International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 56-60 http://medscipublisher.com/index.php/ijmms 58 Figure 3 displays the antiviral activity of PLpro 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 PLpro 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 PLpro over USP7 and USP14. Figure J evaluates the impact of Jun12682 and the control GRL0617 on the stability of SARS-CoV-2 PLpro through differential scanning fluorimetry, indicating that Jun12682 can effectively stabilize the PLpro protein. Overall, these data suggest that Jun12682 is a potent SARS-CoV-2 PLpro inhibitor with potential as a new antiviral drug. Figure 4 illustrates the X-ray crystal structures of SARS-CoV-2 PLpro with multiple biarylbenzamide inhibitors, with a particular emphasis on the atomic model of Jun12682 at the PLpro 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 PLpro. 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 PLpro, confirming the potential efficacy of these molecules in blocking viral functions. Figure 5 presents the pharmacokinetic (PK) characteristics of PLpro 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 3 Antiviral activity of PLpro inhibitors against SARS-CoV-2 variants and mechanistic studies of Jun12682
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