
==== Front
ACS Infect Dis
ACS Infect Dis
id
aidcbc
ACS Infectious Diseases
2373-8227
American Chemical Society

39207884
10.1021/acsinfecdis.4c00504
Article
STING Agonist Induced Innate Immune Responses Drive Anti-Respiratory Virus Activity In Vitro with Limited Antiviral Efficacy In Vivo
https://orcid.org/0000-0003-3740-5442
Broeckel Rebecca *†
Browne Amanda †
Sucoloski Scott †
Cantizani Juan ‡
Simpson Juliet. K. §
Pesiridis Scott ∥
https://orcid.org/0000-0003-3471-651X
Ramanjulu Joshi M. ∥
Stokes Neil ⊥
Luthra Priya *†
† Infectious Diseases Research Unit, GSK R&D, Collegeville, Pennsylvania 19426, United States
‡ Global Health Medicines R&D, GSK R&D, Tres Cantos, Madrid 28760, Spain
§ Target Discovery Research Projects, GSK R&D, Stevenage SG1 2NY, United Kingdom
∥ Immunology Research Unit, GSK R&D, Collegeville, Pennsylvania 19426, United States
⊥ Infectious Diseases Research Unit, GSK R&D, Stevenage SG1 2NY, United Kingdom
* Email: Rebeccabroeckel@gmail.com.
* Email: Priyaluthra13@gmail.com.
29 08 2024
13 09 2024
10 9 33923407
17 06 2024
21 08 2024
20 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

The emergence of SARS-CoV-2 and seasonal outbreaks of other respiratory viruses highlight the urgent need for broad-spectrum antivirals to treat respiratory tract infections. Stimulator of interferon genes (STING) is a key component of innate immune signaling and plays a critical role in protection of the host against viral infections. Previously the STING agonist diABZI-4, a diamidobenzimidazole-based compound, demonstrated protection against SARS-CoV-2 both in vitro and in vivo. However, its broad-spectrum antiviral activity against other respiratory viruses in human airway epithelial cells, which are the primary targets of these infections, is not well established. In this study, we demonstrated that diABZI-4 stimulated robust innate immune responses protecting lung cells against a wide range of respiratory viruses, including influenza A virus (IAV), common cold coronaviruses, SARS-CoV-2, human rhinovirus (HRV), and human parainfluenza virus. diABZI-4 was highly active in physiologically relevant human airway epithelial tissues grown at the air–liquid interface, blocking replication of IAV, SARS-CoV-2, and HRV in these tissues. Furthermore, treatment of macrophages with diABZI-4 resulted in the secretion of cytokines that protected the primary airway epithelial cells from IAV infection. Despite the promising in vitro pan-antiviral activity, intranasal administration of diABZI-4 in mice provided early, but not sustained, inhibition of IAV replication in the lungs. These data highlight the complexities of the relationship between timing of STING agonist-driven inflammatory responses and viral replication dynamics, emphasizing the development challenge posed by STING agonists as potential therapeutics against respiratory viruses.

STING
diABZI
influenza virus
SARS-CoV-2
inflammation
respiratory virus
document-id-old-9id4c00504
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pmcThe rapid emergence of SARS-CoV-2 illustrates the need for the development of antiviral therapeutics with broad-spectrum activity that can treat both current and emerging respiratory infections. The existence of a broad-spectrum therapeutics would allow early intervention to prevent vulnerable populations from hospitalization and resulting downstream complications following infection. Therapeutics that stimulate innate immune responses offer pan-viral inhibitory potential as they work nonspecifically against a wide range of viruses and other pathogens.1

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS), along with the adaptor stimulator of interferon genes (STING), are key components of innate immune signaling involved in detection of cytosolic DNA derived from foreign pathogens and self.2−4 After detecting cytosolic DNA, cGAS synthesizes the secondary messenger cGAMP to stimulate STING, which results in activation of the transcription factors NF-kB and IRF3 and induces cellular processes such as autophagy.5 Importantly, STING activation provokes downstream antiviral immune responses that include production of type I and type III interferons (IFNs) and IFN-stimulated genes (ISGs).3,6 A novel class of noncyclic dinucleotide STING agonists, called diamidobenzimidazole (diABZI) based compounds, were shown potently to activate STING in comparison to the endogenous ligand cGAMP.7 Furthermore, treatment of cells with diABZI-based compounds resulted in induction of innate immune responses capable of inhibiting infection of SARS-CoV-2, HCoV-OC43, HCoV-229E, human rhinovirus A16 (HRV A16), and parainfluenza virus type 3 (PIV3).8−11

Current preclinical approaches for testing therapeutics are expanding toward using complex in vitro models, while minimizing usage of animal models.12 While mouse models have been developed for IAV and SARS-CoV-2, there are species-specific differences in the lung in both infection dynamics and tissue-specific cell compositions between mouse and human.13,14 Lung organoid cultures, complex in vitro lung models, and ex vivo tissue cultures, such as precision cut lung slices (PCLS), offer some advantages over animal models in that primary human tissues are represented and some immune cells may be incorporated for mimicking immune aspects of a native tissue that might not be present in animal disease models.15,16 With regard to STING, there may be species-specific differences in expression, signaling, and immune responses, highlighting the value of human in vitro model systems to inform human dose predictions compared to other animal models. In addition, distinct virus families have been shown to interact with STING in a species-dependent manner as part of their replication cycles, with implications of STING as a species-specific susceptibility factor for certain viral infections.17,18 While human lung model systems have specific translational advantages, they do not fully recapitulate the entire disease progression with an intact, functional immune response. Therefore, the in vitro models and in vivo models need to complement each other until a more advanced human lung model is available.

In this work, we investigated the antiviral activity of the STING agonist diABZI-4 in immortalized human lung cells, primary human airway epithelial cells, and a C57BL/6J mouse model for IAV. We measured the potency of diABZI-4 against IAV, HRV-A16, HCoV-229E, PIV3, and SARS-CoV-2 in human lung fibroblasts and a human lung epithelial cell line. In addition, we demonstrated that cotreatment of primary human nasal and bronchial tissues with as little as 20–60 nM diABZI-4 resulted in consistent inhibition of infection with HRV, SARS-CoV-2, and IAV. Furthermore, these tissues produced STING-driven inflammatory cytokines, such as IFNβ, IL-6, and IP-10 in a dose-dependent manner, detected as early as 3 h post diABZI-4 treatment. Human macrophages, an abundant immune cell type in the lungs, treated with diABZI-4 upregulated costimulatory molecules and secreted type I IFNs, IL-6, TNFα, and IP-10, indicative of activation of STING-dependent pathways. Transfer of media from diABZI-4-treated macrophages to primary bronchial epithelial cells resulted in antiviral protection against IAV. Intranasal treatment of C57BL/6J mice with diABZI-4 resulted in elevated inflammatory responses that protected mice against IAV at day 1 post infection but not at later time points. Together these data show that diABZI-4 elicits antiviral immune responses in both human lung cells alone as well as in primary immune cells capable of protecting against respiratory virus infection in vitro, but those findings do not translate to lasting protection in an IAV mouse model.

