Date Published: December 8, 2015
Publisher: Public Library of Science
Author(s): Tina M. Sali, Kara M. Pryke, Jinu Abraham, Andrew Liu, Iris Archer, Rebecca Broeckel, Julia A. Staverosky, Jessica L. Smith, Ahmed Al-Shammari, Lisi Amsler, Kayla Sheridan, Aaron Nilsen, Daniel N. Streblow, Victor R. DeFilippis, Sara Cherry.
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.
The innate immune system includes an array of sentinel proteins termed pattern recognition receptors (PRRs) that sense and react to microbe- and danger-associated molecular patterns (reviewed in ). These patterns are often constituents or replication intermediates of intracellular (especially viral) pathogens. PRRs respond to this engagement by initiating signaling pathways that bring about the expression or processing of cytokines, chemokines, and effector molecules that both directly block microbial replication and facilitate related adaptive immune processes. As such, PRRs represent an essential first line of immunological defense against infection and are the target of both microbial inhibitory phenotypes as well as pharmacologic manipulation for therapeutic purposes (reviewed in ).
Pharmacologic activation of STING-dependent signaling represents a potentially high-impact therapeutic strategy with applications in diverse clinical areas such as broad-spectrum antivirals, vaccine adjuvants, vascular disruption, and antitumor immunology. This is represented by multiple successes of the utilization of this approach in mouse models of virus infection [29,32,33,90,95], enhancement of vaccine immunogenicity [89,96–98], immune-mediated tumor necrosis [36,38,99], and inhibition of solid tumor angiogenesis [100,101]. Unfortunately, synthetic small molecules identified thus far have only exhibited suitable efficacy in mouse models due to their strict specificity for the murine STING ortholog [39–41]. Here we employed high-throughput screening to identify a novel compound (G10) capable of triggering IRF3/IFN-dependent responses and subsequently blocking replication of CHIKV, VEEV, and SINV in human cells. Follow-up work seeking to pinpoint cellular targets essential to the phenotypic responses utilized a reverse genetics approach by way of CRISPR/Cas9-mediated genome editing. This enabled identification of the STING protein as required for G10’s biological activity thus indicating that the compound is the first described human-specific synthetic small molecule STING agonist.