Thapsigargin is highly effective against COVID-19 virus (SARS-CoV-2)

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Researchers from the University of Nottingham have discovered a novel antiviral property of a drug that could have major implications in how future epidemics / pandemics – including COVID-19 – are managed.

The study, published in Viruses, shows that thapsigargin is a promising broad spectrum antiviral, highly effective against COVID-19 virus (SARS-CoV-2), a common cold coronavirus, respiratory syncytial virus (RSV) and the influenza A virus.

Given that acute respiratory virus infections caused by different viruses are clinically indistinguishable on presentation, an effective broad-spectrum that can target different virus types at the same time could significantly improve clinical management. An antiviral of this type could potentially be made available for community use to control active infection and its spread.

The study is a collaborative project led by Professor Kin-Chow Chang and experts at the University of Nottingham (Schools of Veterinary Medicine and Sciences, Biosciences, Pharmacy, Medicine, and Chemistry), and colleagues at the Animal and Plant Health Agency (APHA), China Agricultural University and the Pirbright Institute.

In this ground-breaking study, the team of experts found that the plant-derived antiviral, at small doses, triggers a highly effective broad-spectrum host-centred antiviral innate immune response against three major types of human respiratory viruses – including COVID-19.

The key features based on cell and animal studies, which make thapsigargin a promising antiviral are that it is:

  • effective against viral infection when used before or during active infection
  • able to prevent a virus from making new copies of itself in cells for at least 48 hours after a single 30-minute exposure.
  • stable in acidic pH, as found in the stomach, and therefore can be taken orally, so could be administered without the need for injections or hospital admission.
  • not sensitive to virus resistance.
  • at least several hundred-fold more effective than current antiviral options.
  • just as effective in blocking combined infection with coronavirus and influenza A virus as in single-virus infection.
  • safe as an antiviral (a derivative of thapsigargin has been tested in prostate cancer).

Professor Chang said: “Whilst we are still at the early stages of research into this antiviral and its impact on how viruses such as COVID-19 can be treated, these findings are hugely significant.

“The current pandemic highlights the need for effective antivirals to treat active infections, as well as vaccines, to prevent the infection. Given that future pandemics are likely to be of animal origin, where animal to human (zoonotic) and reverse zoonotic (human to animal) spread take place, a new generation of antivirals, such as thapsigargin, could play a key role in the control and treatment of important viral infections in both humans and animals.”

Indeed, influenza virus, coronavirus and RSV are global pathogens of humans as well as animals. Thapsigargin represents a lead compound in the development of a new generation of powerful host-centred antivirals (as opposed to conventional antiviral drugs that directly target viruses) that could even be adopted in a holistic “One Health” approach to control human and animal viruses.

Professor Chang adds: “Although more testing is clearly needed, current findings strongly indicate that thapsigargin and its derivatives are promising antiviral treatments against COVID-19 and influenza virus, and have the potential to defend us against the next Disease X pandemic.”


Coronaviruses are enveloped plus-strand RNA viruses with a broad host range, including humans (de Wit et al, 2016; Gorbalenya et al, 2020). The four seasonal human CoVs (HCoV-229E, -NL63, – HKU1, -OC43) generally cause a spectrum of (mild) symptoms that are mainly restricted to the upper respiratory tract (Gerna et al, 2006; Greenberg, 2011; Jevsnik et al, 2012; Nicholson et al, 1997).

In contrast, the three highly pathogenic CoVs that emerged from animal reservoirs over the past two decades are frequently associated with significant disease burden and mortality in humans. The latter include the severe acute respiratory syndrome (SARS) CoV (Drosten et al, 2003; Guan et al, 2004; Rota et al, 2003), SARS-CoV-2 (Zhou et al, 2020; Zhu et al, 2020) and Middle East respiratory syndrome CoV (MERS-CoV) (Zaki et al, 2012).

The current SARS-CoV-2 pandemic highlights the urgent need to identify new antiviral strategies, including drugs that target the host side (Zhu et al., 2020). CoVs impose multiple functional but also structural changes to a wide range of cellular pathways and there is increasing evidence that some of these pathways may be exploited therapeutically (de Wilde et al, 2018; Fung & Liu, 2019).

In common with other cellular stress conditions, including infections by diverse pathogens, CoVs are known to activate the NF-κB, JNK and p38 MAPK pathways and to reprogram host cell transcriptomes (Liao et al, 2011; Mizutani et al, 2005; Poppe et al, 2017).

In addition, they induce the formation of replicative organelles (ROs), an intracellular network of double-membrane vesicles (DMV) that harbors the viral replication/transcription complexes (RTC) and shields these complexes from recognition by cellular defense mechanisms (Snijder et al, 2020). The combination of these and other events leads to cell damage and cell death upon virus budding and release within a few days (de Wilde et al., 2018).

