ABSTRACT
A recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses. One Sentence Summary: A repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.
ABSTRACT
The emergence of SARS-CoV-2 has led to a global health crisis that, in addition to vaccines and immunomodulatory therapies, calls for the identification of antiviral therapeutics. The papain-like protease (PLpro) activity of nsp3 is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG), an orally available and widely available generic drug, inhibits SARS-CoV-2 replication in Vero-E6 cells with an EC50 of approximately 2 µM. 6-TG also inhibited PLpro-catalyzed polyprotein cleavage and de-ISGylation in cells and inhibited proteolytic activity of the purified PLpro domain in vitro. We therefore propose that 6-TG is a direct-acting antiviral that could potentially be repurposed and incorporated into the set of treatment and prevention options for COVID-19.
ABSTRACT
ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.
Subject(s)
Cytokines/metabolism , Signal Transduction , Ubiquitins/metabolism , Animals , HEK293 Cells , Humans , Influenza, Human/immunology , Influenza, Human/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Jurkat Cells , Mice , Mice, Inbred C57BL , Mycobacterium Infections/immunology , Mycobacterium Infections/metabolism , Pathogen-Associated Molecular Pattern Molecules , Typhoid Fever/immunology , Typhoid Fever/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
A recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses. ONE SENTENCE SUMMARY: A repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.