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

Abstract

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 (Mpro) and the papain-like protease (PLpro) 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 Mpro and PLpro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for Mpro and 8 hits for PLpro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC50 = 2.72 ± 0.09 μM for PLpro and IC50 = 7.25 ± 0.15 μM for Mpro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene (IC50 = 3.28 ± 0.29 μM for PLpro and IC50 = 42.8 ± 6.7 μM for Mpro). 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 PLpro 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 PLpro may provide new repurposing opportunities or starting points for chemical optimization.

Dual inhibition of coronavirus Mproand PLproenzymes by phenothiazines and their antiviral activity

“…Other studies have shown M pro activity against host proteins involved in immune response, such as galectin-8 [Pablos et al, 2021; Chen et al, 2023]. The proteolytic and immune modulating functions of these enzymes make M pro and PL pro attractive targets for potential antiviral drug intervention [Owen et al, 2021; Cao et al, 2023; Puhl et al, 2023]…”