The COVID-19 pandemic, caused by SARS-CoV-2, has presented an urgent need for effective antiviral therapies. While main protease (Mpro) has been a focus of drug discovery efforts, the papain-like protease (PLpro) of SARS-CoV-2 also represents a compelling target due to its multiple functions. PLpro is involved in viral polyprotein processing essential for viral maturation, and also interferes with host immune responses by deubiquitinating and deISGylating host proteins. The deISGylation activity specifically inhibits the host's type I interferon antiviral immune response by removing interferon-stimulated gene 15 (ISG15) modifications. This multifaceted role makes PLpro an attractive target for therapeutic intervention. Previous attempts to utilize existing drugs against COVID-19, such as Remdesivir, Hydroxychloroquine, Lopinavir, and Interferon, have yielded limited success in clinical trials. Consequently, a search for novel and effective inhibitors against SARS-CoV-2 PLpro is of paramount importance. This study aims to identify and characterize potent inhibitors of SARS-CoV-2 PLpro, potentially leading to the development of novel antiviral therapies. The focus will be on understanding the binding mechanism of promising inhibitor candidates and identifying key structural features that can be exploited for drug design.

Prior research has extensively investigated the structure and function of PLpro in related coronaviruses such as SARS-CoV and MERS-CoV. Studies have revealed the importance of PLpro in viral replication and its role in suppressing the host's immune response. The SARS-CoV PLpro has been shown to remove ISG15 modifications, thereby interfering with the host's innate immune response. This is consistent with the observed functions in SARS-CoV-2 PLpro. Several studies have explored the development of inhibitors targeting PLpro, but the field has been challenged by the complexity of this enzyme's multiple roles and the need to achieve selectivity over host deubiquitinases. The development of effective PLpro inhibitors could offer a crucial strategy for combating COVID-19, especially in the face of emerging viral variants and potential limitations of other therapeutic strategies.

The researchers employed a multi-faceted approach to identify and characterize inhibitors of SARS-CoV-2 PLpro. This involved a high-throughput screening (HTS) of a library of FDA- and CFDA-approved drugs using a fluorescence resonance energy transfer (FRET) assay to measure PLpro enzymatic activity. The assay utilized a fluorogenic peptide substrate, RLRGG-AMC, representing the C-terminal residues of ubiquitin. Initial screening yielded approximately 30 compounds with over 50% inhibition at 100 µM. Following a second round of validation and filtering for solubility and reactivity issues, seven compounds were selected for IC50 determination. In parallel, the authors selected GRL0617 and a related analog (compound 6) from previous studies on SARS-CoV PLpro inhibitors based on the high sequence similarity between SARS-CoV and SARS-CoV-2 PLpro. The in vitro IC50 values of GRL0617 and compound 6 against SARS-CoV-2 PLpro were determined. To assess the in-cell activity of GRL0617, HEK293T cells were transfected with plasmids encoding PLpro and the ISGylation machinery. Cells were then treated with varying concentrations of GRL0617, and changes in ubiquitination and ISGylation levels were examined using immunoblotting. The antiviral activity of GRL0617 was tested in Vero E6 cells infected with SARS-CoV-2. Viral RNA copy numbers were monitored by qRT-PCR to assess the efficacy of the compound. Cytotoxicity was also evaluated using a cell viability assay. To elucidate the mechanism of GRL0617 inhibition, the authors solved the co-crystal structure of SARS-CoV-2 PLproC111S (a cysteine-to-serine mutant at position 111) in complex with GRL0617. This was achieved through the use of X-ray crystallography. To further investigate the interaction between GRL0617 and PLpro, as well as the interaction between PLpro and its substrates, solution state nuclear magnetic resonance (NMR) spectroscopy was performed, focusing on the binding of ISG15 and ubiquitin to PLpro, in the presence and absence of GRL0617. To investigate the role of the ISG15 C-terminus, truncated ISG15 mutants (ISG15-AC6, ISG15-AC5, and ISG15-AC4) were generated, and their binding to SARS-CoV-2 PLpro and related orthologs was examined using isothermal titration calorimetry (ITC) and NMR. Furthermore, site-directed mutagenesis was employed to examine the role of key residues in the interaction between PLpro and ISG15, with the subsequent enzyme activity being assessed.

The high-throughput screening identified GRL0617 as a promising lead compound, exhibiting an in vitro IC50 of 2.1 µM against SARS-CoV-2 PLpro. GRL0617 also demonstrated effective antiviral activity in cell-based assays, with an EC50 of 21 µM. The co-crystal structure revealed that GRL0617 binds non-covalently to the USP domain of PLpro, distinct from the catalytic triad. NMR studies showed that GRL0617 competes with ISG15 for binding to PLpro, effectively blocking the interaction. Further analysis using truncated ISG15 mutants demonstrated the dominant role of the ISG15 C-terminus in the interaction with PLpro. Structural analysis of the PLpro-ISG15 complex highlighted an extensive hydrogen bond and electrostatic interaction network between the C-terminus of ISG15 and key residues on PLpro's BL2 loop. The C-terminus of ISG15 was found to contribute a disproportionately large portion of the binding energy to the interaction, making the binding pocket a key 'hot spot' for drug development. Mutagenesis studies targeting residues involved in the ISG15 C-terminus binding further supported its importance in the enzymatic activity of PLpro. Comparison with known USP7 and USP14 inhibitors revealed that GRL0617 occupies a similar binding pocket, reinforcing its significance as a drug target.

The findings of this study strongly support the druggability of SARS-CoV-2 PLpro and highlight the potential of targeting the ISG15 C-terminus binding site for antiviral drug development. The identification of GRL0617 as a potent non-covalent inhibitor, combined with the structural and biochemical data, provides a strong foundation for future drug design efforts. The observed dominant role of the ISG15 C-terminus in PLpro binding suggests a potential strategy for developing highly specific inhibitors that avoid off-target effects on host deubiquitinases. The study's success in identifying a potent inhibitor through a combination of HTS and structural biology approaches demonstrates a powerful strategy for accelerating antiviral drug discovery. The identification of a "hot spot" in the PLpro structure adds to this, offering a highly promising avenue for future drug discovery efforts.

This study successfully identified GRL0617 as a potent inhibitor of SARS-CoV-2 PLpro, showcasing the druggability of this viral protease. The detailed structural and biochemical characterization of GRL0617's interaction with PLpro, coupled with the discovery of a key "hot spot" for inhibitor binding, provides valuable insights for future drug design. The dominant role of the ISG15 C-terminus in binding suggests a strategy for developing highly specific inhibitors, possibly with reduced off-target effects. Future research should focus on optimizing GRL0617 and related compounds to enhance their potency and selectivity, ultimately leading to effective antiviral therapies against SARS-CoV-2 and potentially other coronaviruses.

The study utilized a C111S mutant of PLpro for co-crystallization due to challenges with crystallizing the wild-type protein. Although the mutation is located outside the GRL0617 binding site, it is possible that subtle differences in conformation or dynamics could exist between the mutant and the wild-type enzyme. The antiviral assays were conducted in a single cell line (Vero E6). Further studies in other relevant cell types, such as human lung cells, are needed to confirm the efficacy and broader applicability of GRL0617. The study focused primarily on the interaction of PLpro with ISG15. A more comprehensive examination of PLpro's interactions with other relevant substrates and cellular targets may provide a more complete understanding of its biological function and the effects of inhibition.