A light-resuming strategy as a screening method for selecting Sec61 inhibitors down-modulating PD-L1 expression

• Published in: Nature communications Vol. 16; no. 1; pp. 7243 - 14
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.08.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/106; 13/109; 13/31; 49; 49/98; 631/154/1435; 631/1647/2163; 631/80/2023/2022; 82/47; Assaying; B7-H1 Antigen - genetics; B7-H1 Antigen - metabolism; Cell Line, Tumor; Cytosol; Deactivation; Down-Regulation - drug effects; Drug Evaluation, Preclinical - methods; Endoplasmic reticulum; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; HEK293 Cells; Humanities and Social Sciences; Humans; Immune checkpoint; Inhibitors; Ligands; Lighting; multidisciplinary; PD-L1 protein; Protein transport; Protein Transport - drug effects; Proteins; Science; Science (multidisciplinary); SEC Translocation Channels - antagonists & inhibitors; SEC Translocation Channels - metabolism; Translocation; Tumors
• ISSN: 2041-1723; 2041-1723
• 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. Here the authors develop an assay capable of selecting Sec61 inhibitors by exploiting 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.