The virus-induced cellular changes are associated with an activation of the unfolded protein response (UPR) which is evident from a profound transcriptomic endoplasmic reticulum (ER) stress signature, as recently reported for cells infected with HCoV-229E (Poppe et al., 2017).

The ER is critically involved in surveying the quality and fidelity of membrane and secreted protein synthesis, as well as the folding, assembly, transport and degradation of these proteins (Wang & Kaufman, 2016). The accumulation of unfolded or misfolded proteins in the ER lumen leads to ER stress and UPR activation, thereby slowing down protein synthesis and increasing the folding capacity of the ER (Karagoz et al, 2019).

As a result, cellular protein homeostasis can be restored and the cell survives. If this compensatory mechanism fails, ER stress pathways can also switch function and will eventually induce oxidative stress and cell death (Hetz & Papa, 2018; Wang & Kaufman, 2016).

The system relies on three ER membrane-inserted sensors, including the protein kinase R (PKR)-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and cyclic AMP-dependent transcription factor 6α (ATF6α). PERK and IRE1α are Ser/Thr kinases whose conserved N termini are oriented towards the ER lumen (Wu et al, 2014).

In non-stressed cells, the highly abundant major ER chaperone and ER stress sensor binding-immunoglobulin protein BiP (also called 78 kDa glucose- regulated protein, GPR78; heat shock protein family A member 5, HSPA5) binds to PERK and IRE1α, which keeps these two proteins in an inactive monomeric state (Bertolotti et al, 2000; Pobre et al, 2019).

Upon increased binding of BiP to misfolded ER clients, BiP is released from both PERK and IRE1α, resulting in an (indirect) activation of the two kinases by oligomerization and trans(auto)phosphorylation (Carrara et al, 2015; Cui et al, 2011; Kopp et al, 2019).

Active PERK phosphorylates the eukaryotic translation initiation factor 2 (eIF2) subunit α to shut down translation and also activates ATF4, the master transcription factor orchestrating ER stress- induced genes (Han et al, 2013; Urra & Hetz, 2017). Phosphorylated IRE1α activates its own RNase domain to generate spliced (s)XBP1 protein, a multifunctional transcriptional regulator responsible for adaptive responses but also cell death (Chen & Brandizzi, 2013).

The specific function(s) in this response of yet another ER stress-activated transcription factor, ATF3, is less well understood (Mungrue et al, 2009). Generally, the various branches of the UPR act in concert, allowing a multitude of potential outcomes, ranging from the compensation of ER stress and restoration of proteostasis to cell death (Hetz & Papa, 2018).

The activation of ER stress by infectious agents has been widely observed. However, with few exceptions, it remains to be studied how this response is shaped in a microbe-specific manner and whether or not these responses are beneficial or detrimental to the host (Grootjans et al, 2016). Moreover, there is a lack of knowledge on CoV-mediated (de)regulation of ER stress components at the protein level. The latter is important because CoVs, in common with many RNA viruses, are known to cause a global shutdown of host protein synthesis (Hilton et al, 1986).

Here, we report that CoV infection activates UPR signaling and induces ER stress components at the mRNA level but suppresses them at the protein level. Strikingly, the well-known chemical activator of the UPR, thapsigargin, exerts a profound antiviral effect in the lower nanomolar range on three different CoVs in different cell types. A detailed proteomics analysis reveals multiple thapsigargin- regulated pathways and a network of proteins that are suppressed by CoV but (re)activated by chemically stressed infected cells. These results reveal new insight into central factors required for CoV replication and open new avenues for targeted CoV antivirals.

Results

To investigate how CoVs modulate ER stress components at the mRNA compared to the protein level, we determined the expression levels of 166 components of the ER stress pathway KEGG 04141 “protein processing in endoplasmic reticulum” in human HuH7 liver cells, a commonly used cellular model for CoV replication, in response to infections with HCoV-229E and MERS-CoV, respectively.

For untreated HuH7 cells, we obtained mRNA (by RNA-seq) and protein (by LC-MS/MS) expression data for 119 components which revealed a positive correlation between mRNA and protein abundancies (Fig.1A, upper graph). However, in cell lysates obtained at 24 h post infection (p.i.), this effect was largely lost (Fig. 1A, middle and lower graph).

Pearson correlation matrix confirmed a progressive loss of correlation between mRNA levels and protein levels for this pathway over a time course from 3 h to 24 h p.i. (Fig. 1B). Thus, out of 37 (for HCoV-229E) or 56 (for MERS-CoV) ER stress factors that were found to be regulated at the mRNA level, only a few remained (down)regulated at the protein level at late time points (Fig. 1C, Fig. S1).

