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J Biol Chem ; 297(2): 100925, 2021 08.
Article in English | MEDLINE | ID: covidwho-1336599


Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

Famotidine/pharmacology , Histamine Antagonists/pharmacology , SARS-CoV-2/drug effects , Toll-Like Receptor 3/metabolism , A549 Cells , Binding Sites , Caco-2 Cells , Chemokine CCL2/metabolism , Coronavirus 3C Proteases/metabolism , HeLa Cells , Humans , Interferon Regulatory Factor-3/metabolism , Interleukin-6/metabolism , Molecular Docking Simulation , NF-kappa B/metabolism , Protein Binding , SARS-CoV-2/physiology , Signal Transduction , Toll-Like Receptor 3/chemistry , Virus Replication
EMBO J ; 39(18): e106275, 2020 09 15.
Article in English | MEDLINE | ID: covidwho-730426


The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/metabolism , Ubiquitin/metabolism , Animals , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Crystallography, X-Ray , Cytokines/genetics , Drug Evaluation, Preclinical/methods , Drug Repositioning , Fluorescence Polarization , HEK293 Cells , Humans , Kinetics , Models, Molecular , Protease Inhibitors/pharmacology , Protein Conformation , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Ubiquitins/genetics , Vero Cells
Nature ; 587(7835): 657-662, 2020 11.
Article in English | MEDLINE | ID: covidwho-691112


The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

COVID-19/immunology , COVID-19/virology , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Immunity, Innate , SARS-CoV-2/enzymology , SARS-CoV-2/immunology , Animals , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Cytokines/chemistry , Cytokines/metabolism , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Humans , Interferon Regulatory Factor-3/metabolism , Interferons/immunology , Interferons/metabolism , Mice , Models, Molecular , Molecular Dynamics Simulation , NF-kappa B/immunology , NF-kappa B/metabolism , Protein Binding , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Ubiquitination , Ubiquitins/chemistry , Ubiquitins/metabolism