Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a d-Pro-l-Pro motif, cyclo(AFΦrpPRRFQ) (where Φ is l-naphthylalanine, r is d-arginine, and p is d-proline). The structural constraints provided by cyclization and the d-Pro-l-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(Y^pVNFΦrpPRR) (where Y^p is l-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant β-hairpin character surrounding the d-Pro-l-Pro motif. These results demonstrate cyclo(AFΦrpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).

环肽能够以高亲和力和特异性结合具有挑战性的靶标（例如，参与蛋白质-蛋白质相互作用的蛋白质），但由于膜通透性差，通常无法进入细胞内靶标。在这项工作中，我们发现了一种构象受限的环状细胞穿透肽 (CPP)，它包含一个d -Pro- l -Pro 基序，环 (AFΦrpPRRFQ)（其中 Φ 是l -萘丙氨酸，r 是d -精氨酸，p 是d -脯氨酸）。环化和d -Pro- l提供的结构约束-Pro 基序允许合理设计大环尺寸（最多 16 个氨基酸）的细胞渗透性环肽。该策略用于设计一种有效的、细胞可渗透的、生物活性的环状肽基抑制剂 cyclo(Y ^p VNFΦrpPRR)（其中 Y ^p是l-磷酸酪氨酸），以对抗 Grb2 SH2 结构域。多维 NMR 光谱和圆二色性分析表明，环状 CPP 以及 Grb2 SH2 抑制剂主要呈现无规卷曲结构，但在d- Pro- l周围具有显着的 β-发夹特征-Pro 主题。这些结果表明，环（AFΦrpPRRFQ）是一种有效的 CPP，可用于内环（将货物插入 CPP 环）或生物货物的外环递送（将货物连接到 Gln 侧链）。