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

There are very fewsmall-molecule antivirals for SARS-CoV-2thatare either currently approved (or emergency authorized) in the USor globally, including remdesivir, molnupiravir, and paxlovid. Theincreasing number of SARS-CoV-2 variants that have appeared sincethe outbreak began over three years ago raises the need for continualdevelopment of updated vaccines and orally available antivirals inorder to fully protect or treat the population. The viral main protease(M-pro) and the papain-like protease (PLpro)are key for viral replication; therefore, they represent valuabletargets for antiviral therapy. We herein describe an in vitro screenperformed using the 2560 compounds from the Microsource Spectrum libraryagainst M-pro and PLpro in an attempt to identifyadditional small-molecule hits that could be repurposed for SARS-CoV-2.We subsequently identified 2 hits for M-pro and 8 hits forPL(pro). One of these hits was the quaternary ammonium compoundcetylpyridinium chloride with dual activity (IC50 = 2.72 +/- 0.09 mu M for PLpro and IC50 = 7.25 +/- 0.15 mu M for M-pro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene(IC50 = 3.28 +/- 0.29 mu M for PLpro andIC(50) = 42.8 +/- 6.7 mu M for M-pro). Weadditionally tested several kinase inhibitors and identified olmutinib(IC50 = 0.54 +/- 0.04 mu M), bosutinib (IC50 = 4.23 +/- 0.28 mu M), crizotinib (IC50 = 3.81 +/- 0.04 mu M), and dacominitinib (IC50 = IC50 3.33 +/- 0.06 mu M) as PLpro inhibitorsfor the first time. In some cases, these molecules have also beentested by others for antiviral activity for this virus, or we haveused Calu-3 cells infected with SARS-CoV-2. The results suggest thatapproved drugs can be identified with promising activity against theseproteases, and in several cases we or others have validated theirantiviral activity. The additional identification of known kinaseinhibitors as molecules targeting PLpro may provide newrepurposing opportunities or starting points for chemical optimization.