The COVID‐19 pathogen, SARS‐CoV‐2, requires its main protease (SC2MPro) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID‐19 treatment option. Guided by previous medicinal chemistry studies about SARS‐CoV‐1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain β‐(S‐2‐oxopyrrolidin‐3‐yl)‐alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active‐site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS‐CoV‐2‐induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS‐CoV‐2‐induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID‐19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra‐high antiviral potency.

As an essential enzyme of SARS-CoV-2, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M Pro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M Pro inhibition information to assess an M Pro inhibitor. We used this assay to analyze 30 known M Pro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine 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. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. 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. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

A number of inhibitors have been developed for the SARS‐CoV‐2 main protease (M Pro ) as potential COVID‐19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed M Pro inhibitors including MPI1‐9, GC376, 11a, 10–1, 10–2, and 10–3. MPI1‐9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the M Pro active site cysteine and 10–1, 10–2 and 10–3 contain a labile ester to exchange with the M Pro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC 50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular M Pro inhibition potency and anti‐SARS‐CoV‐2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS‐CoV‐2 cell entry. By selectively inhibiting both SARS‐CoV‐2 M Pro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID‐19.

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