Results

STING Activation Confers Pan-Respiratory Virus Activity in Human Lung Cell Lines

A potent STING agonist, diABZI-4, has previously been demonstrated to elicit antiviral responses against herpes simplex virus, IAV, HCoV-OC43, and SARS-CoV-2.11 This molecule has a favorable solubility profile and shows activity across different species with high potency against human and nonhuman primate STING (Figure S1). In this work, we set out to characterize the pan-respiratory antiviral activity and immune activation by diABZI-4 in human lung cells. As STING expression is dependent on cell type, we initially utilized human lung fibroblasts (MRC-5 cells) that have robust STING transcript signatures (Figure 1A). Utilizing STING phosphorylation as a proximal marker for target engagement, we evaluated STING activation in MRC-5 cells. diABZI-4 induced STING activation in a dose-dependent manner (Figure 1B), and this activation led to the transcription of IFNβ, RSAD2, TNFα, IL6, and TRIM22 (Figure 1C). Interestingly, some of these gene signatures remained elevated at 24 h post diABZI-4 treatment, demonstrating the differences in transcript stability kinetics following STING stimulation.

Figure 1 STING agonist diABZI-4 triggers STING activation in human lung fibroblasts. A. TMEM173 baseline transcript levels from untreated HeLa, Hep2, A549, Calu-3, and MRC-5 cell lysates were compared. Relative transcript levels of TMEM173 were compared to transcript levels in A549 cell lysates. B. MRC-5 cells were treated with serial dilutions of diABZI-4 and total STING, phosphorylated STING (p-STING) and β-actin protein levels were measured at 2.5 h post treatment using capillary electrophoresis (CE) immunoassay. C. Host transcripts (IFNβ, RSAD2, TNF, IL-6, and TRIM22) were measured in MRC-5 cell lysates at 5 and 24 h post treatment with 50 and 500 nM diABZI-4 using qRT-PCR, and transcript levels are expressed as fold-change relative to the DMSO control.

We next assessed the pan-viral potential of diABZI-4 against IAV, HRV, PIV3, HCoV-229E, and SARS-CoV-2. Pretreatment of MRC-5 cells 3 h prior to infection with IAV (Figure 2A), HRV (Figure 2B) or HCoV-229E (Figure 2C) and at the time of infection with PIV3 (Figure 2D) inhibited respective virus replication with EC50s in the low nanomolar range (Figure 2F). SARS-CoV-2 infects lung cells using the ACE-2 receptor.19 As Calu-3 cells express the ACE-2 receptor and are highly permissive to SARS-CoV-2, we utilized these cells for SARS-CoV-2 infections.19 diABZI-4 treatment led to inhibition of SARS-CoV-2 (Figure 2D-E). However, the EC50s were 10-fold higher compared to other respiratory viruses, likely due to lower expression of STING in Calu-3 compared to MRC-5 cells (Figure 1A). Interestingly, diABZI-4 treatment only had a limited impact on RSV (data not shown), suggesting different sensitivities of respiratory viruses to STING agonism. Overall, these data demonstrate the potent pan-respiratory virus activity of diABZI-4.

Figure 2 diABZI-4 elicits antiviral activity against respiratory viruses in human cell lines. A. MRC-5 cells were pretreated with serial dilutions of diABZI-4 for 3 h and infected with IAV A/WSN/33 (MOI = 0.01) for 1 h. Inoculum was removed, cells were rinsed with PBS, and media with compound were added back to the plates. At 48 hpi, cells were rinsed with PBS, and cell lysates were collected for transcriptional analysis. Viral RNA levels were measured, and data are represented as a percentage of viral inhibition. The cell viability assays were performed in parallel to the infection assays. Uninfected MRC-5 cells were treated with serial dilutions of diABZI-4, and data were expressed as a percentage to the uninfected, untreated condition. B. HRV A16 infections and treatments were performed as in A., except using an MOI = 0.05. C. MRC-5 cells were pretreated with diABZI-4 for 3 h followed by infection with HCoV-229E (MOI = 0.2). Infection was allowed to proceed for 5 days and cytopathic effect was quantified by measuring cell viability (Cell Titer Glo kit). Data are expressed as percent inhibition to the uninfected condition. D. MRC-5 cells were treated with serial dilutions of diABZI-4 at the time of infection with PIV3 (MOI = 0.33). At 48 hpi, PIV3 antigen was measured in the cells using in situ ELISA, and data are expressed as percent inhibition to the uninfected condition. E. Calu-3 cells were treated with diABZI-4 and infected with SARS-CoV-2 (Wuhan, MOI = 2.5, Omicron, MOI = 1.25). Plates were fixed, permeabilized and stained with Draq5 (Biostatus) and Alexa Fluor 488 Anti-SARS-CoV-2 nucleocapsid protein antibody (Abcam, ab283243) and imaged at 72 hpi. Three images per well were taken in the Opera Phenix (PerkinElmer) using 20X air objective and analyzed in Columbus 2.9.1 (PerkinElmer) to measure the % of infected cells. F. The diABZI-4 EC50 values are reported in the table. EC50 values were calculated using nonlinear regression analysis. G. MRC-5 cells were treated with 500 nM diABZI-4 at 24 h prior to infection, at the time of infection, or 24 h post infection (−24, 0, + 24, respectively) and infected with either IAV or HRV (MOI = 0.01). Cell lysates were harvested at 48 hpi, and viral RNA was measured by qRT-PCR. Fold-change values relative to the DMSO control are reported. Data are representative of multiple experiments, and one independent run is displayed. Statistics were performed on log-transformed data using Ordinary one-way ANOVA followed by Sidak’s multiple comparison test (* p < 0.03; ** p < 0.0021, *** p < 0.0002; **** p < 0.0001).

We next performed time of addition studies to determine the timing of the diABZI-4 treatment relative to infection that was required for antiviral efficacy. Pretreatment at 24 h prior to infection resulted in over a 100-fold reduction in viral titers for both IAV and HRV (Figure 2G, Figure S2A). Treatment of diABZI-4 at 24 h post virus infection resulted in a less robust but still significant reduction of viral titer, suggesting early activation of STING mediated immune responses are optimal for protection against these respiratory viral infections.

diABZI-4 Activates STING and Stimulates Innate Immune Responses and Protects against Respiratory Infection in Primary Human Airway Tissues

We further evaluated the impact of diABZI-4 on physiologically relevant primary human lung airway tissues grown at the air–liquid interface (ALI). Culturing primary cells at ALI allows for epithelial cell differentiation and representation of multiple epithelial cell types, with morphological similarities to the native epithelium.20 diABZI-4 treatment of primary nasal epithelial tissues in the basal media for 3 h led to strong stimulation of STING, with a minimum of 20 nM sufficient for STING phosphorylation (Figure 3A), and secretion of cytokines including IFNβ, IP10, and IL-6 (Figure 3B). The presence of the air-facing surface of these tissues allows in vitro apical exposure that mimics intranasal human respiratory exposures. Therefore, we also examined the impact of diABZI-4 treatment through apical and basal surfaces. Interestingly, both basal and apical treatment for 1 h, or just 0.5 h exposure in basal media, induced a p-STING signal (Figure 3C). These data indicate that a short exposure with diABZI-4 on either surface is sufficient for STING activation.

