key: cord-328073-bqeffvzl
authors: Limonta, Daniel; Dyna-Dagman, Lovely; Branton, William; Makio, Tadashi; Wozniak, Richard W.; Power, Christopher; Hobman, Tom C.
title: Nodosome inhibition as a novel broad-spectrum antiviral strategy against arboviruses and SARS-CoV-2
date: 2020-11-06
journal: bioRxiv
DOI: 10.1101/2020.11.05.370767
sha: 
doc_id: 328073
cord_uid: bqeffvzl

In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses and SARS-CoV-2, the causative agent of COVID-19. Another drug that inhibits the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) which functions downstream of NOD2, also decreased replication of these pathogenic RNA viruses. The broad-spectrum action of nodosome targeting drugs is mediated, at least in part, by enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.

10 diagnosis and lack of effective vaccines against most arboviruses, development of broad-spectrum 193 antivirals against these pathogens should be a high priority. 194 The ongoing pandemic caused by SARS-CoV-2 poses a different set of challenges. Despite 195 concerted efforts to repurpose and find new antiviral drugs (31-33), so far only remdesivir has 196 shown modest efficacy in the acute stages of 35) . While more than 200 CoV-2 vaccine candidates are in accelerated development at preclinical and clinical stages (36), it 198 will likely take another year or more before they are broadly available to the general population as 199 safety and efficacy still need to be evaluated (37, 38) . 200 In this study, we characterized the broad-spectrum antiviral activities of nodosome inhibitors 201 GSK717 and GSK583. These small molecules display robust antiviral action against multiple RNA 202 viruses and may hold promise as pan-flavivirus inhibitors. First, we showed that NOD2 expression 203 promotes ZIKV multiplication in HFAs which are the main target of this flavivirus in the fetal 204 brain (10, 11) . Next, we demonstrated that the NOD2 inhibitor GSK717 blocks infection by and 205 spread of ZIKV in human fetal brain and cell lines. NOD2 inhibition also reduced replication of 206 the related DENV, the alphavirus MAYV and the pandemic coronavirus SARS-CoV-2. Blocking 207 the NOD2 downstream signaling kinase RIPK2 with GSK583 (which does not affect its catalytic 208 activity) significantly inhibited replication of these viral pathogens. 209 Gefitinib is an FDA-approved drug for treatment of lung, breast and other cancers. It works by 210 reducing the activity of the epidermal growth factor receptor (EGFR) tyrosine kinase domains. Of 211 note, this drug also inhibits the tyrosine kinase activity of RIPK2 (39) and has been shown to 212 inhibit replication of DENV and release of pro-inflammatory cytokines from infected human 213 primary monocytes (40). The authors suggested a role for EGFR/RIPK2 in DENV pathogenesis 214 and that gefitinib may be beneficial in the treatment of dengue patients. Similarly, the work here which demonstrated the antiviral activity of NOD2 and RIPK2 inhibitors using tissue explants, 216 primary cells and cell lines, support the potential clinical use of these compounds in mono or co-217 infections by arboviruses as well as coronavirus infections at early and/or advanced stages. 218 As GSK717 and GSK583 were developed primarily for immune-mediated inflammatory 219 conditions, their anti-inflammatory effects may have the added benefit of reducing the 220 hyperinflammatory state associated with flavi-, alpha-and coronavirus diseases (17, 29, 30, 41) . 221 Finally, our findings raise potential concerns regarding adjuvants in viral vaccines that augment 222 NOD2 as an immune strategy (42, 43) since this immune signaling protein is not a restriction factor 223 but rather an enhancement factor for multiple pathogenic RNA viruses. 224 The current study illustrates how the identification of a drug target through transcriptomic analyses 225 of virus-infected cells can lead to novel broad-acting host-directed antiviral strategies with a high 226 barrier of resistance. Increased NOD2 expression may be a novel mechanism of immune evasion 227 that viruses use to evade the innate immune response. Conversely, drugs that block nodosome 228 formation appear to have broad-spectrum antiviral activity by enhancing the interferon response. 229 Collectively, our results warrant consideration of these and related compounds as broad-spectrum 230 antiviral drug candidates for further preclinical development. At 12 hours post-transfection, total RNA was extracted and transcripts levels for IFN-stimulated 259 genes (ISGs) were quantified by qRT-PCR. 13 Human recombinant INF-α assay. HFAs in 96-well plates (Greiner) were treated with or without 261 25-100 U/mL of human recombinant IFN-α (Sigma-Aldrich) for 4-12 hours after which total RNA 262 was isolated and subjected to qRT-PCR in order to measure expression of ISGs. We declare no competing financial interests. The next day, cells were rinsed once with PBS and viruses at an MOI of 0.05 to 5 were added to the wells. Cells were then incubated for 1 (SARS-CoV-2), 2 (arboviruses) or 3 (arboviral coinfections) hours at 37C using fresh media supplemented with 3% fetal bovine serum. Next, the inoculum was removed, and the cells were washed twice with PBS. Complete culture medium was added to each well, and cells were incubated at 37C and 5% CO2. Mock-infected cells were incubated with the culture supernatant from uninfected cells. Fetal brain explant cultures and infections. Human brain tissue from two 15-19-week aborted fetuses was obtained (after written consent) under protocol 1420 of the University of Alberta Human Research Ethics Board. After delivery to the laboratory in ice-cold PBS, the fresh tissue was placed in a 100 mM Petri dish and then dissected with sterile scalpel and forceps into approximately 5 mm x 5 cm x 1 mm blocks (2) . The small tissue blocks were immediately immersed in 24-well plates containing the same media used to culture HFAs (see above).

