title: Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses.
abstract: Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Sali
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Kara M
authors.initials: KM
authors.lastname: Pryke
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Jinu
authors.initials: J
authors.lastname: Abraham
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Liu
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Iris
authors.initials: I
authors.lastname: Archer
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Broeckel
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Staverosky
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Smith
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Iraqi Centre for Cancer and Medical Genetics Research, Baghdad, Iraq.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Al-Shammari
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Lisi
authors.initials: L
authors.lastname: Amsler
authors.affiliation: Veterans Affairs Medical Center, Portland, Oregon, United States of America.
authors.forename: Kayla
authors.initials: K
authors.lastname: Sheridan
authors.affiliation: Veterans Affairs Medical Center, Portland, Oregon, United States of America.
authors.forename: Aaron
authors.initials: A
authors.lastname: Nilsen
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Streblow
authors.affiliation: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.|Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Victor R
authors.initials: VR
authors.lastname: DeFilippis
mesh_terms: D000524:Alphavirus / Q000276:immunology; D018354:Alphavirus Infections / Q000276:immunology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D065632:Chikungunya Fever / Q000276:immunology*; D002646:Chikungunya virus / Q000276:immunology; D057166:High-Throughput Screening Assays; D006801:Humans; D015151:Immunoblotting; D050838:Interferon Regulatory Factor-3 / Q000276:immunology; D008565:Membrane Proteins / Q000819:agonists* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013843:Thiazines / Q000494:pharmacology*; D000099197:STING Protein
chemical_list: D000998:Antiviral Agents; D050838:Interferon Regulatory Factor-3; D008565:Membrane Proteins; D013843:Thiazines; C532857:STING1 protein, human; D000099197:STING Protein; C530855:Sting1 protein, mouse; C000606717:human-specific STING agonist G10