Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic PeptideLibraries
Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic PeptideLibraries
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DOI：
10.1021/jacs.2c00216
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发表时间：
2022-03-23
影响因子：
15
通讯作者：
Wu, Chuanliu
中科院分区：
文献类型：
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作者：
Dong, Huilei;Li, Jinjing;Wu, Chuanliu
由于二硫键配对的困难，天然富含二硫键的肽（DRP）的工程化受到了极大的阻碍。利用折叠导向基序和非经典巯基氨基酸的新型DRP易于折叠，具有预期的二硫键连接性，代表了一类用于开发肽配体和治疗剂的新支架。然而，支架的有限多样性，特别是非规范氨基酸的使用[例如，青霉胺（Penicillamine，Pen）]的存在，极大地阻碍了这些DRPs的进一步开发和应用。在这里，我们设计并合成了具有类似于Pen硫醇的空间位阻硫醇基团的非规范双硫醇基序，以指导肽折叠成特定的双环和三环结构。通过与遗传密码重编程相结合的PURE系统，这些双巯基基序可以被整合到肽中，这使得首次能够在体外表达具有两个非典型和正交二硫键的双环肽，我们进一步构建了由mRNA编码的双环肽文库，利用该文库成功地筛选出了与蛋白质具有纳摩尔亲和力的新的双环肽配体。因此，本研究提供了一种新的、通用的、稳健的方法，用于发现非天然多肽衍生的具有新结构和功能的新型DRPs，这将极大地有益于多肽药物发现领域。
The engineering of naturally occurring disulfide-rich peptides (DRPs) has been significantly hampered by thedifficulty of manipulating disulfide pairing. New DRPs that takeadvantage of fold-directing motifs and noncanonical thiol-bearingamino acids are easy-to-fold with expected disulfide connectivities,representing a new class of scaffolds for the development ofpeptide ligands and therapeutics. However, the limited diversity ofthe scaffolds and particularly the use of noncanonical amino acids[e.g., penicillamine (Pen)] that are difficult to be translated byribosomes greatly hamper the further development and applicationof these DRPs. Here, we designed and synthesized noncanonicalbisthiol motifs bearing sterically obstructed thiol groups analogousto the Pen thiol to direct the folding of peptides into specific bicyclic and tricyclic structures. These bisthiol motifs can beribosomally incorporated into peptides through a commercially available PURE system integrated with genetic code reprograming,which enables, for thefirst time, the in vitro expression of bicyclic peptides with two noncanonical and orthogonal disulfide bonds.We further constructed a bicyclic peptide library encoded by mRNA, with which new bicyclic peptide ligands with nanomolar affinityto proteins were successfully selected. Therefore, this study provides a new, general, and robust method for discovering de novoDRPs with new structures and functions not derived from natural peptides, which would greatly benefit thefield of peptide drugdiscovery.