1 Experimental Data Analysis

In the study, the discovery and evaluation of Jun12682 were accomplished through a series of meticulous experimental steps. In vitro experiments demonstrated that Jun12682 has very strong inhibitory activity against PL^pro, with an inhibitory constant (Ki) of only 37.7 nanomolar, and an effective concentration for 50% of maximal effect (EC50) value of 1.1 micromolar in the FlipGFP PL^pro cell assay, indicating its high efficacy in inhibiting the key enzyme activity of the virus. Further in vivo experiments conducted in a mouse model showed that Jun12682 significantly improved the survival rate of mice infected with SARS-CoV-2, mitigated the virus-induced weight loss, and effectively reduced the viral load in the lungs. These experimental data collectively validate the value of Jun12682 as a potential anti-SARS-CoV-2 therapeutic drug.

Figure 1 displays the X-ray crystal structure of the hybrid covalent inhibitor Jun11313, designed based on XR8-24 and Cp7, with SARS-CoV-2 PL^pro. In structure (B), the interactions between Jun11313 and the PL^pro binding site are represented by hydrogen bonds (black dashed lines), with the spatial structure of Jun11313 shown in green ball-and-stick model. Figure (C) overlays the structure of PL^pro with Jun11313 against the XR8-24 complex, highlighting the amino acid residues related to the binding of both compounds. Structure (D) further presents an overlay of the binding modes of Jun11313, XR8-24, and ubiquitin on PL^pro. Structure (E) provides the generic chemical structure of the designed biarylphenyl PL^pro inhibitors and highlights key interactions. Lastly, the flowchart (F) shows the optimization process for PL^pro inhibitors, with Jun12682 being selected as the leading candidate for in vivo studies.

Figure 1 X-ray crystal structure of the covalent inhibitor Jun11313 with SARS-CoV-2 PLpro and structure-based design of biarylphenyl SARS-CoV-2PLpro inhibitors

Figure 2 showcases a series of representative compounds of biarylbenzamide class SARS-CoV-2 PL^pro inhibitors. Item A in the illustration represents the positive control GRL0617; items B, C to F are PL^pro inhibitors containing thiophene and pyrazole. These compounds were characterized for their half-maximal inhibitory concentration (IC50) and inhibitory constant (Ki) through FRET-based enzymatic activity assays, and their cytotoxicity (CC50) in Vero E6 cells was evaluated. Moreover, the EC50 values for FlipGFP PL^pro were tested using 293T cells transfected with PL^pro and FlipGFP. The antiviral activity (EC50) of these compounds against SARS-CoV-2 was assessed in Caco-2 cell lines expressing hACE2 and hTMPRSS2. The figure also provides data on the half-life (T1/2) of PL^pro inhibitors in mouse liver microsomes, with all data presented as the mean ± standard deviation of three technical replicates.

Figure 2 Representative biarylbenzamide series of SARS-CoV-2 PLpro inhibitors

Figure 3 displays the antiviral activity of PL^pro inhibitors against different variants of SARS-CoV-2 and the mechanistic studies of Jun12682. Figures A, B, and C show the inhibitory effects of nirmatrelvir, Jun11941, and Jun12682 on the original WA1 strain, the Delta variant, the Omicron variant, and resistant strains, respectively. Jun12682 exhibits significant antiviral activity against all viral strains, with particularly strong effects against the Omicron variant. Figures D to G present the quantitative results of Jun12682 inhibiting SARS-CoV-2 PL^pro enzymatic activity, including the inhibition of ubiquitin (Ub-AMC) and interferon-stimulated gene 15 (ISG15-AMC) hydrolysis, demonstrating its potent inhibitory action. Figures H and I are screening for inhibition against human proteases USP7 and USP14, where Jun12682 shows selective inhibition of PL^pro over USP7 and USP14. Figure J evaluates the impact of Jun12682 and the control GRL0617 on the stability of SARS-CoV-2 PL^pro through differential scanning fluorimetry, indicating that Jun12682 can effectively stabilize the PL^pro protein. Overall, these data suggest that Jun12682 is a potent SARS-CoV-2 PL^pro inhibitor with potential as a new antiviral drug.

