Directed evolution of cyclic peptides for inhibition of autophagy.
Directed evolution of cyclic peptides for inhibition of autophagy.
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循环肽的定向演变以抑制自噬。
DOI：
10.1039/d0sc03603j
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发表时间：
2021-01-13
期刊：
影响因子：
8.4
通讯作者：
Millward SW
中科院分区：
文献类型：
--
作者：
Gray JP;Uddin MN;Chaudhari R;Sutton MN;Yang H;Rask P;Locke H;Engel BJ;Batistatou N;Wang J;Grindel BJ;Bhattacharya P;Gammon ST;Zhang S;Piwnica-Worms D;Kritzer JA;Lu Z;Bast RC Jr;Millward SW
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抗性和休眠的发展中起着重要作用。遗憾的是，临床试验中的两种自噬抑制剂氯喹（CQ）和羟氯喹（HCQ）在治疗剂量下具有不良的药代动力学和高毒性。这引起了人们对靶向自噬抑制剂的开发的强烈兴趣，以使疾病对治疗重新敏感，对正常组织的影响极小。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LC 3的大环肽。所得肽结合LC 3A和LC 3B-自噬体成熟机制的两个基本组成部分-具有中纳摩尔亲和力，并在体外破坏LC 3与其结合伙伴之间的蛋白质-蛋白质相互作用（PPI）。最有前途的LC 3结合SUPR肽在低微摩尔浓度下进入细胞质，如通过氯代烷烃渗透试验（CAPA）测量的，并以浓度依赖性方式抑制饥饿介导的GFP-LC 3斑点形成。LC 3结合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，表明通过成功破坏细胞培养物中的LC 3 PPI降低了自噬通量。在转移性卵巢癌的小鼠模型中，用LC 3结合SUPR肽和卡铂治疗导致在治疗四周后几乎完全抑制肿瘤生长。这些结果表明，SUPR肽mRNA展示可用于开发靶向和破坏体外和体内自噬机制的细胞穿透大环肽。使用SUPR肽mRNA展示来进化用于自噬抑制的细胞可渗透的大环肽。
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. SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.
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