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.

A MicroRNA Screen Identifies the Wnt Signaling Pathway as a Regulator of the Interferon Response during Flavivirus Infection

“…4C). To quantitate the secretion of type I IFNs, supernatants from miRNA-transfected, SeV-treated cells were collected and used to treat IFN-stimulated responsive element (ISRE)-luciferase human reporter cells (39). In agreement with the observed effects of miR-34a on IRF3 activation, high levels of type I IFNs were detected in the supernatants of miR-34a-transfected cells (Fig.…”

“…To examine whether IRF3-dependent induction of the IFN response is required for the antiviral role of miR-34a, IRF3 knockout (IRF3KO) HeLa cells were produced by using clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing (39). IRF3 protein levels (Fig.…”

“…IRF3 knockout cell production. IRF3 knockout HeLa cells were produced by using CRISPR-Cas9 technology as previously described (39,65). HeLa cells were transduced with lentiviruses expressing IRF3-targeting guide RNA and Cas9 endonuclease, and positive transductants were selected with 3 g/ml puromycin.…”

A cell-based high throughput screening assay for the discovery of cGAS-STING pathway agonists

“…G10, a small molecule human STING agonist (Sali et al, 2015), served as a control compound and was purchased from Aobious. Double strand DNA 90 (dsDNA-90) was prepared as previously described (Guo et al, 2017).…”

“…G10, or 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, is a recently identified human STING-specific agonist by high throughput screening. G10 had been demonstrated to induce an antiviral response in human fibroblasts against alphaviruses, but its in vivo biological activity and pharmacological property remain to be determined (Sali et al, 2015). …”