Figure 3 diABZI-4 induces p-STING and pro-inflammatory cytokines in primary human nasal epithelial cells grown at an air–liquid interface. A. Primary human nasal epithelial tissues grown at ALI were treated with serial dilutions of diABZI-4 in the basal media, and STING, p-STING, and β-actin protein levels were measured at 3 h post treatment using capillary Western blot. B. Cytokines from the basal media of tissues treated in A were measured at 3 h post diABZI-4 treatment using an MSD assay kit. Data are represented as fold-change to uninfected, untreated ALI tissues. C. Primary nasal epithelial tissues grown at ALI were treated with 5 μM diABZI-4 on the apical surface for 1 h, basal media 1 h, or basal media for 0.5 h. Following treatment, the ALI tissues were transferred to fresh drug-free media. p-STING, total STING, and β-actin levels were measured at 3 h post diABZI-4 treatment by capillary Western blot.

We tested the potential antiviral effects of diABZI-4 in primary nasal and bronchial epithelial ALI tissues. Treatment of nasal ALI tissues with diABZI-4 in the basal media at the time of infection inhibited IAV (Figure 4A). These results were consistent in primary bronchial ALI tissues (Figure 4B), demonstrating that diABZI-4 has antiviral efficacy across multiple regions of the respiratory epithelium. diABZI-4 treatments of these tissues resulted in inhibition of HRV (Figure 4C, Figure S2B) and SARS-CoV-2 (Figure 4D-E) replication in a dose-dependent manner. These data are consistent with published studies showing diABZI molecules impair SARS-CoV-2 replication in lung epithelial cells.8,10,21 We also noted that HRV and SARS-CoV-2 were highly sensitive to diABZI-4 treatment, with over 2–3 log reductions in virus replication. However, IAV was inhibited by diABZI-4 by about 10–15-fold, suggesting that respiratory viruses have different sensitivities to STING agonists. Treatment of cells with 500–2000 U/mL IFN at the time of infection led to less than a log reduction of IAV and HRV viral titers (Figure 4A-C), although greater levels of inhibition would be expected if cells were pretreated with IFN. The expected mechanism by which STING activation blocks infection is through the induction of type I and type III IFNs.8,11 These viruses are known to have differential sensitivities to type I and type III IFNs, which might contribute in part to the differences in antiviral activities observed.22,23 In addition to immune-mediated antiviral mechanisms, STING activation may impact essential virus-host interactions or trigger other cellular processes, resulting in reduced level of replication. For example, HRV recruits STING into its replication organelle, and the activation of STING mis-localizes it so that it is unavailable for integration into the HRV replication body.9,18,24 Together these data demonstrate that diABZI-4 is a potent inhibitor for respiratory virus infection in primary airway epithelial cells, and further analysis of the epithelial cell immune response will elucidate potential mechanisms of inhibition.

Figure 4 diABZI-4 engagement in primary human airway epithelial cells inhibits replication of IAV, HRV, and SARS-CoV-2. A. Nasal and B. bronchial primary ALI tissues were treated with serial dilutions of diABZI-4, DMSO, 5 μM oseltamivir, or recombinant IFNβ at the time of infection with IAV A/PR8/34 (MOI = 0.2). IAV levels were measured at 24 hpi in the cell lysates and quantified by qRT-PCR. Relative vRNA levels were expressed as fold change to the DMSO-treated sample. C. Nasal ALI tissue was treated with serial dilutions of diABZI-4, DMSO, 5 μM rupintrivir (Rup), or 2000 U/mL IFNβ at the time of infection, and cells were infected with HRV A16 (MOI = 0.1). At T = 48 hpi, cells were lysed and viral transcripts were quantified using qRT-PCR. Relative vRNA levels to DMSO are reported. D. Primary bronchial epithelial ALI inserts from two donors were infected with SARS-CoV-2 (MOI = 10) and treated with diABZI-4 or 5 μM remdesivir at the time of infection. At 72 hpi, SARS-CoV-2 transcripts were measured in cell lysates by qRT-PCR, and relative vRNA levels are reported as fold change to the DMSO samples. The experiment was repeated in primary nasal epithelial inserts (right). Data are represented as fold change to the remdesivir-treated samples. E. SARS-CoV-2 NP levels were visualized by immunofluorescence of the ALI insert stained with Alexa Fluor 488 anti-SARS-CoV-2 nucleocapsid protein antibody (Abcam, ab283243) at 72 hpi using a 20x objective in Opera phenix (PerkinElmer). Maximum projection of 20 planes per field were represented for each insert. Statistics were performed on log-transformed data using Ordinary one-way ANOVA (ns = not significant, * p < 0.033, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001).

We further investigated STING driven immune responses in these infected tissues by examining the secretion of cytokines over time. Though virus infection slightly stimulated cytokine secretion, as observed in the DMSO only condition, diABZI-4 treatment increased the secretion of cytokines over time in IAV-infected primary nasal and bronchial tissues (Figure 5A-B). Elevated levels of IFNα and IFNβ, IP-10, IL-6, IL-4, IL-7, IL-9, IL-1RA, MIP-1α, G-CSF, and TNFα were observed in these conditions. The concentration-dependent cytokine response also strongly correlated with the observed antiviral activity. Exposure of IAV-infected primary airway tissues with only 20 nM diABZI-4 was sufficient to stimulate cytokines and resulted in over a 1-log reduction in viral titers. In the HRV-infected nasal tissues, diABZI-4 treatment resulted in elevated levels of IFNβ, IP-10, IL-6, IL-1RA, MIP-1α, and IL-4 (Figure 5C). Thus, diABZI-4 exposure in primary airway tissues resulted in secretion of proinflammatory cytokines in the presence of infection with IAV and HRV, and this treatment ultimately limited viral replication.

Figure 5 diABZI-4 treated primary nasal and bronchial tissues secrete similar cytokines following infection with IAV and HRV. Primary ALI tissues were infected and treated with serial dilutions of diABZI-4. A. Nasal, and B. bronchial cytokine profiles were measured from basal media samples at 4, 16, and 24 hpi from tissues infected with IAV A/PR8/34 (MOI = 0.2). C. Nasal tissues were infected with HRV A16 (MOI = 0.1) and treated with diABZI-4. Cytokines were measured in the basal media samples at T = 24 and 48 hpi. Cytokine data are represented as fold-change to uninfected, untreated conditions at each time point.

