Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, Queensland 4072, Australia.
ACS Chem Biol. 2021 Feb 19;16(2):414-428. doi: 10.1021/acschembio.0c00988. Epub 2021 Feb 3.
Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein-protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT-KD3 and cR10-KD3, able to activate the p53 pathway in cancer cells. cTAT-KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1-12 μM. cR10-KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 μM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.
肽类正在被开发为靶向抗癌药物，以调节参与癌症进展的细胞质蛋白-蛋白相互作用。然而，它们作为治疗剂的应用常常受到其低细胞膜通透性和/或无法到达细胞质靶标的限制。与细胞穿透肽缀合已成功用于提高高亲和力结合肽的细胞质递送，但细胞摄取并不总是导致靶向途径的调节。为了克服这一限制，我们通过将 KD3（一种非细胞渗透性但强效和特异的 p53：MDM2 和 p53：MDMX 相互作用的肽抑制剂）与一组环状细胞穿透肽缀合，开发了“鱼钩肽”。我们研究了它们与 MDM2 和 MDMX 的结合亲和力、细胞进入机制以及在重新激活 p53 途径中的作用。我们鉴定了两种鱼钩肽，cTAT-KD3 和 cR10-KD3，它们能够激活癌细胞中的 p53 途径。cTAT-KD3 通过内吞途径进入细胞，从内涵体中逃逸，并在浓度为 1-12 μM 的范围内激活乳腺癌（MCF7）、肺癌（A549）和结肠癌（HCT116）癌细胞系中的 p53 途径。cR10-KD3 通过直接跨膜易位到达细胞质，并在所有测试的细胞系中以 1 μM 的浓度激活 p53 途径。我们的工作表明，当与稳定的细胞穿透肽缀合时，非渗透性抗癌肽可以递送到细胞质中并抑制细胞内癌症途径。机制研究表明，直接易位导致毒性降低、在较低浓度下更高的细胞质递送以及对测试细胞系的膜的依赖性降低，而不是发生具有内涵体逃逸的内吞途径。鱼钩策略可以挽救从高通量筛选中鉴定出的高亲和力肽结合物，并将其转化为靶向抗癌治疗剂，但研究其细胞摄取和细胞死亡机制对于确认靶向癌症途径的调节至关重要。
