Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides <i>via</i> mRNA Display

Hacker, David E.; Hoinka, Jan; Iqbal, Emil S.; Przytycka, Teresa M.; Hartman, Matthew C. T.

doi:10.1021/acschembio.6b01006

Cited by 61 publications

(75 citation statements)

References 81 publications

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“…There are several reports of mRNA display that involve modification by posttranslational treatment including cyclization mediated by bis-NHS crosslinking, bis-alkylation, lanthionine formation, CuAAC, or disulfide bond(s) assisted by protein disulfide isomerase, or conjugation of a small molecules (Fiacco et al, 2016; Guillen Schlippe et al, 2012; Hacker, Hoinka, Iqbal, Przytycka, & Hartman, 2017; Hofmann et al, 2012; Li & Roberts, 2003; Millward, Fiacco, Austin, & Roberts, 2007; Yamaguchi et al, 2009). The present method is similar to those, with the striking differences that our library modifications (oligosaccharides) are much larger and are derived from precious azide-modified sugar reagents synthesized in-house.…”

Section: Discussion Summary and Conclusionmentioning

confidence: 99%

Directed Evolution of Glycopeptides Using mRNA Display

Horiya

Bailey

Krauss

2017

Methods in Enzymology

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Directed evolution is a useful method for the discovery of nucleic acids, peptides or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogenous mixture of glycoforms at each site on the protein. Although yeast, mammalian and Escherichia coli cells can be engineered to produce a homogenous glycoform at all glycosylation sites, there are just a few specific glycan structures that can readily be accessed in this manner. Recently, we reported a novel system for the directed evolution of glycopeptide libraries, which could in principle be decorated with any desired glycan. Our method combines in vitro peptide selection by mRNA display with unnatural amino acid incorporation and chemical attachment of synthetic oligosaccharides. Here, we provide an updated and optimized protocol for this method, which is designed to create glycopeptide mRNA display libraries containing ~1013 sequences and select them for target binding. The target described here is the HIV broadly-neutralizing monoclonal antibody 2G12; 2G12 binds to cluster of high-mannose oligosaccharides on the HIV envelope glycoprotein gp120 and glycopeptides that mimic this epitope may be useful in HIV vaccine applications. This method is expected to be readily applicable for other types of glycans and targets of interest in glycobiology.

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Section: Discussion Summary and Conclusionmentioning

confidence: 99%

Directed Evolution of Glycopeptides Using mRNA Display

Horiya

Bailey

Krauss

2017

Methods in Enzymology

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“…a) Scheme showing the key features of the mRNA display technology; b) bicyclization of mRNA displayed peptides by the method of Sako et al; [36] c) bicyclization of mRNA displayed peptide library by the method of Hacker et al; [37] and d) bicyclization of SICLOPPS-synthesized peptides by the method of Bionda and Fusan. [39] …”

Section: Figurementioning

confidence: 99%

“…[38] Bicyclizationw as achieved by additionally incorporating azidohomoalaninea nd propargyl glycine residues into the peptides equence, followed by Cu I -mediated azide-alkyne cycloaddition.M ore recently,H acker et al exploited the orthogonality between alkylation of cysteines ide chains with bis(bromomethyl)benzene and click chemistry to produce bicyclic peptides that are either mannacle shaped or theta-bridged (Figure 7c). [39] Unlike previous bicyclic peptidel ibraries, in which the conformation of bicyclic peptides was pre-determined by the small-molecule scaffold, the bicyclization methodo fH ackere tal. is scaffold free, therefore allowing for different loop attachment points and access to multiple bicyclic topologies.…”

Section: Biological Synthesisofb Icyclic Peptide Librariesmentioning

confidence: 99%

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Bicyclic Peptides as Next‐Generation Therapeutics

Rhodes

Pei

2017

Chemistry A European J

138

107

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Bicyclic peptides have greater conformational rigidity and metabolic stability than linear and monocyclic peptides and are capable of binding to challenging drug targets with antibody-like affinity and specificity. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes, receptors, and protein-protein interaction targets. Bicyclic peptides have been developed as potential therapeutics against a wide range of diseases, drug targeting agents, imaging/diagnostic probes, and research tools. In this minireview, we provide a summary of the recent progresses on the synthesis and applications of bicyclic peptides.

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“…Cyclized peptide ligands feature superior bioactivity and biochemical stability compared to their linear counterparts. [1][2][3][4] An extensive body of literature exists on cyclic peptides with antibody-like affinity for protein targets, 5,6 the ability to permeate cells and deliver a therapeutic payload, 7,8 or act as modulators of intracellular and extracellular protein-protein interactions. 9,10 These studies emphasize the usefulness and the need for efficient methods to develop cyclic peptides with strong biorecognition activity.…”

Section: Introductionmentioning

confidence: 99%

Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

Bowen

Schneible

Labar

et al. 2020

Preprint

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Abstract. We present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 μM for YAP and 0.84 μM for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.

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Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display

Cited by 61 publications

References 81 publications

Directed Evolution of Glycopeptides Using mRNA Display

Directed Evolution of Glycopeptides Using mRNA Display

Bicyclic Peptides as Next‐Generation Therapeutics

Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

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