ABSTRACT As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an M Pro -eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of M Pro inhibitors. In comparison to an antiviral assay in which M Pro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular M Pro inhibition information for assessment and optimization of M Pro inhibitors. We used this assay to analyze 30 literature reported M Pro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of M Pro , GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit M Pro . Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 μM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular M Pro inhibition potency implicating their roles in interfering with key steps other than just the M Pro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit M Pro . Kinetic recharacterization of these two compounds rules out their possibility as M Pro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC 50 and EC 50 values respectively below 1 μM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M Pro inhibition IC 50 value of 31 nM that matches closely to its strong antiviral effect with an EC 50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics