Wallbrecher Rike, Chène Patrick, Ruetz Stephan, Stachyra Therese, Vorherr Thomas, Brock Roland
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Novartis Institutes for Biomedical Research, Basel, Switzerland.
Br J Pharmacol. 2017 Aug;174(16):2613-2622. doi: 10.1111/bph.13834. Epub 2017 Jul 6.
Helix stapling enhances the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cytoplasmic targeting. We assessed the extent to which the pharmacokinetic characteristics are a function of the staple for a peptide inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein and differ from those of the standard cationic cell-penetrating peptide nona-arginine.
Stapled peptides and linear counterparts were synthesized in free and fluorescently labelled forms. Activity was determined in biochemical time-resolved Förster resonance energy transfer experiments and cellular high-content assays. Cellular uptake and intracellular trafficking were visualized by confocal microscopy.
Peptides showed sub-nanomolar potency. For short-time incubation, uptake efficiencies for the stapled and linear peptides were similar and both were taken up less efficiently than nona-arginine. Only for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides showed an enhanced cytoplasmic and nuclear accumulation after long-term incubation. This was also observed for the linear counterparts, albeit to a lesser degree. For HeLa cells, which lack target expression, no such accumulation was observed.
Cytosolic and nuclear accumulation was not an intrinsic property of the stapled peptide, but resulted from capture by the target Hdm2 after endo-lysosomal release. Considering the rather poor uptake of stapled peptides, further development should focus on increasing the efficiency of uptake of these peptides.
螺旋钉合可增强以螺旋构象与靶蛋白相互作用的肽的活性。这些钉合结构还被认为会改变分子的药代动力学并促进胞质靶向。我们评估了对于一种抑制p53与人双微体2（Hdm2）蛋白相互作用的肽而言，药代动力学特征在多大程度上是钉合结构的函数，以及其与标准阳离子细胞穿透肽九聚精氨酸的药代动力学特征有何不同。
合成了游离形式和荧光标记形式的钉合肽及其线性对应物。在生化时间分辨荧光共振能量转移实验和细胞高内涵分析中测定活性。通过共聚焦显微镜观察细胞摄取和细胞内转运。
肽表现出亚纳摩尔级的效力。对于短时间孵育，钉合肽和线性肽的摄取效率相似，且二者的摄取效率均低于九聚精氨酸。仅对于表达Hdm2靶蛋白的SJSA-1细胞，长期孵育后钉合肽显示出增强的胞质和核内积累。线性对应物也观察到了这种积累，尽管程度较轻。对于缺乏靶标表达的HeLa细胞，未观察到这种积累。
胞质和核内积累不是钉合肽的固有特性，而是内溶酶体释放后被靶标Hdm2捕获的结果。考虑到钉合肽摄取相当差，进一步的开发应侧重于提高这些肽的摄取效率。
