Wallbrecher Rike, Depré Luc, Verdurmen Wouter P R, Bovée-Geurts Petra H, van Duinkerken Richard H, Zekveld Mariët J, Timmerman Peter, Brock Roland
Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre , Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
Bioconjug Chem. 2014 May 21;25(5):955-64. doi: 10.1021/bc500107f. Epub 2014 Apr 16.
Cell-penetrating peptides (CPPs) possess the capacity to induce cell entry of themselves and attached molecular cargo, either by endocytosis or by direct translocation. Conformational constraints have been described as one means to increase the activity of CPPs, especially for direct crossing of the plasma membrane. Here, we explored the structure-activity relationship of bicyclic peptides for cell entry. These peptides may be considered minimal analogues of naturally occurring oligocyclic peptide toxins and are a promising scaffold for the design of bioactive molecules. Increasing numbers of arginine residues that are primarily contributing to cell-penetrating activity were introduced either into the cycles, or as stretches outside the cycles, at both ends or at one end only. In addition, we probed for the impact of negatively charged residues on activity for both patterns of arginine substitution. Uptake was investigated in HeLa cells by flow cytometry and confocal microscopy. Overall, uptake efficiency showed a positive correlation with the number of arginine residues. The subcellular distribution was indicative of endocytic uptake. One linear stretch of arginines coupled outside the bicycle was as effective in promoting uptake as substituting the same number of arginines inside the bicycles. However, the internally substituted analogues were more sensitive to the presence of negatively charged residues. For a given bicyclic peptide, uptake was more effective than for the linear counterpart. Introduction of histidine and tryptophans further increased uptake efficiency to comparable levels as that of nonaarginine despite the larger size of the bicyclic backbone. The results demonstrate that both arginine clustering and spatial constraints are uptake-promoting structural principles, an observation that gives freedom in the introduction of cell-penetrating capacity to structurally constrained scaffolds.
细胞穿透肽（CPPs）具有通过内吞作用或直接转运诱导自身及附着的分子货物进入细胞的能力。构象限制已被描述为提高CPPs活性的一种手段，特别是对于直接穿过质膜而言。在此，我们探索了双环肽进入细胞的构效关系。这些肽可被视为天然存在的寡环肽毒素的最小类似物，是设计生物活性分子的有前景的支架。主要有助于细胞穿透活性的精氨酸残基数量不断增加，这些残基要么被引入环中，要么作为环外的延伸段，位于两端或仅一端。此外，我们探究了带负电荷残基对两种精氨酸取代模式活性的影响。通过流式细胞术和共聚焦显微镜在HeLa细胞中研究了摄取情况。总体而言，摄取效率与精氨酸残基数量呈正相关。亚细胞分布表明是通过内吞作用摄取。连接在双环外侧的一段线性精氨酸在促进摄取方面与在双环内部取代相同数量的精氨酸一样有效。然而，内部取代的类似物对带负电荷残基的存在更敏感。对于给定的双环肽，摄取比线性对应物更有效。引入组氨酸和色氨酸进一步提高了摄取效率，使其达到与九聚精氨酸相当的水平，尽管双环主链的尺寸更大。结果表明，精氨酸聚集和空间限制都是促进摄取的结构原则，这一观察结果为在结构受限的支架中引入细胞穿透能力提供了自由度。
