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Cell ; 182(2): 417-428.e13, 2020 07 23.
Article in English | MEDLINE | ID: covidwho-342735


Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Betacoronavirus/chemistry , Betacoronavirus/enzymology , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/metabolism , Alanine/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Catalytic Domain , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Models, Chemical , Models, Molecular , RNA, Viral/metabolism , SARS-CoV-2 , Transcription, Genetic , Virus Replication
Nat Struct Mol Biol ; 27(6): 529-532, 2020 06.
Article in English | MEDLINE | ID: covidwho-222247


The antineoplastic drug carmofur is shown to inhibit the SARS-CoV-2 main protease (Mpro). Here, the X-ray crystal structure of Mpro in complex with carmofur reveals that the carbonyl reactive group of carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 µM) and is a promising lead compound to develop new antiviral treatment for COVID-19.

Betacoronavirus/enzymology , Cysteine Endopeptidases/chemistry , Fluorouracil/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Animals , Betacoronavirus/drug effects , COVID-19 , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus Infections/virology , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Fluorouracil/chemistry , Fluorouracil/pharmacology , Models, Molecular , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism