title: The small molecules AZD0530 and dasatinib inhibit dengue virus RNA replication via Fyn kinase.
abstract: In this study, we characterized the antiviral mechanism of action of AZD0530 and dasatinib, two pharmacological inhibitors of host kinases, that also inhibit dengue virus (DV) infection. Using Northern blot and reporter replicon assays, we demonstrated that both small molecules inhibit the DV2 infectious cycle at the step of steady-state RNA replication. In order to identify the cellular target of AZD0530 and dasatinib mediating this anti-DV2 activity, we examined the effects of RNA interference (RNAi)-mediated depletion of the major kinases known to be inhibited by these small molecules. We determined that Fyn kinase, a target of both AZD0530 and dasatinib, is involved in DV2 RNA replication and is probably a major mediator of the anti-DV activity of these compounds. Furthermore, serial passaging of DV2 in the presence of dasatinib led to the identification of a mutation in the transmembrane domain 3 of the NS4B protein that overcomes the inhibition of RNA replication by AZD0530, dasatinib, and Fyn RNAi. Although we observed that dasatinib also inhibits DV2 particle assembly and/or secretion, this activity does not appear to be mediated by Src-family kinases. Together, our results suggest that AZD0530 and dasatinib inhibit DV at the step of viral RNA replication and demonstrate a critical role for Fyn kinase in this viral process. The antiviral activity of these compounds in vitro makes them useful pharmacological tools to validate Fyn or other host kinases as anti-DV targets in vivo.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Melissanne
authors.initials: M
authors.lastname: de Wispelaere
authors.forename: Amy J
authors.initials: AJ
authors.lastname: LaCroix
authors.forename: Priscilla L
authors.initials: PL
authors.lastname: Yang
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D052117:Benzodioxoles / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D000069439:Dasatinib; D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D004121:Dimethyl Sulfoxide; D057809:HEK293 Cells; D006801:Humans; D009173:Mycophenolic Acid; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology*; D034622:RNA Interference; D012367:RNA, Viral / Q000096:biosynthesis*; D012093:Replicon / Q000235:genetics; D013844:Thiazoles / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology
chemical_list: D000911:Antibodies, Monoclonal; D052117:Benzodioxoles; D017931:DNA Primers; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D012367:RNA, Viral; D013844:Thiazoles; C515233:saracatinib; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D009173:Mycophenolic Acid; D000069439:Dasatinib; D004121:Dimethyl Sulfoxide