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 evalua...
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| Main Authors: | , , , , , , , , |
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| Format: | Book |
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Taylor & Francis Group,
2024-12-01T00:00:00Z.
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| Summary: | 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. |
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| Item Description: | 10.1080/14756366.2024.2387417 1475-6374 1475-6366 |