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Abstract
The 2003-2005 SARS epidemic and currently ongoing COVID-19 pandemic highlight the threat that zoonotic coronaviruses pose to humans. Both SARS-CoV-1 and SARS-CoV-2 belong to a closely related subgroup of betacoronaviruses known as 2b that primarily infect bats throughout southeast Asia. Coronaviruses encode a papain-like protease (PLpro) that is critical their success. PLpros are required for assembly of the viral replicase complex, without which the RNA genome cannot be replicated. Additionally, PLpros suppress the host’s innate immune response through reversal of post-translational modifications by ubiquitin (Ub), and the Ub-like protein (Ubl) interferon (IFN)-stimulated gene product 15 (ISG15). Due to the importance of PLpros they are common targets of drug discovery research, and some small molecules have been designed with the ability to inhibit SARS-CoV-1 PLpro activity. The PLpros of subgroup 2b coronaviruses are highly conserved, sharing upwards of 80 percent sequence identity. Here we characterize the activity of the PLpros of SARS-CoV-2 as well as subgroup 2b virus isolated from the greater horseshoe bat, BtSCoV-Rf1.2004, against Ub and ISG15 from various species. We then investigate the ability of naphthalene-based compound to noncovalently inhibit the PLpros of SARS-CoV-1, SARS-CoV-2, and BtSCoV-Rf1.2004, and their potential for use as pan subgroup 2b coronavirus therapeutics. To this end we obtained the X-ray crystal structures BtSCoV-Rf1.2004 in complex with two different series of naphthalene-based compounds for use in structure-based drug design. Furthermore, we structurally characterize Ubl domain of the immunoregulatory protein oligoadenylate synthetase-like (OASL), and analyze its interactions with various coronavirus PLpros as well as the ovarian tumor domain proteases (OTUs) of multiple nairoviruses. This is the first comparative investigation into the activity and inhibition of subgroup 2b PLpros. It validates the use of naphthalene-based inhibitors against novel subgroup 2b coronaviruses, and provides insight that will useful in optimizing these compounds for use as therapeutics.