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Peptide Cyclization

mRNA display, a crucial platform technology for discovering cyclic peptide ligands, has emerged as a leading strategy capable of producing natural-product-like macrocyclic peptide binders with antibody-like affinities. At CD BioSciences, we offer comprehensive drug development services based on our mRNA display technology, supporting your cyclic peptide drug development needs.

Fig 1. General scheme for cyclic peptide selection by mRNA display.

Peptide Cyclization in mRNA Display

Peptide backbone cyclization is generally not feasible in mRNA display due to the necessity of the puromycin linker at the peptide C terminus. As an alternative, various chemical strategies have been developed to form a cyclic peptide by reacting two side chains or the N terminus with a side chain of the translated peptide. This cyclization reaction must be highly efficient across a variety of ring sizes to ensure that all peptides in a library are cyclic and to prevent the undesirable accumulation of linear peptides.

Fig 2. Post-translational thioether macrocyclization is extensively used for the generation of cyclic peptides for mRNA display. (Sohrabi, et al., 2020)

In mRNA display, the cyclization conditions must be relatively mild to ensure that both the peptide and the RNA remain intact and unaltered. The following examples all involve cyclization through one or more side chains. Since the C-terminus of the peptide is not free in mRNA display, creating backbone-cyclized peptides is particularly challenging.

A classical approach of a disuccinimidyl glutarate linker was used to crosslink the N-terminal amino group of the peptide with the side chain amino group of a downstream lysine residue.

Chemistry for the generation of macrocyclic peptide.

An approach in which two cysteine residues are crosslinked by meta-substituted dibromoxylene as an exogenous crosslinking reagent can be used to construct cyclic peptides.

Chemistry for the generation of macrocyclic peptide.

Reduction of the disulfides with tris(2-carboxyethyl)phosphine (TCEP) initiated the intramolecular 1,4 addition to the dehydroalanine, forming the lanthionine moiety.

Chemistry for the generation of macrocyclic peptide.

Two orthogonal cyclization chemistries, the Cu-catalyzed azide-alkyne cycloaddition and thioether cyclization, can be used to produce bicyclic peptides through ribosomal synthesis

Fig 3. Chemistry for the generation of macrocyclic peptide mRNA display libraries. (Peacock H, Suga H., 2021)

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Various chemical methods can be employed to generate cyclic peptides. CD BioSciences utilizes the most common approach to create a library of macrocyclic peptides. This method involves forming disulfide bonds between two cysteine residues. The principle behind this approach is that the cysteine residues can be bridged using a dibromoxylene linker, leading to the formation of two thioethers when reacting with the cysteine side chains.

Our Workflow

Transcribe the DNA library. Ligate the transcribed RNA with puromycin oligonucleotides.

Cyclize through bis-alkylation of the Cys residues (incubation with dibromoxylene can achieve efficient cyclization), then reverse transcribe.

Sequence
Design Puromycin
Ligation PURE System
Translation Cyclization Affinity
Selection Our Services

First, design the DNA sequences for the DNA library. The DNA library encodes peptides with random amino acids, flanked by two Cys residues.

Translate the library in a fully reconstituted translation system (PURE) to form an mRNA-peptide fusion library.

Incubate each library separately with magnetic beads carrying the target for selection.

Initiation factors (IF1, IF2, IF3).
Elongation factors (EF-Tu, EF Ts, EF-G), the
Release factors (RF1, RF2, RF3).
Ribosome recycling factor
20 aminoacyl-tRNA synthetases
Methionyl-tRNA formyltransferase
Pyrophosphatase.

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At CD BioSciences, we are dedicated to transforming drug development through innovative mRNA display technology. To learn more about how we can support your drug development efforts with our mRNA display services, please contact us. Our team is ready to discuss your project requirements and design a customized solution that meets your needs.

References

Peacock, H., Suga, H. Discovery of de novo macrocyclic peptides by messenger RNA display. Trends in Pharmacological Sciences. 2021, 42(5): 385-397.
Sohrabi, C., et al. Methods for generating and screening libraries of genetically encoded cyclic peptides in drug discovery. Nature Reviews Chemistry. 2020, 4(2): 90-101.

For Research Use Only. Not For Clinical Use.

Peptide Cyclization

Peptide Cyclization in mRNA Display

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