In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

木瓜样蛋白酶（Papain-like protease, PLpro）是极具潜力的抗冠状病毒靶点。然而，现有PLpro检测方法的局限性以及已验证活性化合物的稀缺性，极大阻碍了PLpro抑制剂的研发进程。本研究开发了一种基于生物发光共振能量转移（Bioluminescence Resonance Energy Transfer, BRET）的新型细胞内PLpro检测分析法，并将其用于新型冠状病毒（Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2）PLpro抑制剂的评估与发现。该检测方法在检测细胞内PLpro活性降低方面展现出卓越的灵敏度，同时在抑制剂评价与高通量筛选中表现出极高的可靠性与优异性能。通过该检测体系，研究人员鉴定出三种蛋白酶抑制剂为新型PLpro抑制剂，其结构与此前已知的PLpro抑制剂存在显著差异。后续的酶学实验与基于分子对接的配体-蛋白质相互作用分析结果显示，色瑞替尼可直接靶向抑制PLpro，与PLpro的底物结合口袋具有高度的几何互补性；而CA-074甲酯则会在细胞内发生水解，暴露出可与BL2凹槽内G266位点形成氢键所必需的游离羧羟基，进而实现对PLpro的抑制作用。