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

ABSTRACT: The papain-like protease (PL^pro) 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 PL^pro 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 PL^pro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL^pro inhibitors in the BSL-2 setting. X-ray crystal structure of PL^pro 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 PL^pro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL^pro assay is a suitable surrogate for screening PL^pro inhibitors in the BSL-2 setting.

SUBMITTER: Ma C

PROVIDER: S-EPMC8265724 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay.

Project description:The papain-like protease (PL pro ) of SARS-CoV-2 is a validated antiviral drug target. PL pro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PL pro inhibitors with IC 50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC 50 values ranging from 0.56 to 0.90 µM. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PL pro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PL pro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC 50 values of 8.89 and 8.32 µM, respectively, which were 3-fold more potent than GRL0617 (EC 50 = 25.1 µM). The X-ray crystal structures of PL pro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PL pro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PL pro assay might be a suitable surrogate for screening PL pro inhibitors in the BSL-2 setting.

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