title: Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.
abstract: Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Hong
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Guangyi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Josh
authors.initials: J
authors.lastname: Taylor
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Lucas K
authors.initials: LK
authors.lastname: Smith
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Fung
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Stevens
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Natalia
authors.initials: N
authors.lastname: Dyatkina
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Marija
authors.initials: M
authors.lastname: Prhavc
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Antitsa D
authors.initials: AD
authors.lastname: Stoycheva
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Serebryany
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Jyanwei
authors.initials: J
authors.lastname: Liu
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: David B
authors.initials: DB
authors.lastname: Smith
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Yuen
authors.initials: Y
authors.lastname: Tam
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Qingling
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States of America; Children's Healthcare of Atlanta, Atlanta, Georgia, United States of America.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Moore
authors.affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Fearns
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Sushmita M
authors.initials: SM
authors.lastname: Chanda
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Blatt
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Symons
authors.affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.
authors.forename: Leo
authors.initials: L
authors.lastname: Beigelman
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D018357:Respiratory Syncytial Virus Infections / Q000188:drug therapy* / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000187:drug effects* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
chemical_list: C000606051:2'-fluoro-4'-chloromethyl-cytidine triphosphate; D000998:Antiviral Agents; D012367:RNA, Viral; D014764:Viral Proteins; D003570:Cytidine Triphosphate; D012321:DNA-Directed RNA Polymerases