The COVID-19 pandemic, caused by SARS-CoV-2, presented an urgent need for effective antiviral therapeutics. While remdesivir offered some treatment, the lack of specific antivirals highlighted a critical gap. SARS-CoV-2, a betacoronavirus, shares significant genomic similarity (~80% nucleotide identity) with SARS-CoV, and their main proteases (Mpro) exhibit high amino acid sequence identity (96%). This high degree of conservation in Mpro structure and function across related coronaviruses suggested that targeting this protease could be a viable strategy for developing broad-spectrum antiviral agents. The authors leveraged this structural homology, focusing on existing compounds with known activity against SARS-CoV, to explore their potential against SARS-CoV-2. The study's primary objective was to identify and characterize potent small molecule inhibitors targeting the SARS-CoV-2 Mpro, ultimately contributing to the development of novel therapeutics for COVID-19. The importance of this research stemmed from the global health crisis and the desperate need for effective treatment options beyond remdesivir.

The research builds upon previous work on SARS-CoV and other coronaviruses, notably focusing on the main protease (Mpro) as a drug target. Several studies had demonstrated the effectiveness of targeting Mpro in related coronaviruses. The authors specifically mention prior research on the design and synthesis of inhibitors for SARS-CoV Mpro, including chloropyridyl ester derivatives and dipeptide-type inhibitors. The high degree of conservation between SARS-CoV and SARS-CoV-2 Mpro structures, as evidenced by sequence alignments and structural superposition, provided a strong rationale for investigating these existing compounds' efficacy against SARS-CoV-2. The review implicitly points to the limitations of existing treatments, including remdesivir, and the need for more effective and less toxic antiviral options. Furthermore, the review highlights the importance of understanding the molecular interactions of potential inhibitors with Mpro to optimize their efficacy and safety profiles.

The study employed a multi-faceted approach combining in vitro assays with structural analysis. Two compounds, GRL-1720 and 5h, were selected and synthesized based on prior success against SARS-CoV Mpro. Their inhibitory activity against SARS-CoV-2 Mpro was initially assessed using a continuous fluorescence assay with a FRET-based substrate. Kinetic parameters, including IC50 and Ki values, were determined for both compounds. The antiviral activity of GRL-1720, 5h, and remdesivir was evaluated using VeroE6 cells. These assays employed RNA-qPCR to quantify viral RNA levels, cytopathic assays to assess cell damage, and immunocytochemistry to visualize viral infection at the cellular level. The cytotoxicity of the compounds was determined using Cell Counting Kit-8 (CCK-8) assays on VeroE6, Calu-3, PBMCs, and HBTECs. Air-liquid interface (ALI) cultures of human airway epithelial cells were also used to assess the effects of the compounds under more physiologically relevant conditions. X-ray crystallography was used to determine the three-dimensional structure of the SARS-CoV-2 Mpro in complex with 5h, revealing the molecular interactions responsible for inhibition. Mass spectrometry analysis (nanoLC-ESI-QTOF-MS) was conducted to investigate the nature of the interaction between GRL-1720/5h and Mpro, specifically exploring the possibility of covalent bonding. Finally, differential scanning fluorimetry (DSF) was used to assess the thermal stability of Mpro in the presence and absence of the compounds to further characterize their binding interactions. Synergistic effects of combining 5h and remdesivir were evaluated using both RNA-qPCR and immunocytochemistry assays.

Both GRL-1720 and 5h demonstrated potent inhibitory activity against SARS-CoV-2 Mpro in enzymatic assays. GRL-1720 showed irreversible covalent inhibition, while 5h exhibited reversible covalent inhibition. Cell-based assays revealed that both compounds effectively blocked SARS-CoV-2 infectivity in VeroE6 cells, with 5h exhibiting superior potency (EC50 of 4.2 ± 0.7 µM compared to 15 ± 4 µM for GRL-1720). Notably, 5h completely inhibited SARS-CoV-2 infection at 20 µM without detectable cytotoxicity, unlike remdesivir, which showed viral breakthrough at this concentration. Immunocytochemistry confirmed the potent antiviral activity of both compounds, demonstrating a significant reduction in the number of infected cells. The combination of 5h and remdesivir exhibited synergistic antiviral activity, achieving a significantly greater reduction in viral RNA copies than either compound alone. X-ray crystallography revealed that 5h forms a covalent bond with Cys-145 of Mpro and interacts with multiple active site residues through hydrogen bonds and hydrophobic interactions. Mass spectrometry confirmed the covalent binding of GRL-1720 with Mpro, but indicated that the covalent bond formed by 5h was reversible. DSF experiments showed that 5h increased the thermal stability of Mpro, further supporting its strong interaction. In contrast, compounds like shikonin, nelfinavir, and atazanavir, previously reported to have anti-SARS-CoV-2 activity, showed no significant antiviral effect in this study, highlighting the importance of rigorous assessment of cytotoxicity alongside antiviral activity.

The study successfully identified compound 5h as a potent and non-cytotoxic inhibitor of SARS-CoV-2 Mpro. Its superior potency compared to GRL-1720 and remdesivir, along with its lack of cytotoxicity, suggests its potential as a promising lead compound for the development of novel COVID-19 therapeutics. The synergistic effect observed when combining 5h with remdesivir indicates that a combination therapy approach, targeting both Mpro and RdRp, could be highly effective. The structural analysis provides valuable insights into the molecular mechanisms underlying the inhibitory activity of 5h, guiding further optimization efforts. The findings emphasize the critical need to differentiate between true antiviral activity and the potentially misleading effects of cytostatic or cytotoxic compounds. The discrepancies observed with previously reported compounds highlight the importance of rigorous validation using multiple assays. The study strongly supports the continued development of compound 5h, potentially in combination with other antivirals, for the treatment of COVID-19.

This research successfully identified compound 5h as a potent and safe inhibitor of SARS-CoV-2 main protease. Its superior performance compared to existing treatments, along with the observed synergism when combined with remdesivir, makes it a promising candidate for therapeutic development. The detailed structural and biochemical analyses provide a strong foundation for future optimization and the exploration of combination therapies. Future research should focus on preclinical studies to evaluate the efficacy and safety of 5h in animal models and ultimately in human clinical trials. Furthermore, exploring modifications to 5h to enhance its potency and pharmacokinetic properties should be a priority.

The study primarily used in vitro cell culture models. While these provide valuable initial data, the results might not fully translate to the complex in vivo environment. The study focused on a single SARS-CoV-2 strain; additional research is needed to assess the efficacy of 5h against other emerging variants. The synergistic effect observed with remdesivir needs further exploration to determine the optimal dosage and administration regimen for combination therapy. Although mass spectrometry suggested a reversible covalent interaction for compound 5h, further investigation into the exact nature of this bond may be warranted.