Figure 3 Antiviral activity of PLpro inhibitors against SARS-CoV-2 variants and mechanistic studies of Jun12682

Figure 4 illustrates the X-ray crystal structures of SARS-CoV-2 PL^pro with multiple biarylbenzamide inhibitors, with a particular emphasis on the atomic model of Jun12682 at the PL^pro binding site. This model reveals the hydrogen bonds (black dashed lines), van der Waals contacts (red dashed lines), and π-π interactions (light green dashed lines) between Jun12682 and PL^pro. Additionally, for each compound, the inhibitory constant Ki from FRET enzymatic assays and the antiviral activity EC50 value against SARS-CoV-2 in Caco-2 cells are provided. The "polder map" of the inhibitors, displayed as a gray mesh, reveals the precise morphology of binding to PL^pro, confirming the potential efficacy of these molecules in blocking viral functions.

Figure 4 X-ray crystal structures of SARS-CoV-2 PLpro with biarylphenyl PLpro inhibitors

Figure 5 presents the pharmacokinetic (PK) characteristics of PL^pro inhibitors in vitro and in vivo and the in vivo antiviral efficacy of Jun12682. Figures A and B show the plasma concentration of different compounds in mice. Notably, Jun12682 exhibits favorable pharmacokinetic properties after oral administration (Figure C). Figure D summarizes the in vitro pharmacokinetic parameters of Jun12682, such as microsomal stability and CYP inhibition. Figures E to G and H to J designed treatment experiments for 5 days and 3 days, respectively, to evaluate the efficacy of Jun12682 against the SARS-CoV-2 N501Y variant infection in a mouse model, including changes in body weight and survival rates. Mice treated with Jun12682 showed lower viral loads (Figure K), reduced pathological damage (Figures N and O), and decreased levels of inflammatory factors (Figure M) compared to untreated mice. These results collectively suggest that Jun12682 has potential as an oral antiviral drug against SARS-CoV-2.

Figure 5 In vitro and in vivo PK profiling of PLpro inhibitors and in vivo antiviral efficacy of Jun12682

2 Analysis of Research Findings

In this study, the research team made a breakthrough confirmation that the papain-like protease (PL^pro) of SARS-CoV-2 is an effective drug target. Through detailed structural analysis, they discovered the Val70Ub site on PL^pro for the first time, suggesting its potential as a drug-binding site. This discovery provides a foundation for designing novel antiviral drugs. Using a structure-guided approach, the team successfully developed a series of non-covalent PL^pro inhibitors targeting this new site. Among these inhibitors, Jun12682 was particularly noted for its exceptional efficacy. It not only demonstrated potent PL^pro inhibitory activity in vitro but also proved to effectively inhibit SARS-CoV-2 replication in a mouse model, showing its great potential as a therapeutic drug. The discovery of Jun12682 validates the strategy of effectively blocking SARS-CoV-2 replication by targeting the Val70Ub site on PL^pro, and it opens new directions for future drug development against SARS-CoV-2 and potential variants of coronaviruses. This breakthrough holds significant importance for current COVID-19 treatment research and offers new insights and strategies for the global scientific community in exploring more effective methods for viral treatment.

3 Evaluation of the Research

This study, by integrating advanced methods from structural biology and medicinal chemistry, successfully identified Jun12682 as a potential drug candidate against the SARS-CoV-2 virus, demonstrating the research team's exceptional capability in scientific innovation. Through precise analysis of the structure and function of the PL^pro enzyme, the research not only deepened our understanding of viral biology but also showed how to translate complex scientific knowledge into practical therapeutic strategies. The discovery of Jun12682 validates the feasibility, both theoretically and practically, of therapies targeting the SARS-CoV-2 PL^pro enzyme, offering new hope and possibilities for combating the current pandemic and potential future coronavirus variants.

4 Conclusions

Jun12682, as a novel PL^pro inhibitor, has shown significant anti-SARS-CoV-2 activity both in vitro and in vivo, offering not only a new potential means to combat the COVID-19 pandemic but also new directions and strategies for the development of therapeutic drugs for coronavirus diseases. This work highlights the importance of PL^pro as a drug target and demonstrates the power of structure-guided design methods in modern drug discovery.

5 Access the Full Text

Bin Tan et al., Design of a SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model. Science 383, 1434-1440 (2024). DOI:10.1126/science.adm9724

Acknowledgments

I sincerely thank Science for its open access policy provided to its wide readership. This policy allows readers like me to download, read, analyze, and share this outstanding research achievement without any fees. By doing so, Science not only significantly promotes the dissemination of scientific knowledge but also opens a precious resource of knowledge to researchers, students, and those passionate about science worldwide. The concept of open access greatly advances the pace of scientific development and plays a crucial role in enhancing the public's understanding and interest in scientific work. I express my gratitude again to Science for its contribution and look forward to discovering more exciting content in the scientific world through this platform in the future.