Inhibition of coronavirus infection by a synthetic STING agonist in primary human airway system

Abstract

The newly emerged severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) coronavirus initiated a pneumonia outbreak (COVID-19) that rapidly spread worldwide and quickly became a public health emergency of international concern; However to date, except Remdesivir, there are no clinically approved specific or effective medicines to prevent or treat COVID-19. Therefore, the development of novel treatments against coronavirus infections caused by the current SARS-CoV-2 virus, as well as other highly pathogenic human coronaviruses, represents an urgent unmet need. Stimulator of interferon genes (STING) plays a central role in host defense mechanisms against microbial infections. STING activation leads to the induction of both type I interferon and autophagy responses, which elicit strong inhibitory effect against the infections caused by a broad range of microbial pathogens. However, whether STING activation can impact infections from SARS-CoV-2 or other coronaviruses remains largely unknown. In this study, we investigated the anti-coronavirus activity triggered by STING activation. We discovered that dimeric amidobenzimidazole (diABZI), a synthetic small molecule STING receptor agonist, showed potent anti-coronavirus activity against both the common cold human coronavirus 229E (HCoV-229E) and SARS-CoV-2 in cell culture systems. In addition, we demonstrated that the antiviral activity of diABZI was dependent on the interferon pathway in HCoV-229E infected normal human fibroblast lung cells (MRC-5) and reconstituted primary human airway air-liquid interface (ALI) cultures. Furthermore, low-dose of diABZI treatment at 0.1 μM effectively reduced the SARS-CoV-2 viral load at the epithelial apical surface and prevented epithelial damage in the reconstituted primary human bronchial airway epithelial ALI system. Our findings have thus revealed the therapeutic potential of STING agonists, such as diABZI, as treatments for SARS-CoV-2 and other human coronavirus infections.

Keywords: Coronavirus; HCoV-229E; SARS-CoV-2; STING agonist; diABZI.

Fig. 1

STING agonist inhibits HCoV-229E replication on MRC-5. (A) The chemical structures of ABZI-based STING receptor agonist diABZI. (B) MRC-5 cells were infected with HCoV-229E at MOI of 0.01 and treated with indicated concentrations of diABZI or RDV for 72 h. The CPE was determined by a CCK-8 assay and the percent inhibition relative to DMSO treatment was plotted. (C) The cell viability was assessed by measuring CCK-8 in non-infected MRC-5 in the presence of DMSO and indicated concentrations of diABZI or RDV. (D) The HCoV-229E viral RNA were isolated from infected MRC-5 at MOI of 0.01 for 48hrs treatment with indicated concentrations of diABZI or DMSO and measured by real-time PCR. The EC₅₀ values were calculated using Prism software. The data are presented as the means ± SDs of triplicate samples from one experiment and are representative of at least three independent experiments.

Fig. 2

STING agonist represses HCoV-229E amplification on human SAEC ALI. (A–C) Human SAEC ALI cells were differentiated and seeded in 24-well plates. The cells were infected with HCoV-229E at MOI of 0.01 on apical and treated with different doses of the indicated compounds in basal medium. (A) After 72 h, the slides were subjected to the H&E stains. (B) After 72 h, the infectious supernatant collected from apical cells by PBS wash were subjected to viral titration assay on MRC-5. (C) After 48 h, the viral RNAs were isolated from the infectious supernatant collected from apical cells by PBS wash and measured by real-time PCR. The data are presented as the means ± SDs of triplicate samples from one experiment and are representative of at least three independent experiments.

Fig. 3

The combination study for STING agonist diABZI with RDV. (A) (Top) Representative antiviral dose response curves of diABZI in combination with RDV against HCoV-229E. Serial dilutions of diABZI with a range of indicated concentration of RDV. (Bottom) In vitro antiviral activity shift in 50% and 90% of diABZI with fixed concentration of RDV. (B) A representative heat-map (Left) and 3-dimensional drug interaction landscape plotting synergy scores analyzed using SynergyFinder 2.0. The data are presented as the means ± SDs of triplicate samples from one experiment and are representative of at least three independent experiments.

Fig. 4

Time-of-drug-addition study for STING agonist diABZI. (A) MRC-5 cells were infected with HCoV-229E at an MOI of 0.01 and treated with different doses of diABZI at the indicated times. After 72 h, the CPE was determined by a CCK-8 assay and the percent inhibition relative to DMSO treatment was plotted. (B) MRC-5 cells were pre-treated with different doses of diABZI at the indicated times, followed by infection with HCoV-229E at an MOI of 0.01 after PBS wash to remove the residue drug. After 72 h, the CPE was determined by a CCK-8 assay and the percent inhibition relative to DMSO treatment was plotted. (C) SAEC ALI were infected with HCoV-229E on apical cell at an MOI of 0.01 and treated with the indicated concentrations of compounds at different time points. After 48 h, the viral RNAs were isolated from the infectious supernatant collected from apical cells by PBS wash and measured by real-time PCR. The CPE was determined by a CCK-8 assay and the percent inhibition relative to DMSO treatment was plotted. The data are presented as the means ± SDs of triplicate samples from one experiment and are representative of at least three independent experiments.

Fig. 5

The mechanism of action study for STING agonist diABZI on MRC5 and reconstituted human ALI system. (A) MRC-5 cells were infected with HCoV-229E at an MOI of 0.01 and treated with the indicated concentrations of diABZI in the presence or absence of 5 μM TBKi (BX795) or 10 μM CQ for 72 h. (B) SAEC ALI were infected with HCoV-229E on apical cell at an MOI of 0.01 and treated with the indicated concentrations of diABZI in the presence or absence of 10 μM TBKi (BX795) or 10 μM CQ for 72 h in basal medium. The infectious supernatants collected from apical cells by PBS wash were subjected to viral titration assay on MRC-5. (C) SAEC ALI were infected with HCoV-229E on apical cell at an MOI of 0.01 and treated with the indicated concentrations of diABZI in the presence or absence of 10 μM TBKi (BX795) or 10 μM CQ for 72 h in basal medium. After 48 h, the viral RNAs were isolated from the infectious supernatant collected from apical cells by PBS wash and measured by real-time PCR. The data are presented as the means ± SDs of triplicate samples from one experiment and are representative of at least three independent experiments. *P < 0.05, **P < 0.01.

Fig. 6

The STING agonist exhibits anti-SARS-CoV-2 ability on MucilAir™. (A) The diagram of assay procedure. (B–C) MucilAir™ were infected with SARS-CoV-2 at an MOI 0.1 at apical cell, followed by treatment with indicated concentrations of compounds for 48 h and 72 h. At indicated time point, (B) SARS-CoV-2 viral RNA quantification and (C) transepithelial electrical resistance (TEER) assay were performed for treatment of diABZI or RDV. The data are presented as the means ± SD of triplicate samples from one experiment and are representative of at least three independent experiments. *P < 0.05, **P < 0.01.