The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P.R. China.
State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao 266237, P.R. China.
J Am Chem Soc. 2022 Mar 23;144(11):5116-5125. doi: 10.1021/jacs.2c00216. Epub 2022 Mar 15.
The engineering of naturally occurring disulfide-rich peptides (DRPs) has been significantly hampered by the difficulty of manipulating disulfide pairing. New DRPs that take advantage of fold-directing motifs and noncanonical thiol-bearing amino acids are easy-to-fold with expected disulfide connectivities, representing a new class of scaffolds for the development of peptide ligands and therapeutics. However, the limited diversity of the scaffolds and particularly the use of noncanonical amino acids [e.g., penicillamine (Pen)] that are difficult to be translated by ribosomes greatly hamper the further development and application of these DRPs. Here, we designed and synthesized noncanonical bisthiol motifs bearing sterically obstructed thiol groups analogous to the Pen thiol to direct the folding of peptides into specific bicyclic and tricyclic structures. These bisthiol motifs can be ribosomally incorporated into peptides through a commercially available PURE system integrated with genetic code reprograming, which enables, for the first 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 affinity to proteins were successfully selected. Therefore, this study provides a new, general, and robust method for discovering de novo DRPs with new structures and functions not derived from natural peptides, which would greatly benefit the field of peptide drug discovery.
天然富含二硫键的肽（DRP）的工程设计受到二硫键配对操作难度的严重阻碍。利用折叠导向模体和非典型含硫氨基酸的新型 DRP 很容易折叠，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型支架。然而，支架的多样性有限，特别是使用核糖体难以翻译的非典型氨基酸[例如，青霉胺（Pen）]，极大地阻碍了这些 DRP 的进一步发展和应用。在这里，我们设计并合成了带有空间位阻硫醇基团的非典型双硫醇模体，类似于 Pen 硫醇，可将肽定向折叠成特定的双环和三环结构。这些双硫醇模体可以通过与遗传密码重编程集成的商业上可获得的 PURE 系统掺入肽中，这首次实现了具有两个非典型和正交二硫键的双环肽的体外表达。我们进一步构建了一个由 mRNA 编码的双环肽文库，用该文库成功地筛选到了对蛋白质具有纳摩尔亲和力的新型双环肽配体。因此，这项研究提供了一种新的、通用的、强大的方法，用于发现具有新结构和功能的新型从头设计的 DRP，这将极大地有益于肽药物发现领域。
