Vitale Fulvia, Scerra Gianluca, Marrone Laura, Di Micco Anna, Cannata Serio Magda, Intasiri Amarawan, Amodio Giuseppina, Cirillo Vittorio, Remondelli Paolo, Luongo Antonietta, Bonavita Raffaella, Caporaso Maria Gabriella, Perez Franck, Renna Maurizio, Bell Thomas W, Romano Simona, D'Agostino Massimo
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
Institute Curie, PSL Research University, Paris, France.
Nat Commun. 2025 Aug 6;16(1):7243. doi: 10.1038/s41467-025-62439-w.
The perturbation of protein translocation into the secretory pathway using Sec61 translocon inhibitors is a novel and promising strategy for tackling many pathological situations, including cancer and viral infections. However, a highly sensitive and direct screening platform for selecting Sec61 inhibitors is unavailable. Here, we develop a "resuming luminescence upon translocation interference" (RELITE) assay capable of selecting Sec61 inhibitors in a single round of screening. This assay exploits the inactivation of firefly luciferase, once translocated into the endoplasmic reticulum (ER), and the possibility of diverting and "re-lighting" luciferase into the cytosol by a Sec61 inhibitor. Using this method, we select small molecules capable of hampering the protein expression of the PD-L1 immune checkpoint by interfering with its ER translocation and delivering it for degradation. In conclusion, our screening method will greatly facilitate the selection of Sec61 inhibitors for down-modulating the expression of many disease-relevant proteins.
使用Sec61易位子抑制剂干扰蛋白质转运到分泌途径中是一种新颖且有前景的策略，可用于应对多种病理情况，包括癌症和病毒感染。然而，目前尚无用于筛选Sec61抑制剂的高灵敏度直接筛选平台。在此，我们开发了一种“转运干扰后恢复发光”（RELITE）检测方法，能够在一轮筛选中筛选出Sec61抑制剂。该检测方法利用了萤火虫荧光素酶一旦转运到内质网（ER）就会失活，以及Sec61抑制剂将荧光素酶转移并“重新点亮”到细胞质中的可能性。利用这种方法，我们筛选出了能够通过干扰PD-L1免疫检查点蛋白的内质网转运并促使其降解来阻碍其蛋白质表达的小分子。总之，我们的筛选方法将极大地促进Sec61抑制剂的筛选，以下调许多与疾病相关蛋白质的表达。