Immune Cells Treated with diABZI-4 Protect Against Respiratory Virus Infection of Primary Airway Epithelial Tissue

The primary airway epithelial cells treated with diABZI-4 can elicit innate immune responses (Figure 5A-C) that, in isolation, protect the tissue against respiratory virus infection. These airway epithelial tissues are part of a broader tissue environment that includes immune cells, such as airway and interstitial macrophages.25,26 It is known that some highly pathogenic avian IAV isolates can infect immune cells such as macrophages and dendritic cells,27,28 but it is not known whether the diABZI-4 can protect macrophages from IAV replication. To test whether diABZI-4 treatment results in both immune activation and protection from IAV replication, induced pluripotent stem cell-derived macrophages (iMacs), were treated with 50 nM diABZI-4 for 24 h with and without IAV (MOI = 0.1) (Figure 6A). diABZI-4 treatment led to a 3.8-fold reduced frequency of nucleoprotein (NP)+ cells compared to the DMSO vehicle control, suggesting diABZI-4 inhibits viral replication in the iMacs. diABZI-4- treated iMacs also had significantly elevated levels of CD80 and CD86 compared to controls (Figure 6B). In IAV-infected conditions, diABZI-4 treatment resulted in a trend of increased CD80 and CD86 levels compared to infected DMSO controls, but these trends were not significant. Furthermore, 50 nM diABZI-4 treatment resulted in elevated pro-inflammatory cytokines including IL-6, TNFα, IFNα and IFNβ, and IP-10 at 24 h post-treatment (Figure 6C). Together these results show that diABZI-4 treatment of iMacs for 24 h is sufficient to activate the iMacs and cause the accumulation of pro-inflammatory factors in the media.

Figure 6 iMacs treated with diABZI-4 are protected against IAV and show an activated phenotype. A. iPSC-derived macrophages (iMacs) were infected with IAV A/PR8/34 (MOI = 0.1) and treated with 50 nM diABZI-4 or vehicle (DMSO) in MucilAir media containing M-CSF. At 24 hpi, iMacs were analyzed for IAV levels by measuring the percent of NP+ (IAV) cells by flow cytometry. B. CD80 (left) and CD86 (right) MFI was detected at 24 hpi in the macrophage cultures using flow cytometry. Data are compiled from three independent experiments and normalized to the uninfected, DMSO-treated macrophage condition. Statistics were performed using Kruskal–Wallis multiple comparison test (* p < 0.0332). C. Secreted cytokines in the media were measured at 24 hpi by MSD and plotted as fold change to uninfected and untreated samples.

We then assessed whether the cytokines, such as type I and III IFNs, produced by diABZI-4-treated macrophages could indirectly protect airway tissues. To test this, the iMacs were first treated with 50 nM diABZI-4 for 24 h and the conditioned media was transferred to the basal chamber of the airway epithelial cells 24 h prior to infection with IAV (Figure 7A). As expected, the diABZI-4-treated iMac media protected the ALI tissues from IAV, resulting in a significant reduction of secreted IAV as well as decreased cell-associated vRNA in the cell lysates at 24 hpi, (Figure 7B–C). Furthermore, subsequent mixing of diABZI-4-treated iMac media with the Janus kinase 1 and 2 (JAK 1 and 2) inhibitor, ruxolitinib, reversed the antiviral effect of the diABZI-4 (Figure 7B–C), suggesting that the JAK/STAT pathway is critical for diABZI-4 to confer antiviral activity against IAV. The basal media of the ALI tissues treated with diABZI-4-conditioned media had elevated pro-inflammatory cytokines, including IFNα2a, IL-1RA, MCP-1, and MIP-1α (Figure 7D). These pro-inflammatory cytokines were elevated in samples that were treated with ruxolitinib, indicating that the antiviral activity is not directly linked to diABZI-4 driven production of inflammatory cytokines. Rather, these data imply that the protective activity effected by diABZI-4 is likely mediated by type I and type III IFN that signals through JAK/STAT pathways to produce downstream antiviral effector proteins.

Figure 7 diABZI-4-induced secreted factors by macrophages are sufficient for protection against IAV infection in primary bronchial epithelial tissues. A. A diagram of the experimental design is shown. iMacs were treated for 1 h with 50 nM diABZI-4. Media was removed and residual compound was rinsed 3 times from the iMacs. iMacs were replenished with MucilAir media containing M-CSF for 24 h. The iMac-conditioned media was collected at 24 h post treatment, and media was applied to the basal compartment of the bronchial epithelial ALI tissues in the presence or absence of 10 μM ruxolitinib. Bronchial epithelial cells were pretreated for 24 h with the diABZI-4-conditioned media and then infected with IAV A/PR8/34 (MOI = 0.2). At 24 hpi, IAV was measured in the B. apical rinse samples using an NA-XTD kit, and C. cell-associated vRNA levels by qRT-PCR. Statistical analysis was performed on log-transformed data using one-way ANOVA followed by Dunnett’s multiple comparison test in B or Sidak’s multiple comparison test in C (** p < 0.0021; **** p < 0.0001). D. Cytokines from the basal media were collected at 24 hpi and analyzed using a 20-plex MSD kit. Relative cytokine levels are compared to basal media from uninfected, untreated bronchial ALI tissue.

Intranasal Administration of diABZI-4 Elicits a Rapid Induction of Inflammation, But Does Not Result in Lasting Efficacy In Vivo

We showed that STING agonists elicit innate immune responses in human cells and macrophages that protect against infection in a JAK/STAT-dependent manner. However, these in vitro systems do not fully recapitulate the native human lung environment, which may be better simulated by an in vivo model to gain a fuller understanding of the dynamics of the immune responses to diABZI-4 treatment and to address the timing and duration of treatment required.

diABZI-4 was previously demonstrated to show functional activity in C57BL/6J mice,8,11 but it was unknown whether there may be differences in the production of type I IFN across different strains of mice. Whole blood from C57BL/6J mice and Balb/c mice was incubated with a concentration range (30 to 0.117 μM) of diABZI-4, and after 4 h, IFNβ secretion was measured in the serum. IFNβ was detected from serum from either mouse strain in a concentration-dependent manner, with comparable EC50 across strains (Figure 8A). Therefore, Balb/c and C57BL/6J mice both possess STING protein that is functionally responsive to diABZI-4 at similar concentration ranges, and either strain is suitable for downstream in vivo modeling using diABZI-4.

