s):

Protein Interaction ◽

Interaction Site ◽

Site Prediction ◽

Interaction Site Prediction

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Improvement on Permeability of Cyclic Peptide/Peptidomimetic: Backbone N-Methylation as A Useful Tool

Marine Drugs ◽

10.3390/md19060311 ◽

2021 ◽

Vol 19 (6) ◽

pp. 311

Author(s):

Yang Li ◽

Wang Li ◽

Zhengshuang Xu

Keyword(s):

Cyclic Peptides ◽

Cyclic Peptide ◽

Three Dimensional ◽

Conformational Space ◽

Cell Permeability ◽

Relative Importance ◽

Intrinsic Properties ◽

Surface Areas ◽

Synthetic Methodologies ◽

Three Dimensional Configuration

Peptides have a three-dimensional configuration that can adopt particular conformations for binding to proteins, which are well suited to interact with larger contact surface areas on target proteins. However, low cell permeability is a major challenge in the development of peptide-related drugs. In recent years, backbone N-methylation has been a useful tool for manipulating the permeability of cyclic peptides/peptidomimetics. Backbone N-methylation permits the adjustment of molecule’s conformational space. Several pathways are involved in the drug absorption pathway; the relative importance of each N-methylation to total permeation is likely to differ with intrinsic properties of cyclic peptide/peptidomimetic. Recent studies on the permeability of cyclic peptides/peptidomimetics using the backbone N-methylation strategy and synthetic methodologies will be presented in this review.

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Novel Cyclic Peptides from Lethal Amanita Mushrooms through a Genome-Guided Approach

Journal of Fungi ◽

10.3390/jof7030204 ◽

2021 ◽

Vol 7 (3) ◽

pp. 204

Author(s):

Shengwen Zhou ◽

Xincan Li ◽

Yunjiao Lüli ◽

Xuan Li ◽

Zuo H. Chen ◽

...

Keyword(s):

Mass Spectrometry ◽

Cyclic Peptides ◽

Cyclic Peptide ◽

Amino Acid Residues ◽

Fragment Ions ◽

Peptide Pair ◽

A Genome ◽

Peptide Toxins ◽

Poisonous Mushrooms ◽

Large Peptide

Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms.

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