In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

中文翻译：

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用于抑制自噬的环肽的定向进化

近几十年来，越来越清楚的是，自噬的诱导在许多癌症类型的治疗耐药性和休眠的发展中起着重要作用。不幸的是，临床试验中的两种自噬抑制剂氯喹 (CQ) 和羟氯喹 (HCQ) 在治疗剂量下具有较差的药代动力学和高毒性。这引起了人们对开发靶向自噬抑制剂的浓厚兴趣，以在对正常组织影响最小的情况下使疾病对治疗重新敏感。我们利用扫描非天然​​蛋白酶抗性 (SUPR) mRNA 展示来开发靶向自噬蛋白 LC3 的大环肽。体外. 最有前途的 LC3 结合 SUPR 肽以低微摩尔浓度进入胞质溶胶，如通过氯代烷渗透测定 (CAPA) 测量的，并以浓度依赖性方式抑制饥饿介导的 GFP-LC3 斑点形成。结合 LC3 的 SUPR 肽使铂耐药卵巢癌细胞对顺铂治疗重新敏感，并触发衔接蛋白 p62 的积累，这表明通过成功破坏细胞培养中的 LC3 PPI 来降低自噬通量。在转移性卵巢癌的小鼠模型中，使用结合 LC3 的 SUPR 肽和卡铂治疗 4 周后几乎完全抑制了肿瘤生长。这些结果表明 SUPR 肽 mRNA 展示可用于开发靶向和破坏自噬机制的细胞穿透大环肽体外和体内。