Figure 8 Intranasal delivery of diABZI-4 results in robust activation of pro-inflammatory cytokines and ISGs in the lungs with limited protection from IAV challenge. Whole blood derived from Balb/c or C57BL/6J mice was pooled and treated with serial dilutions of diABZI-4 (2-fold serial dilutions, 30 μM to 0.12 μM). A. At 4 h post stimulation, the serum was collected and IFNβ levels were measured using MSD. Data were normalized to % response, and the EC50 values were calculated using nonlinear regression analysis. B–C. C57BL/6J mice (n = 4 female mice/group) were treated intranasally with 0.1 mg/kg diABZI-4, and lungs were collected at 0, 0.125, 1, 2, 4, and 8 days post treatment for host transcript and cytokine analysis. B. IFIT1, MX1, IL-6, CXCL10, ISG15, IFNL2/3, IFNβ, STAT1, TNF, and STING transcripts were measured using qRT-PCR from lung tissue homogenates. Data were normalized to mouse b-actin, and data are represented as fold change to the average of the T = 0 samples. C. Cytokines were measured from serum and lung homogenates using a mouse MSD assay kit. Data are represented as fold-change to the T = 0 samples. D. Concentrations of diABZI-4 in lung homogenates and serum are shown from mice treated IN with 0.1 mg/kg diABZI-4. E-F. Mice were intranasally treated with 20 μL 0.1 mg/kg diABZI-4 or 1.2% DMSO vehicle 1 day prior to infection. Mice were infected with 400 TCID50 of IAV A/PR8/34 in a 40 μL volume. On day 1, 3, and 7 post infection, lungs were collected and IAV transcripts (E) and cytokines (F) were measured from lung tissue homogenates. Each group is representative of 8 mice, including 4 males and 4 females per treatment group per time point. Statistics were performed using two-way ANOVA followed by Sidak’s multiple comparison test (ns = not significant, * p < 0.03). LOD is limit of detection.

Previous work demonstrated that intranasal (IN) delivery of diABZI-4 protected mice against SARS-CoV-2 and IAV challenge when administered at or near the time of infection.11 While this treatment resulted in expression of ISGs at 3 h after administration, the full dynamic profile of host transcripts and cytokines in lungs following treatment was not obtained. For a complete characterization of the induction and resolution of lung ISG expression and cytokines, C57BL/6J mice were treated IN with 0.1 mg/kg diABZI-4, and host transcripts as well as cytokines were measured at 0, 0.125, 1, 2, 4, and 8 days post treatment. Consistent with previous results,11 peak induction of host ISG and cytokine transcripts including IFIT1, MX1, IL-6, CXCL10, ISG15, IFNL, IFNβ, STAT1, and TNFα was observed at 3 h post treatment (0.125 days) (Figure 8B). Selected transcripts remained elevated throughout the 8 day study duration, but their expression declined from an early peak. Cytokine levels peaked at 3 h (IFNβ) or day 1 (IFNα, IFNγ, IL-1β, IL-6, IP-10, MCP-1, TNFα) (Figure 8C). Lung levels of IFNα, IFNγ, IL-1β, IL-6, MCP-1, and TNFα were decreasing but remained elevated up to day 4, and dropped to near-baseline by day 8. Interestingly, IN delivery led to very limited cytokine activation in serum, suggesting that targeted delivery of an STING agonist results in a limited systemic response. Levels of diABZI-4 were detected in lung at 3 h post treatment, and although the diABZI-4 levels started to decline at 2 days post treatment, the levels were maintained up to 8 days post treatment (Figure 8D). Minimal levels of diABZI-4 were detected in the serum, correlating with the cytokine results, showing limited systemic activity following IN delivery.

We next tested whether the elevated cytokines and ISGs in the lungs could translate into desirable protection against IAV infection. C57BL/6J mice were prophylactically treated with IN with 0.1 mg/kg diABZI-4. One day later, mice were infected with 400 TCID50 of IAV A/PR8/34. Lung-associated-IAV levels were significantly reduced at day 1 post infection in mice treated with diABZI-4 (Figure 8E), but IAV levels were equivalent across vehicle and diABZI-4 treatment groups at day 3 and 7 post infection. This suggests that diABZI-4 had antiviral activity at early times post infection, but the protective effects were not maintained over the course of infection. Cytokine levels in the lungs were elevated at day 1 in the diABZI-4 treatment group, but at days 3 and 7 post infection, cytokines were elevated to similar levels across treatment groups (Figure 8F). These data show that diABZI-4 treatment resulted in induction of cytokines and transient protection against IAV replication at day 1 post infection, but this protection was not sufficient to eliminate virus spread or virus-driven inflammation at day 3 and day 7.

Discussion

diABZI-4 is a potent and selective STING agonist that is functional across the species. This work showed the tight correlation between STING activation and broad-spectrum antiviral activity against respiratory viruses in vitro. In primary human ALI tissues, treatment with as little as 20–60 nM diABZI-4 at the time of infection resulted in consistent antiviral activity against IAV, HRV, and SARS-CoV-2. This strongly correlated with STING activation as measured by p-STING levels, and a subsequent increase in cytokines at 3 h post-treatment. Immune cells treated with 50 nM diABZI-4 secreted cytokines that protected primary airway tissues from IAV infection. These data provide evidence that macrophages exposed to diABZI-4 in the lung could produce inflammatory cytokines and chemokines that protect primary airway tissues from respiratory infection. In contrast to the in vitro data, the in vivo models show that prophylactic administration of diABZI-4 results in limited, transient protection from IAV. The in vivo mouse model incorporates an intact lung environment where additional cell types, such as infiltrating immune cells, may influence replication kinetics and diABZI-4 driven inflammatory responses.

Previous studies showed that single intranasal administration of STING agonists provided protection against SARS-CoV-2.8,11 The intranasal delivery of diABZI-4 in mice did not cause excessive inflammation, but there were elevated levels of CD69+ myeloid cells, DCs, and γδ T cells in the lungs at 12 h post treatment.11 No significant immune cell recruitment and no gross pathology was observed out to day 5 post treatment. The level of innate immune stimulation by diABZI-4 required for protection from infection in mice against various respiratory viruses such as SARS-CoV-2 has not been directly correlated and could be further explored for finely mapping the minimum effective dose required for protection and the duration of time, by which a protective response is expected. Similarly, the level of protection may vary across different respiratory infections because limited, partial protection was observed against IAV in comparison to complete protection of mice against SARS-CoV-2.

Too high a dose of a STING agonist may raise safety concerns because excessive STING activation may result in tissue damage, with too much inflammation leading to a cytokine storm. Previously, repeat administration of diABZI-1 to C57BL/6J mice via the endotracheal route was shown to induce neutrophilic inflammation, epithelial injury, and inflammatory cytokines such as IL-6 and TNFα in a dose-dependent manner.29 Some of these effects may be alleviated by altering the delivery mechanisms and formulations of the STING agonist using technologies such as nanoprecipitation, hydrogels, and self-assembly.30,31 These different approaches and targeted delivery to the tissue of interest may alter the kinetics of the release of the compound and local exposure to the target tissue, thus potentially preventing a toxic systemic response.

