Discovery of PL^pro and M^pro Inhibitors for SARS-CoV-2

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (M^pro) and the papain-like protease (PL^pro) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against M^pro and PL^pro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for M^pro and 8 hits for PL^pro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC₅₀ = 2.72 ± 0.09 μM for PL^pro and IC₅₀ = 7.25 ± 0.15 μM for M^pro). A second inhibitor of PL^pro was the selective estrogen receptor modulator raloxifene (IC₅₀ = 3.28 ± 0.29 μM for PL^pro and IC₅₀ = 42.8 ± 6.7 μM for M^pro). We additionally tested several kinase inhibitors and identified olmutinib (IC₅₀ = 0.54 ± 0.04 μM), bosutinib (IC₅₀ = 4.23 ± 0.28 μM), crizotinib (IC₅₀ = 3.81 ± 0.04 μM), and dacominitinib (IC₅₀ = IC₅₀ 3.33 ± 0.06 μM) as PL^pro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PL^pro may provide new repurposing opportunities or starting points for chemical optimization.