9-aminominocycline potentiates the efficacy of EIDD-1931 and PF-332 by targeting the papain like protease enzyme of SARS-CoV-2

The 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), and RNA-dependent RNA polymerase (RdRp) are key enzymes in SARS-CoV-2 replication and serve as critical targets for an antiviral drug. Currently, Paxlovid® and Lagevrio™ specifically target 3CLpro and RdRp, respectively, for COVID-19 treatment. However, no antivirals target for the SARS-CoV-2 PLpro enzyme, essential for viral replication and suppression of the host antiviral immune response. This study identified 9-aminominocycline (9-AMN) as a potent inhibitor of SARS-CoV-2 PLpro. Unlike the parent compound minocycline, 9-AMN inhibits PLpro's proteolytic and deubiquitinase activities by approximately 90%, with IC₅₀ values of 4.15 µM and 4.55 µM, respectively, while showing no effect on the enzymatic activity of 3CLpro or RdRp. Enzyme kinetics reveal that 9-AMN functions as a mixed PLpro inhibitor and binds to its active site, disrupting its function as predicted by computer modeling. Furthermore, 9-AMN demonstrates, efficacy against the Delta and Omicron variants, with EC₅₀ values of 1.04 µM and 2.35 µM, respectively. When combined with EIDD-1931 (an active form of molnupiravir) or nirmatrelvir (PF-332), 9-AMN exhibits synergistic effects, significantly reducing the doses required to inhibit the Omicron variant. In conclusion, 9-AMN inhibits SARS-CoV-2 replication, and PLpro activity, highlighting its potential as a promising candidate for COVID-19 treatment strategies.