STING agonists elicit type I and type III IFN responses, but the dynamics of the IFN induction in combination with other pro-inflammatory cytokines could exacerbate local inflammation during respiratory infection. Prophylactic type I IFNs are generally protective against acquisition of respiratory infections in animal models, but delayed delivery of type I IFNs after infection is established may result in increased lung damage, proinflammatory cytokines, and morbidity.32−36 Therefore, the timing of ABZI-4 delivery relative to infection needs to be properly controlled so as to avoid any unintended exacerbations.

Compared with SARS-CoV-2, IAV may be less sensitive to diABZI-4-driven innate immune responses, particularly in the C57BL/6J mouse model. C57BL/6J mice are highly susceptible to IAV infection, but they lack a functional Mx1 gene that encodes for an important IFN-effector against IAV.37,38 Prophylactic administration of type I IFN resulted in enhanced protection against IAV in Mx1 transgenic mice compared to standard laboratory mice missing functional Mx1.36,39 Antiviral efficacy of a RIG-I agonist was also shown to be limited and short-lived in standard C57BL/6J mice, but more potent and long-lasting antiviral effects mediated by the RIG-I agonist were observed in C57BL/6J transgenic mice expressing functional Mx1.40 These studies suggest that enhanced antiviral efficacy with diABZI-4 could be achieved with IAV using Mx1 transgenic mice.

Our study provides molecular and cellular characterization of host immune modulation by diABZI-4 and its antiviral activity against respiratory viruses. We further used highly translatable human airway lung epithelial cells as an ideal model to determine compound potency and perform mechanistic studies in the context of respiratory virus infections. Though they lack other cell types, utilizing lung epithelial cell and immune cell coculture systems can enable the optimization of drug responses in a disease relevant model and may reduce animal studies in the follow-up in vivo work. STING agonist elicited potent innate immune responses that showed efficacy against a broad range of respiratory infections, particularly in vitro. Striking a balance between triggering protective immunity without inducing too much systemic inflammation is crucial for protection against viral infection. The rapid dynamics of viral replication in the lungs and concerns about exacerbating existing virus-driven inflammatory responses are key challenges in the development of STING agonists as treatments for respiratory infections.

Materials and Methods

Cells and Viruses

MRC-5 cells (ATCC CCL-171), Hep-2 cells (ATCC CCL-23), HeLa cells (ATCC CRL-1958), Calu-3 cells (ATCC HTB-55), VeroE6 cells (ATCC CRL1586), and A549 (ATCC CCL-185) cells were grown in DMEM supplemented with 10% Fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Pen-strep). MDCK cells (ATCC NBL-2) were grown in MEM supplemented with 10% FBS and 1% Pen-strep. Calu-3 cells (ATCC HTB-55) were grown in EMEM, Earle’s BSS supplemented with MEN nonessential amino acids (NEAA), 1X, Glutamax 2 mM, 10% FBS and 1% Pen-strep. Primary differentiated human nasal and bronchial epithelial tissues (Epithelix) were maintained in an air–liquid interface in MucilAir Culture Medium (Epithelix). All cells were grown in a humidified incubator at 37 °C with 5% CO2. Influenza A viruses (A/PR8/34 and A/WSN/33) were propagated in MDCK cells in MEM supplemented with nonessential amino acids, 2 mg/mL TPCK-treated trypsin, and 7.5% bovine serum albumin. Other viruses were propagated in complete DMEM. HRV A16 was propagated on HeLa cells at 33 °C with 5% CO-2. PIV3 was propagated on LLC-MK2 cells at 37 °C. HCoV-229E (35 °C) was propagated on MRC-5 cells. Experimental work with SARS-CoV-2 was carried out following standard operating procedures in compliance with biosafety level 3 regulations (BSL3). SARS-CoV-2 isolates hCoV-19/USA-WA1/2020 and hCoV-19/USA/MD-HP20874/2021 (Lineage B.1.1.529; Omicron Variant) were used to infect VeroE6 cells and generate an infectious stock that was frozen in OPTIPRO (Gibco) supplemented with 0.5% Gelatin (Sigma). Viruses were titrated on their corresponding cell lines, and TCID50 end point titers were calculated using the Spearman-Karber method.

Compound Treatments and Infections

All compounds were sourced internally at GSK. diABZI-4, oseltamivir, rupintrivir, remdesivir and ruxolitinib solids were resuspended to 10 mM solutions in DMSO and further diluted in media at the reported concentrations. For infection assays, cells were seeded overnight in tissue culture plates. The following day, cells were infected and treated with compound as specified. Recombinant IFNβ (R&D Systems, 8499-IF-010) was resuspended and added to the media as indicated. For infection of the ALI tissues, cells were rinsed 2–3 times with PBS to remove excess mucous. Inoculum was added in a small volume to the surface of the inset for 1–3 h, then removed and the apical surface was rinsed 2–3 times with PBS to remove excess inoculum. Infection was allowed to proceed as indicated in the experiment. Compound was added to the basal media or apical surface as specified in the experiment. IAV levels were measured in apical rinse samples using the NA-XTD Influenza Neuraminidase Assay Reagent Set (Applied Biosciences 4457534) according to the manufacturer’s protocol. Briefly, the apical rinse samples were mixed with NA-XTD assay substrate and buffer for 30 min followed by the addition of the NA-XTD accelerator. Then luminescence values were collected on the PHERAstar FSX plate reader (BMG). Raw luminescence values were directly plotted.

