Boceprevir, Calpain Inhibitors II and XII, and GC-376 Have Broad-Spectrum Antiviral Activity against Coronaviruses

Abstract

As the COVID-19 pandemic continues to unfold, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (M^pro) inhibitors including boceprevir, calpain inhibitors II and XII, and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. In this study, we further characterized the mechanism of action of these four compounds using the SARS-CoV-2 pseudovirus neutralization assay. It was found that GC-376 and calpain inhibitors II and XII have a dual mechanism of action by inhibiting both viral M^pro and host cathepsin L in Vero cells. To rule out the cell-type dependent effect, the antiviral activity of these four compounds against SARS-CoV-2 was also confirmed in type 2 transmembrane serine protease-expressing Caco-2 cells using the viral yield reduction assay. In addition, we found that these four compounds have broad-spectrum antiviral activity in inhibiting not only SARS-CoV-2 but also SARS-CoV, and MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral M^pro, which was supported by the thermal shift-binding assay and enzymatic fluorescence resonance energy transfer assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 might be promising starting points for further development against existing human coronaviruses as well as future emerging CoVs.

Keywords: COVID-19; GC-376; SARS-CoV-2; boceprevir; calpain inhibitor; main protease.

Figure 1.

Chemical structures of SARS-CoV-2 M^pro inhibitors boceprevir, calpain inhibitors II and XII and GC-376.

Figure 2.

Inhibitory activity of GC-376, calpain inhibitors II and XII, and boceprevir in the SARS-CoV-2 pseudovirus replication assay. Effect of E-64d (A), camostat (B), GC-376 (C), calpain inhibitor II (D), calpain inhibitor XII (E), and boceprevir (F) on SARS-CoV-2 pseudovirus amplification in the presence of absence of 2 μM P-gp inhibitor CP-100356. (G). Inhibition of cathepsin L in the FRET-based enzymatic assay. EC₅₀ curve fittings using log (concentration of inhibitors) vs percentage of inhibition with variable slopes were performed in Prism 8. Data are mean ± standard deviation of two replicates.

Figure 3.

Viral yield reduction assay of SARS-CoV-2 in Caco-2 cells. (A-D) Raw data showing the SARS-CoV-2 viral titers in the presence of different concentrations of testing compounds. (G-F) Curve fitting for the antiviral EC₅₀ values. Data are mean ± standard deviation of three independent replicates.

Figure 4.

Effect of GC-376, calpain inhibitors II and XII, and boceprevir on melting temperature (T_m) of MERS-CoV M^pro (A), SARS-CoV M^pro (B), and HCoV-OC43 M^pro (C). Data were plotted with ΔT_m vs log₁₀ (concentrations of compound) using Boltzmann Sigmoidal equation in Prism 8. Data are mean ± standard deviation of two replicates. (D). Melting temperature shift (ΔT_m) of MERS-CoV, SARS-CoV, and HCoV-OC43 M^pro in the presence of indicated concentrations of boceprevir, calpain inhibitors II and XII, and GC-376. ^aN.T. = not tested.

Figure 5.

Data fittings of the proteolytic reaction progression curves of MERS-CoV M^pro (left column), SARS-CoV M^pro (middle column) and HCoV-OC43 M^pro (right column) in the presence or the absence of GC-376 (A); calpain inhibitor II (B); calpain inhibitor III (C); and boceprevir (D). In the kinetic studies, 60 nM MERS-CoV M^pro, or 5 nM SARS-CoV M^pro or 3.3 nM HCoV-OC43 M^pro was added to a solution containing various concentrations of compounds and 20 μM FRET substrate to initiate the reaction. Detailed methods were described in “Materials and methods” section. Data are mean ± standard deviation of two replicates. (E) Enzymatic inhibition of boceprevir, Calpain inhibitor II and XII, and GC-376 against various CoV M^pros. ^aData from reference.

Figure 6.

Dose-dependent inhibitory effect of GC-376, calpain inhibitors II and XII, and boceprevir on HCoV-NL63 viral RNA synthesis in Vero E6 cells using RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC₅₀ curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.

Figure 7.

Combination therapy of remdesivir with GC-376 (A); Calpain inhibitor II (B); Calpain inhibitor XII (C); and Boceprevir (D). Data are mean ± standard deviation of three replicates.