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Viruses ; 13(12)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1572668


Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.

Antimetabolites/pharmacology , Antiviral Agents/pharmacology , RNA Viruses/drug effects , Adenosine Triphosphate/metabolism , Amides/pharmacology , Animals , Cell Line , Drug Synergism , Guanosine Triphosphate/metabolism , Humans , Methylthioinosine/pharmacology , Mutation/drug effects , Phosphoribosyl Pyrophosphate/metabolism , Pyrazines/pharmacology , RNA Viruses/classification , RNA Viruses/genetics , RNA, Viral/drug effects , RNA, Viral/genetics , Virus Replication/drug effects
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1343703


The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

Nidovirales/enzymology , Nucleotides/metabolism , Protein Domains , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Coenzymes/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Diphosphates/pharmacology , Diphosphonates/pharmacology , Guanosine Triphosphate/metabolism , Manganese , Models, Molecular , Nidovirales/chemistry , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Uridine Triphosphate/metabolism
Life Sci ; 248: 117477, 2020 May 01.
Article in English | MEDLINE | ID: covidwho-2799


AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.

Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Coronavirus Infections/drug therapy , Guanosine Monophosphate/analogs & derivatives , Pneumonia, Viral/drug therapy , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Ribavirin/chemistry , Sofosbuvir/chemistry , Viral Proteins/antagonists & inhibitors , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Alphacoronavirus/enzymology , Alphacoronavirus/genetics , Amino Acid Sequence , Antiviral Agents/metabolism , Betacoronavirus/genetics , COVID-19 , Catalytic Domain , Computational Biology/methods , Coronavirus Infections/virology , Drug Repositioning/methods , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Humans , Molecular Docking Simulation , Pneumonia, Viral/virology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Ribavirin/metabolism , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Sofosbuvir/metabolism , Thermodynamics , Uridine Triphosphate/chemistry , Uridine Triphosphate/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism