SARS-CoV-2 M pro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening.

Autor:	Delgado R; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Vishwakarma J; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Moghadasi SA; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA., Otsuka Y; Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA., Shumate J; Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA., Cuell A; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Tansiongco M; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Department of Chemistry, Trinity University, San Antonio, TX 78212, USA., Cooley CB; Department of Chemistry, Trinity University, San Antonio, TX 78212, USA., Chen Y; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Dabrowska A; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Basu R; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA., Anindita PD; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 308232, Singapore; Institute of Structural Biology, Nanyang Technological University, Singapore, 639798, Singapore., Luo D; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 308232, Singapore; Institute of Structural Biology, Nanyang Technological University, Singapore, 639798, Singapore., Dosa PI; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA., Harki DA; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA., Bannister T; Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA., Scampavia L; Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA., Spicer TP; Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA., Harris RS; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, TX 78229, USA. Electronic address: rsh@uthscsa.edu.
Jazyk:	angličtina
Zdroj:	SLAS discovery : advancing life sciences R & D [SLAS Discov] 2024 Sep; Vol. 29 (6), pp. 100181. Date of Electronic Publication: 2024 Aug 22.
DOI:	10.1016/j.slasd.2024.100181
Abstrakt:	Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, M pro (also called 3C-like protease, 3CL pro ), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, M pro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified M pro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported M pro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known M pro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts. Competing Interests: Declaration of competing interest The M(pro) gain-of-signal system is the subject of U.S. Provisional Application Serial No. 63/108,611, filed on November 2, 2020, with RSH and SAM as inventors. The other authors declare that there are no additional competing interests. (Copyright © 2024. Published by Elsevier Inc.)
Databáze:	MEDLINE
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