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

TLDR

The COVID‑19 pandemic continues to increase morbidity and mortality daily. The study aims to characterize the mechanism of action of four SARS‑CoV‑2 main protease inhibitors—boceprevir, calpain inhibitors II and XII, and GC‑376—using pseudovirus neutralization assays. The compounds act by inhibiting viral Mpro, as shown by thermal shift‑binding and fluorescence resonance energy transfer assays, and their antiviral activity was confirmed in pseudovirus neutralization and Caco‑2 viral yield reduction assays. The four inhibitors exhibit potent, broad‑spectrum antiviral activity against SARS‑CoV‑2, SARS‑CoV, MERS‑CoV, and human coronaviruses 229E, OC43, and NL63, inhibit both Mpro and host cathepsin L, and show additive effects with remdesivir, indicating their potential as therapeutic leads.

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 (Mpro) 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 Mpro 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 Mpro, 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.