The COVID‐19 pathogen, SARS‐CoV‐2, requires its main protease (SC2M^Pro) 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 (SC1M^Pro), we have designed and synthesized a series of SC2M^Pro inhibitors that contain β‐(S‐2‐oxopyrrolidin‐3‐yl)‐alaninal (Opal) for the formation of a reversible covalent bond with the SC2M^Pro active‐site cysteine C145. All inhibitors display high potency with K_i values at or below 100 nM. The most potent compound, MPI3, has as a K_i value of 8.3 nM. Crystallographic analyses of SC2M^Pro 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 SC2M^Pro inhibitors with ultra‐high antiviral potency.

Significance: Guided by a computational docking analysis, we experimentally characterized about 30 FDA/EMA-approved drugs on their inhibition of essential main protease of SARS-CoV-2, the pathogen of COVID-19. From this study, we discovered that bepridil, an antianginal medication, is potent against SARS-CoV-2. The antiviral analysis of bepridil indicated that it had low micromolar EC ₅₀ values in inhibiting SARS-CoV-2 in two highly permissive mammalian cell lines. Due to the urgent matter of COVID-19, our current study encourages further preclinical investigations of bepridil in animal models to clear its path for clinical uses in COVID-19 patients.

Significance: Effective therapies are urgently needed for COVID-19. We rapidly (within a week) identified a fully human monoclonal germline-like antibody (ab1) from phage-displayed libraries that potently inhibited mouse ACE2-adapted SARS-CoV-2 replication in wild-type BALB/c mice and native virus in transgenic mice expressing human ACE2 as well as in hamsters when administered before virus challenge. It was also effective when administered after virus infection of hamsters, although at lower efficacy than when used prophylactically. Ab1 was highly specific and did not bind to human cell membrane-associated proteins. It also exhibited good developability properties including complete lack of aggregation. Ab1 has potential for prophylaxis and therapy of COVID-19 alone or in combination with other agents.