Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, 113-0033, Japan.
Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Brisbane, QLD 4072, Australia.
J Am Chem Soc. 2021 Nov 10;143(44):18481-18489. doi: 10.1021/jacs.1c07574. Epub 2021 Nov 1.
Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 10 members based on the cyclotide scaffold of trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.
环肽是一类具有复杂结构的植物来源肽，其结构由头到尾的环肽骨架和半胱氨酸结核心形成。这些结构特征是环肽天然生物活性的基础，也是它们作为药物先导物的有益特性，包括高蛋白酶稳定性和细胞通透性。然而，它们固有的结构复杂性给环肽工程带来了挑战，特别是在获得足够化学多样性的文库以设计有效和选择性的环肽变体方面。在这里，我们报告了一种使用 mRNA 展示的策略，使我们能够从基于胰蛋白酶抑制剂-II (MCoTI-II) 环肽支架的包含超过 10 个成员的文库中选择有效的基于环肽的 FXIIa 抑制剂。最有效和选择性的抑制剂 cMCoFx1 对 FXIIa 的抑制常数为 pM，对相关丝氨酸蛋白酶的选择性大于三个数量级，实现了内源性凝血途径的特异性抑制。cMCoFx1 和 FXIIa 的共晶结构揭示了在几个位置的相互作用，跨越了接触界面，传达了高亲和力结合，突出了这些环肽是有吸引力的半胱氨酸结支架，可用于治疗开发。