qRT-PCR and Imaging

RNA was isolated from cell lysates using the RNeasy Plus Kit (Qiagen, 74134) according to the manufacturer’s protocol. cDNA was generated using a Veriti 96-well fast thermocycler (ThermoFisher) from the isolated RNA using a Superscript VILO Master Mix (Life Technologies, 11755–500) kit. The cDNA was diluted and used as a template for the qPCR reaction using the TaqMan Universal PCR Master Mix (ThermoFisher, 4364340) and the corresponding 20x TaqMan gene expression assay. Human assays are as follows: HPRT housekeeping assay ID Hs01003267_m1, IFNβ assay ID Hs0077958_s1, RSAD2 assay ID Hs00369813_m1, TNF assay ID Hs00174128_m1, IL6 assay ID Hs00174131_m1, and TRIM22 assay ID Hs01001179_m1. Mouse assays are as follows: MX1 assay ID Mm00487796_m1, IFNL assay ID Mm04204155_gh, TMEM173 assay ID Mm01158117_m1, STAT1 assay ID Mm01257286_m1, TNF assay ID Mm00443258_m1, CXCL10 assay ID Mm00445235_m1, ISG15 assay ID Mm01705338_s1, IFIT1 assay ID Mm07295796_m1, and B-Actin assay ID Mm02619580_g1. Virus assays are as follows: IAV assay ID Vi99990011 and HRV assay ID Vi99990017_po.. For SARS-CoV-2 experiments, total RNA isolation was performed with the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit (Applied Biosystems, A4838) using Autopure-96 (AllSheng). Extracted RNA was mixed with Applied Biosystems TaqPath 1-Step Multiplex Master Mix No ROX (Applied Biosystems, A28522) and 20x TaqMan gene expression SARS-CoV-2 assay ID 4331182 Vi07921935_s1. The RT-PCR reaction was run on the QuantStudio 7 Flex instrument (Life Technologies) and analyzed using the Quantstudio Real-time PCR software. Relative gene quantification analysis was performed using standard analysis methods by normalization to the housekeeping gene and expressed as fold-change to the control.41 For imaging analysis, SARS-CoV-2 infected tissues were fixed at the indicated time point with 4% paraformaldehyde. Cells were permeabilized using 0.1% Triton-x 100 in PBS. Cells were labeled with DAPI (Sigma) and Alexa Fluor 488 Anti-SARS-CoV-2 nucleocapsid protein antibody (Abcam, ab283243). Images were obtained on an Opera Phenix (PerkinElmer) using a 20x air objective.

Cell Viability

Compound cytotoxicity was measured by incubating the cells with compound in 96-well plates for the indicated duration without virus. Cell viability was measured by incubating cells with Cell Titer Glo (Promega, G7573) reagent for 10 min to allow for cell lysis. Luminescence was acquired on a PHERAstar FSX plate reader (BMG). Cell viability was calculated as a percentage of the sample reading divided by the average reading of the untreated samples. Viability percentage was calculated according to the following equation:

Viral Cell Killing

Cells were incubated with virus and compound, as specified in the figure legend, until a significant cytopathic effect was observed. Cytopathic effect was measured using the Cell Titer Glo reagent, as specified above.

Virus Inhibition Calculation

Virus inhibition was calculated using data generated using RT-qPCR, viral cytopathic effect, or imaging to allow for a comparison of diABZI-4 antiviral efficacy across different virus families and assay readouts. Percent virus inhibition was calculated by using the following equation:

Quantification of Secreted IAV (Plaque Assay)

MDCK cells were incubated with 10-fold serial dilutions of IAV culture supernatant samples for 1 h, and plates were overlaid with cell culture medium containing carboxymethyl cellulose (CMC). Plates were incubated for 7 days. Plaques were visualized by fixing plates with paraformaldehyde and staining them with crystal violet.

Quantification of Secreted HRV (TCID50/mL)

HeLa cells were incubated with 10-fold serial dilutions of the indicated apical wash samples in 96-well opaque plates. Plates were incubated at 33 °C for 5 days to allow for cell lysis. Cytopathic effect was measured using Cell Titer Glo reagent, as specified above. TCID50 end point titer was calculated using the Spearman-Karber method.

In-Situ PIV3 ELISA

MRC-5 cells were infected with PIV3 and treated with the compound as specified. Virus was inactivated, and cells were fixed with 4% paraformaldehyde. Endogenous peroxidases were inactivated by incubating cells with 0.3% H2O2 (Fisher Bioreagents) for 30 min at 37 °C followed by washing with PBS. Plates were incubated with 1:5000 dilution of mouse anti-PIV3 Hemagglutinin-Neuraminidase antibody (Fitzgerald Industries) in 5% milk at 37 °C for 1 h followed by washing with 0.02% TWEEN-20 (Sigma-Aldrich). Plates were incubated with a 1:2000 dilution of anti-Mouse-HRP secondary antibody at 37 °C for 1 h followed by washing. Plates were incubated with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific) at 37 °C for 30 min followed by ELISA Stop Solution (Thermo Scientific). Absorbance was measured using a PHERAstar FSX plate reader.

Cytokine Measurements

Basal media samples were collected at the indicated time and frozen at −80 °C. Cytokine measurements were performed in undiluted basal media samples using the Meso Scale Discovery U-PLEX Viral Combo 1 kit (MSD, K15343K) according to manufacturer’s instructions. Mouse cytokine measurements of IFNβ, IFNγ, IL-1β, IL-6, IL-12p70, IP-10, MCP-1, and TNF-α were performed using a Meso Scale Discovery U-PLEX Custom Biomarker Group 1 Assay kit (MSD). Plate data were obtained on an MSD Sector Reader and analyzed by using Discovery Workbench software. Mouse IFNα was measured from lung homogenates using the VeriKine-HS Mouse IFN Alpha ELISA kit (PBL assay science) according to the manufacturer’s instructions.

Immune Cells and Flow Cytometry

iPSC myeloid precursors were differentiated into macrophages for 6–7 days by incubation with RPMI 1640 media, 10% FBS, and 100 ng/mL M-CSF (Peprotech 300–25–100UG). For flow cytometry analysis, cells were labeled with the viability dye Live/Dead Yellow (Invitrogen L34959) and FcR Blocking Reagent (Miltenyi Biotec 130–059–901). Subsequently, cells were labeled with fluorescent antibodies CD80-BV650 (Biolegend clone 2D10) and CD86 PerCP/Cy5.5 (Biolegend clone IT2.2). Cells were fixed, permeabilized, and incubated with an intracellular mix containing IAV-NP FITC (ThermoFisher MA1–7322). Data were collected on a CytoFlex S (Beckman Coulter) cytometer and analyzed by using FlowJo software (BD).

Protein Isolation and Capillary Western Immunoassay

MRC-5 cells were lysed in 200 μL of ice cold 1X RIPA Lysis buffer (Millipore, 20–188). Whole cell lysates were clarified by microcentrifuge (14,000 rpm, 5 min, 4 °C), and supernatants were transferred to new tubes. Sample concentrations were not measured or normalized. Primary human epithelial tissues were lysed in 300 μL of ice cold 1X RIPA Lysis buffer (Millipore, 20–188) supplemented with 1X Protease/Phosphatase Inhibitor Cocktail (Cell Signaling, 5872), incubated on ice for 5 min, and lifted from the transwell membrane by scraping with a pipet tip. Whole cell lysates were clarified by microcentrifuge (14,000 rpm, 5 min, 4 °C) and supernatants were transferred to new tubes. Protein concentrations were measured via a BCA protein assay (Pierce, no. 23227). Sample concentrations were normalized to 0.75 mg/mL. Proteins were analyzed via the 12–230 kDa Separation Module (ProteinSimple, SM-W004) on a Jess immunoassay system (ProteinSimple, San Jose, CA, USA). Phosphorylated STING was determined using p-STING (Ser366) rabbit mAb (Cell Signaling, 50907) at 1:250 in Milk free Diluent (Proteinsimple, 043–524); total STING was determined using STING (D2P2F) rabbit mAb (Cell Signaling, 13647) at 1:250 in Assay Diluent 2; and β-Actin was determined using β-Actin (D6A8) rabbit mAb (Cell Signaling, 8457) at 1:50 in Assay Diluent 2. Bound antibodies were detected with the antirabbit HRP detection module (Proteinsimple, DM-001) and quantified via the Compass for SW software.