Brain explant tissue was infected overnight with 10 6 PFU/mL of PRVABC-59 ZIKV strain. The next day, explants were washed once with PBS and then fresh media was added. The explant cultures were maintained in a humidified 37C incubator containing 5% CO2 for up to 5 days under different experimental treatments.

Tissue and cellular RNA purification, cDNA synthesis, and qRT-PCR. Total RNA was extracted from cultured cells or fetal brain tissue using NucleoSpin RNA (Macherey-Nagel GmbH & Co) kits. Samples were then treated with RNase-free DNase (Macherey-Nagel GmbH & Co) before a portion (0.5-1 µg total RNA) was subjected to reverse transcription using ImProm-II Reverse Transcriptase (Promega). Cellular transcripts and viral RNA were quantitated by qRT-4 PCR using PerfeCTa SYBR Green SuperMix (Quanta BioSciences) in a CFX96 Touch Real-Time PCR Detection System instrument (Bio-Rad) under the following cycling conditions: 40 cycles of 94C for 30 s, 55C for 60 s, and 68C for 20 s. Gene expression (fold change) was calculated using the 2 (-ΔΔCT) method with human β-actin mRNA transcript as the internal control.

The following forward and reverse primer pairs were used for PCR: shown. Values are expressed as the mean of three independent experiments. Error bars represent standard errors of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001, by the Student t test. 

Calu-3 and Huh7 cells infected with SARS-CoV-2 (MOI=0.1) were also treated with GSK583 for 24 hours before collecting the cell supernatants for viral titer determination. As a positive control of the flaviviral inhibition in infected A549 cells (MOI=0.5-5), we used the anti-flavivirus nucleoside analog

Vero E6 cells (MOI=0.1) was used as positive control at 0.1-10 µM or DMSO as vehicle

Cells were washed three times in PBS and then permeabilized/blocked in Blocking buffer with 0.2% Triton-X100 and 3% BSA in PBS for 1 hour at room temperature followed by washing with PBS containing 0.3% BSA. Incubations with primary antibodies diluted 1:500 (mouse anti-Flavivirus Group Antigen 4G2, Millipore), 1:200 (mouse monoclonal anti-chikungunya capsid kindly donated by Dr

Blocking buffer were carried out at room temperature for 1.5 hour followed by three washes in Washing buffer (0.02% Triton-X100 with 0.3% BSA and PBS)

Samples were then incubated with secondary antibodies (1:1000) in Blocking buffer containing 1

Secondary antibodies (Invitrogen) were Alexa Fluor 488 anti-mouse. Confocal images of cells on coverslips were acquired using an Olympus 1x81 spinning disk confocal microscope (Tokyo, Japan) and images were analyzed using Volocity 6.2.1 software. A set of confocal imaging was (20X) of ACE2-SK-N-SH infected with SARS-CoV-2 (MOI=1) and treated with GSK583 or DMSO for 24 hours. After fixation, coronavirus-infected cells were stained using mouse monoclonal antibody to SARS spike protein as primary antibody and secondary antibodies were

Confocal images were acquired using a spinning disk confocal microscope with Volocity 6.2.1 software. (D) Cells were counted in 10 different fields for quantitation of infected cells after GSK583 or DMSO treatment. Values are expressed as the mean of three independent experiments. Error bars represent standard errors of the mean

GSK583 suppresses SARS-CoV-2 release at subtoxic concentrations. Calu-3 (A) and

Huh7 (B) cells were infected with SARS-CoV-2 at the MOI of 0.1 and treated with GSK583 or DMSO as control for 24 hours after which cell supernatants were collected for plaque assay. Viral titers as relative fold is shown. Cellular ATP measurements using the CellTiter-Glo assay kit are shown at the 24-hour time point after GSK583 or DMSO treatment in uninfected ACE2-SK-N-SH (C), Calu-3 (D), and Huh7 (E) cells. Values are expressed as the mean of three independent experiments. Error bars represent standard errors of the mean

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