Understanding how natural and engineered peptides enter cells would facilitate the elucidation of biochemical mechanisms underlying cell biology and is pivotal for developing effective intracellular targeting strategies. In this study, we demonstrate that our peptide stapling technique, fluorine-thiol displacement reaction (FTDR), can produce flexibly constrained peptides with significantly improved cellular uptake, particularly into the nucleus. This platform confers enhanced flexibility, which is further amplified by the inclusion of a D amino acid, while maintaining environment-dependent α helicity, resulting in highly permeable peptides without the need for additional cell-penetrating motifs. Targeting the ERα-coactivator interaction prevalent in estrogen receptor-positive (ER+) breast cancers, we showcased that FTDR-stapled peptides, notably SRC2-LD, achieved superior internalization, including cytoplasmic and enriched nuclear uptake, compared to peptides stapled by ring-closing metathesis (RCM). These FTDR-stapled peptides utilize different mechanisms of cellular uptake, including energy-dependent transport such as actin-mediated endocytosis and macropinocytosis. As a result, FTDR peptides exhibit enhanced anti-proliferative effects despite their slightly decreased target affinity. Our findings challenge existing perceptions of cell permeability, emphasizing the possibly incomplete understanding of the structural determinants vital for cellular uptake of peptide-like macromolecules. Notably, while α helicity and lipophilicity are positive indicators, they alone are insufficient to determine high cell permeability, as evidenced by our less helical, more flexible, and less lipophilic FTDR-stapled peptides.

中文翻译：

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灵活的氟-硫醇置换吻合肽，具有增强的膜渗透性，用于雌激素受体/共激活因子相互作用。


了解天然和工程肽如何进入细胞将有助于阐明细胞生物学背后的生化机制，并且对于开发有效的细胞内靶向策略至关重要。在这项研究中，我们证明了我们的肽装订技术，氟-硫醇置换反应 （FTDR），可以产生柔性约束的肽，显着改善细胞摄取，特别是进入细胞核。该平台赋予了更高的灵活性，通过包含 D 氨基酸进一步放大了这种灵活性，同时保持了环境依赖性 α 螺旋性，从而获得高渗透性肽，而无需额外的细胞渗透基序。针对雌激素受体阳性 （ER+） 乳腺癌中普遍存在的 ERα-共激活因子相互作用，我们展示了 FTDR 吻合肽，特别是 SRC2-LD，与闭合环复分解 （RCM） 装订的肽相比，实现了优越的内化，包括细胞质和富集的核摄取。这些 FTDR 装订肽利用不同的细胞摄取机制，包括能量依赖性转运，例如肌动蛋白介导的内吞作用和巨胞饮作用。因此，尽管 FTDR 肽的靶标亲和力略有降低，但它们表现出增强的抗增殖作用。我们的研究结果挑战了现有的细胞通透性看法，强调了对细胞摄取肽样大分子至关重要的结构决定因素可能不完整的理解。值得注意的是，虽然α螺旋性和亲脂性是积极的指标，但仅凭它们不足以确定高细胞通透性，我们的螺旋度较低、柔韧性较高且亲脂性较低的 FTDR 装订肽就证明了这一点。