Mice and Compound Treatments

Specific pathogen free (SPF) female and male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were ordered to arrive between 8 and 9 weeks of age and were 9–10 weeks old at the time study procedures started. Animals were housed 4 to a box in IVC innovive boxes on alpha dry bedding with standard 12-h light/dark cycles, room temperature of 68 to 79 and humidity of 30% - 70%. They were allowed access to Lab DIET-Rodent diet 5001 food and bottled water ad libitum. The animals were randomized on arrival into groups of N = 4 (with 4 female mice/box and 4 male mice/box). All procedures were performed in accordance with IACUC protocol and approved by the GSK Institutional Animal Care and Use Committee and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care (AAALAC), the United States Department of Health and Human Services and all local and federal animal welfare laws. GSK is committed to the replacement, reduction, and refinement of animal studies (3Rs). Our strategy is anchored in the contemporary definitions of the 3Rs.

C57BL/6J mice were treated intranasally with a 20 μL volume of 0.1 mg/kg diABZI-4 in a 1.2% solution of DMSO in PBS, pH 7.0. At the indicated time post compound treatment, animals were humanely euthanized, and lungs were collected. For infections, mice were inoculated with 400 TCID50 of IAV A/PR8/34 in a 40 μL volume. For host transcriptional analysis, lung tissue was weighed and homogenized in buffer RLT plus using a Qiagen Tissue Lyser II, and RNA was extracted from 30 mg equivalent volume of homogenate using the Qiagen RNeasy Plus mini kit according to the manufacturer’s protocol. For cytokine and PK analysis, lung was homogenized in PBS, and homogenates were normalized to 40 mg/mL in RIPA buffer containing protease and phosphatase inhibitors (for cytokine analysis) or normalized at a ratio of 4 mL of PBS to 1 g of tissue homogenate. Cytokines were measured from lung homogenates and serum using a MSD as described above. PK analysis was performed using standard liquid chromatography–mass spectrometry (LC-MS) methods.

HEK293T IRES Reporter and PBMC Assays

The protein coding sequences of human STING (Entrez Gene ID: 340061), mouse STING (Entrez Gene ID: 72512), and cynomolgus monkey STING (Entrez Gene ID: 102142250) were cloned into pCDNA3.1d (Invitrogen) with a carboxy-terminal hemagglutinin tag. The human STING HAQ variant (R71H, G230A, R293Q) was generated by mutagenesis using the QuickChange Lighting Multi kit (Agilent Technologies). The protein coding nucleotide sequence of human cGAS (Entrez Gene ID: 115004) was subcloned into pDEST8 (Invitrogen) with an amino terminal flag-his6 tagged fusion protein.

STING dependent IRF3 activation was measured using an HEK293T cell based ISRE-luciferase reporter gene transcription assay. The ligand dependent activation of STING was measured and compared to the maximum reporter gene activity in the presence of human cGAS protein expression. Briefly, 20,000 HEK293T cells/well in a 384-well plate were transfected with 100 ng DNA containing 20 ng STING (human, human HAQ variant, mouse or cyno) expressed from a pCDNA3.1 vector, 20 ng ISRE-Luc reporter (Agilent Technologies), and 85 ng pcDNA3.1 in 5 μL DMEM (Gibco) following the FuGene6 (Promega) protocol using a 6:1 DNA:FuGene6 ratio. For maximum activation control wells, 1 ng of cGAS expressed from a pDEST8 (Invitrogen) vector was added. The reactions were scaled to accommodate 500 or 25 wells for the cGAS containing maximum activation controls. Reactions were plated in 384-well white tissue culture plates (Greiner Bio one) containing a 11-point dose titration of diABZI-4 (50 μM – 0.2 nM final) by Multi-Drop Combi equipped with a standard cartridge, 50 μL/well at slow speed. Plates were sealed (AeraSeal) and incubated at room temperature for 10 min before 24 h incubation at 37 °C. Plates were removed and equilibrated to room temperature for 30 min before processing the luciferase response using Steady-Glo luciferase assay system (Promega). Raw luminescence was measured on a ViewLux (PerkinElmer) and expressed as a percent activation using the following normalization equation, N = 100–100*(U–C2)/(C1–C2) where U is the experimental unknown value, C1 is the average of 16 no response controls, and C2 is the average of 16 100% cGAS activation controls. Curve fitting was performed using the following equation y = A+((B-A)/(1+(10∧x/10∧C)∧D)) where A is the minimum response, B is the maximum response, C is the log10EC50, and D is the Hill slope. The results for each compound are recorded as pEC50 values (-C) in molar.

The functional activity of diABZI-4 in human PBMCs was conducted as previously described.7

Briefly, serial dilutions (11 × 3-fold serial dilutions starting at 50 μM) of diABZI-4 was added to PBMCs. The level of IFNβ secreted was measured after a 3h incubation at 37 °C using a human IFNβ electrochemiluminescence kit (Meso Scale Diagnostics). The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.

Statistics

All results are represented as the mean ± SEM. Statistical analysis was performed as specified in the figure legends by using GraphPad Prism 9.4 software. Multiple comparisons were performed using Ordinary one-way ANOVA and statistical significance is reported in the figure legends.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00504. Figure S1 diABZI-4 treatment induced IFNβ release from PBMCs and HET293T cells overexpressing STING, and Figure S2 diABZI-4 inhibition of infectious IAV and HRV (PDF)

Supplementary Material

id4c00504_si_001.pdf

The authors declare no competing financial interest.

Acknowledgments

We thank Rebecca Dunning for performing MSD analysis on selected media samples, and Tammy Lambert and Maria Klass for running MSD on the mouse tissues and serum. We also thank the animal care staff for their support in the animal studies, and Brian Peck for the pharmacokinetics bioanalysis. We thank the iPSC team for providing the iPSC precursors for the macrophage experiments. The following reagents were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-19/USA-WA1/2020, NR-52281, Human Lung Carcinoma Cells (A549) Expressing Human Angiotensin-Converting Enzyme 2, NR-53821 and SARS-Related Coronavirus 2, Isolate hCoV-19/USA/MD-HP20874/2021 (Lineage B.1.1.529; Omicron Variant), NR-56461, contributed by Andrew S. Pekosz.

Abbreviations Used

STING stimulator of interferon genes

diABZI diamidobenzimidazole

IAV influenza A virus

HRV human rhinovirus

PIV human parainfluenza virus
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