title: Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase.
abstract: ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Jerome
authors.identifier: 0000-0001-8369-907X
authors.initials: J
authors.lastname: Deval
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Fung
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Stevens
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Jordan
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Gromova
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Taylor
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Hong
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Jia
authors.initials: J
authors.lastname: Meng
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Guangyi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Natalia
authors.initials: N
authors.lastname: Dyatkina
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Marija
authors.initials: M
authors.lastname: Prhavc
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Symons
authors.affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.
authors.forename: Leo
authors.initials: L
authors.lastname: Beigelman
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001552:Benzazepines / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004338:Drug Combinations; D024882:Drug Resistance, Viral / Q000235:genetics*; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000821:virology; D015870:Gene Expression; D006801:Humans; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D017354:Point Mutation; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: C000606051:2'-fluoro-4'-chloromethyl-cytidine triphosphate; C000601049:AZ-27 compound; D000998:Antiviral Agents; D001552:Benzazepines; D004338:Drug Combinations; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D009536:Niacinamide; D003570:Cytidine Triphosphate; D012321:DNA-Directed RNA Polymerases