title: Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases.
abstract: Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2'-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2'-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA jdeval@aliosbiopharma.com zjin@aliosbiopharma.com.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Tucker
authors.affiliation: Indiana University, Bloomington, Indiana, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Lin
authors.affiliation: Indiana University, Bloomington, Indiana, USA.
authors.forename: C Cheng
authors.initials: CC
authors.lastname: Kao
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Ken
authors.initials: K
authors.lastname: Shaw
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Tan
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Symons
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Ishani
authors.initials: I
authors.lastname: Behera
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Rajwanshi
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Dyatkina
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Guangyi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Leo
authors.initials: L
authors.lastname: Beigelman
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA jdeval@aliosbiopharma.com zjin@aliosbiopharma.com.
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D058507:Host Specificity; D006801:Humans; D007700:Kinetics; D051379:Mice; D029322:Norovirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D011719:Pyrazines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000577:Amides; D000998:Antiviral Agents; D011719:Pyrazines; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014764:Viral Proteins; C513402:2'-C-methylcytidine; D003562:Cytidine; D012321:DNA-Directed RNA Polymerases; C462182:favipiravir