Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Chunlong Ma; Brett Hurst; Yanmei Hu; Tommy Szeto; Bart Tarbet; Jun Wang

Coronaviruses have caused three epidemics/pandemics in the past twenty years including SARS, MERS, and COVID-19. With the ongoing pandemic of COVID-19, scientists and researchers around the globe are racing to find effective vaccines and antiviral drugs. 24 The viral polymerase inhibitor remdesivir holds the greatest promise and it is currently being evaluated in several clinical trials. 25, 26 The HIV drug combination lopinavir and ritonavir recently failed in a clinical trial for COVID-19 with no significant therapeutic efficacy was observed. 27 To address this unmet medical need, we initiated a drug repurposing screening to identify potent inhibitors against the SARS-CoV-2 M pro from a collection of FDA-approved protease inhibitors. The M pro has been shown to be a validated antiviral drug target for SARS and MERS. 28 As the SARS-CoV-2 M pro shares a high sequence similarity with SARS and to a less extent with MERS, we reasoned that inhibiting the enzymatic activity of SARS-CoV-2 M pro will similarly prevent viral replication. 7, 9 Noticeable findings from our study include: 1) Boceprevir (28) All potent SARS-CoV-2 M pro inhibitors contain reactive warheads such as α-ketoamide (boceprevir (28) , calpain inhibitor XII (62)) or aldehyde (MG-132 (43), calpain inhibitor II (61)) or aldehyde prodrug, the bisulfite (GC-376 (64)). This result suggests that reactive warheads might be essential for SARS-CoV-2 M pro inhibition. The compounds identified in this study represent the most potent and selective hits reported so far, and are superior than recently reported SARS-CoV-2 M pro inhibitors ebselen, N3, and 13b (Table 5 ). Aside from the above positive results, we also showed that ritonavir (9) and lopinavir (10) In summary, this study identified several potent SARS-CoV-2 M pro inhibitors with potent enzymatic inhibition as well as potent cellular antiviral activity. Further development based on these hits might lead to clinically useful COVID-19 antivirals. They can be used either alone or in combination with polymerase inhibitors such as remdesivir as a means to achieve potential synergic antiviral effect as well as to suppress drug resistance. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.20.051581 doi: bioRxiv preprint were maintained in RPMI 1640 medium. Both medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cells were kept at 37°C in a 5% CO2 atmosphere. The USA_WA1/2020 strain of SARS-CoV-2 obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). 2Apro and 3Cpro were expressed in the pET28b(+) vector as previously described 15, 32, 33 .