Institute for Chemical Research , Kyoto University , Uji , Kyoto 611-0011 , Japan.
Mol Pharm. 2018 Mar 5;15(3):1332-1340. doi: 10.1021/acs.molpharmaceut.7b01130. Epub 2018 Feb 21.
All-hydrocarbon stapled peptides make up a promising class of protein-protein interaction regulators; their potential therapeutic benefit arises because they have a high binding affinity and specificity for intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides. We found that the rate of cellular uptake of unstapled peptides (i.e., those bearing olefinic non-natural amino acids that are not subjected to olefin metathesis) was higher than that for the corresponding stapled peptides. Additionally, the insertion of these olefinic non-natural amino acids into peptide sequences significantly increased their rate of cellular uptake. According to the high-performance liquid chromatography retention times, the overall hydrophobicity of unstapled peptides was greater than that of stapled peptides, followed by that of the original peptides without olefinic non-natural amino acids. There was not a close correlation between helical content and the rate of cellular uptake of these peptides. Therefore, the increase in overall hydrophobicity resulting from the introduction of non-natural amino acids, rather than the structural stabilization resulting from staple formation, is the key driver promoting cellular uptake. Macropinocytosis, a form of fluid-phase endocytosis, was involved in the cellular uptake of all six peptides.
全碳氢 stapled 肽是一类很有前途的蛋白质-蛋白质相互作用调节剂；它们具有高结合亲和力和特异性，对细胞内分子有潜在的治疗益处。这些肽的细胞渗透效力是其生物活性的关键决定因素。然而，决定其细胞摄取的因素仍然是一个活跃的研究领域。在这项研究中，我们评估了 stapled（或交联）形成对六种已知全碳氢 stapled 肽的细胞摄取的影响。我们发现，未 stapled 肽（即带有不进行烯烃复分解的烯键非天然氨基酸的肽）的细胞摄取速率高于相应 stapled 肽。此外，将这些烯键非天然氨基酸插入肽序列会显著增加其细胞摄取速率。根据高效液相色谱保留时间，未 stapled 肽的整体疏水性大于 stapled 肽，其次是没有烯键非天然氨基酸的原始肽。这些肽的细胞摄取速率与螺旋含量之间没有密切的相关性。因此，引入非天然氨基酸导致的整体疏水性增加，而不是 stapled 形成导致的结构稳定性增加，是促进细胞摄取的关键驱动因素。巨胞饮作用，一种液相内吞作用形式，参与了所有六种肽的细胞摄取。
