Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay

  • ACS Cent Sci. 2021 Jul 28;7(7):1245-1260. doi: 10.1021/acscentsci.1c00519.
Chunlong Ma  1 ,  Michael Dominic Sacco  2 ,  Zilei Xia  1 ,  George Lambrinidis  3 ,  Julia Alma Townsend  4 ,  Yanmei Hu  1 ,  Xiangzhi Meng  5 ,  Tommy Szeto  1 ,  Mandy Ba  1 ,  Xiujun Zhang  2 ,  Maura Gongora  2 ,  Fushun Zhang  5 ,  Michael Thomas Marty  4 ,  Yan Xiang  5 ,  Antonios Kolocouris  3 ,  Yu Chen  2 ,  Jun Wang  1
Affiliations
  • 1. Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, United States.
  • 2. Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, United States.
  • 3. Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupolis-Zografou, 15771 Athens, Greece.
  • 4. Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States.
  • 5. Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
PMID: 34341772 DOI: 10.1021/acscentsci.1c00519
Abstract

The papain-like Protease (PLpro) of SARS-CoV-2 is a validated Antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PLpro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and Antiviral activity compared to GRL0617, which was reported as a SARS-CoV PLpro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular Antiviral activity of PLpro inhibitors in the BSL-2 setting. X-ray crystal structure of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617. Overall, the PLpro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PLpro assay is a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

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