\n
$\"Phage$ <\/a>
Figure 1: Phage Display Library of Cyclopeptides with Random Sequences<\/figcaption><\/figure><\/div>\n\n\n
The Fasan laboratory has established a novel strategy by utilizing amber stop codon technology to introduce O-(2-bromoethyl)-tyrosine<\/a>, enabling the cyclization of peptides on phage. This tyrosine can spontaneously react with proximal cysteine<\/a> residues after translation to form irreducible thioether bonds (Figure 2).<\/p>\n\n\n
\n
$\"Phage$ <\/a>
Figure 2: Phage Library Synthesis and Target Validation<\/figcaption><\/figure><\/div>\n\n\n
The Bogyo laboratory introduced an unbiased phage display screening strategy to identify irreversible covalent inhibitors of protein targets. By incorporating cysteine-reactive vinyl sulfone or serine-reactive diphenylphosphinyl ester heads linked to 1,3-dichloroacetone (DCA) into the cyclotide library, cyclopeptides can react with two cysteine residues in the library through DCA-modified heads (Figure 3).<\/p>\n\n\n
\n
$\"Phage$ <\/a>
Figure 3: Phage Library of Cyclopeptides with Random Sequences Reacting with 1,3-Dichloroacetone Heads to Generate Libraries Targeting TEV and FphF<\/figcaption><\/figure><\/div>\n\n\n
\n
Split-Intein Circular Ligation <\/strong>o<\/strong>f Peptides <\/strong>a<\/strong>nd Proteins (SICLOPPS)<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
SICLOPPS is a novel method that screens cyclopeptides in live cells by utilizing the functional splicing of inteins, self-cleaving protein domains that excise their side-chain external protein domains. In this technique, the cyclopeptide of interest is inserted between split inteins in the form of IC-X-IN, where “X” represents the selected cyclopeptide sequence. After translation, the two intein fragments self-assemble, splicing out the cyclopeptide between them, releasing cyclopeptides with native amide bonds (Figure 4).<\/p>\n\n\n
\n
$\"Mechanism$ <\/a>
Figure 4: Mechanism of SICLOPPS Inhibiting HIF-1\u03b1\/HIF-1\u03b2 Proteins<\/figcaption><\/figure><\/div>\n\n\n
\n
mRNA Display<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\nmRNA display is another effective in vitro screening method. Random nonstandard peptides integrated discovery (RaPID) is a mRNA display method promoted by Hiroaki Suga’s laboratory in 2006. This unique strategy utilizes flexible tRNA synthetases, allowing for the incorporation of various non-natural amino acids, including D-amino acids<\/a>, N-methyl amino acids, and \u03b2-amino acids, significantly expanding the chemical space for screening. Moreover, genetic code reprogramming of N-terminal formylated Met can be replaced with amino acids, such as N-chloroacetyl-L-Tyr, which can spontaneously cyclize with proximal cysteine residues to generate cyclopeptide libraries. Recently, Suga’s lab used the RaPID system to identify effective and selective inhibitors of factor XIIa (FXIIa), a critical target for developing antithrombotic drugs (Figure 5).<\/p>\n\n\n
\n
$\"mRNA$ <\/a>
Figure 5: mRNA Display for Identifying Inhibitors of FXIIa and K-Ras<\/figcaption><\/figure><\/div>\n\n\n
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Structure-Guided Design<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
In recent research from Grossmann’s lab, cyclopeptide-designed inhibitors were developed to mimic the \u03b2-sheet characteristics of E-cadherin, targeting the interaction between Tcf4 and \u03b2-catenin. \u03b2-catenin is a transcriptional co-activator and a key component of the Wnt signaling pathway. Overactivation of this specific oncogenic target leads to various cancers, making it a target for therapeutic intervention. The binding constants of cyclopeptides were determined using in vitro fluorescence polarization (FP) assays. In this assay, competitive binding is detected through changes in fluorescence polarization when competitive ligands displace the fluorescently labeled Tcf4 ligand from \u03b2-catenin (Figure 6).<\/p>\n\n\n
\n
$\"Large$ <\/a>
Figure 6: Large Macrocyclic Inhibitors of Tcf4\/\u03b2-Catenin<\/figcaption><\/figure><\/div>\n\n\n
Challenges <\/strong>of<\/strong> Cyclopeptides<\/strong><\/strong><\/h2>\n\n\n\n
Although cyclopeptides are generally more stable than their linear counterparts, they can still degrade rapidly in vivo. One commonly used approach to enhance cyclopeptide proteolytic stability is introducing additional ring constraints to form bicyclic cyclopeptides. Another common approach is introducing non-natural amino acids, such as D-amino acids. As new screening technologies continue to emerge, such as the multiple analytical techniques of affinity selection-mass spectrometry (AS-MS), they may address the challenging issue of pushing cyclopeptides to the market.<\/p>\n\n\n\n
Reference:<\/p>\n\n\n\n
\n
Li, X., Craven, T. W., Levine, P. M., Cyclic Peptide Screening Methods for Preclinical Drug Discovery, J. Med. Chem.<\/em>, 2022, 65<\/em>, 18, 11913-11926.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"
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