To determine the functional consequences of this opposing regulation at the mRNA and protein levels in CoV-infected cells, we focused on HCoV-229E and assessed key regulatory features of the ER stress pathway as shown in Fig. 2A. As a reference, we included samples from cells exposed to thapsigargin, a compound that has been widely used to study prototypically activated ER stress mechanistically (Bertolotti et al., 2000; Oslowski & Urano, 2011). This setup included experiments, in which thapsigargin and virus were added simultaneously to the cell culture medium (followed by a further incubation for 24 h) or thapsigargin was added to the cells at 8 h p.i. for 16 h (Fig. 2B).

The presence of thapsigargin in the growth medium resulted in a major drop in viral titers by more than 150-fold (from 9.18*106 to 5.7*104 pfu / ml) which was paralleled by reduced amounts of viral RNA isolated from thapsigargin-treated, HCoV-229E-infected infected cells at 24 h p.i. (Fig. 2C).

Immunofluorescence analysis of HCoV-229E-infected cells treated with thapsigargin confirmed the impaired formation of functional viral replication/transcription complexes (RTCs) as shown by the reduced levels of both double-stranded RNA (an intermediate of viral RNA replication) and nonstructural protein (nsp) 8 (an essential part of the viral RTC) (Fig. 2D).

A strong suppression of viral replication was also demonstrated by the reduced protein levels observed for the nucleocapsid (N) protein (a major coronavirus structural protein) as well as nonstructural proteins (nsp) 8 and 12, both of which representing essential components of the viral replication complex (Snijder et al, 2016) (Fig. 2E, F). In all cases, the antiviral effect of thapsigargin remained readily detectable when the compound was added at 8 h p.i, suggesting that it does not prevent viral entry but rather suppresses intracellular pathways required for efficient RNA replication and/or particle formation and release or activates unknown antiviral effector systems (Fig. 2C-F).

Next, we investigated ER stress signaling under these conditions. Both virus and thapsigargin were confirmed to activate the PERK branch of ER stress (Fig. 2E, F), as shown by the retarded mobility of PERK in SDS gels (indicating multisite phosphorylation) and by phosphorylation of the PERK substrate eIF2α at Ser51 (Fig. 2E, F). Unlike thapsigargin treatment, HCoV-229E infection led to a weak but significant decrease of PERK (mean 71±15 %) and eIF2α (mean 67±13 %) levels compared to the controls.

Infection also caused an approximately twofold (mean 42±22 %) reduction in BiP expression (Fig. 2E, F). In contrast, long-term thapsigargin treatment (for 16 h or 24 h) caused a 3–4- fold increase in BiP expression, also in HCoV-229E-infected cells, thus reversing the suppression by viral infection (Fig. 2E, F). Similarly, thapsigargin treatment for 16 h or 24 h caused a 1.5–2-fold increase in IRE1α expression (but not phosphorylation), again also in infected cells (Fig. 2E, F). In this set of experiments, ATF3 proved to be the only protein that was induced by virus alone (Fig. 2E, F), while the expression levels of ATF4 remained largely unchanged (Fig. 2E, F).

These data show that both CoV infection and chemicals like thapsigargin activate ER stress through the same proximal PERK pathway, although they affect downstream cellular outcomes differentially. The restoration of BiP and IRE1α levels by long-term thapsigargin treatment further suggests that the CoV-induced block of inducible host factors is not irreversible and can be reprogrammed by a (presumably protective) thapsigargin-mediated response. Our comparative analyses of viral replication and host response lead us to conclude that chemically and virus-induced forms of ER stress, although proceeding through the same core PERK pathway, do not simply potentiate each other but rather (somewhat counterintuitively) counteract each other.

To explore a potential pharmacological exploitation of this effect, we assessed the cytotoxicity of the combined thapsigargin treatment and virus infection, because both conditions are known to promote cell death.

At 24 h p.i., cell viability of HCoV-229E-infected HuH7 cells was only marginally reduced (mean 90.02±12.32 %) (Fig. 2G, upper graph). After 24 h of incubation, thapsigargin decreased cell viability in a dose-dependent manner with a CC50 of 10.7 µM in line with previous reports (Fig. 2G, middle graph, Fig. 2H) (Sehgal et al, 2017; Tombal et al, 2000). The combination of thapsigargin and HCoV-229E infection did not cause additional cytotoxicity as shown by a nearly identical CC50 of

9.7 µM (lower graph, Fig. 2G, Fig. 2H). At 1 µM thapsigargin, i.e., a concentration shown to completely abolish viral protein translation and replication (see above), the cell viability of cells infected with HCoV-229E and treated with thapsigargin was 76.6±7.9 %, suggesting that thapsigargin exerts its antiviral effects at concentrations well below its cytotoxic concentrations (Fig. 2G, H).

To further characterize the metabolic state of the cells under the conditions used in these experiments, we investigated protein de novo synthesis. Newly produced proteins were quantified by in vivo puromycinylation tagging of nascent protein chains followed by immunoblotting using anti-puromycin antibodies. HCoV-229E was found to shut down protein biosynthesis by 90.3±5.4% while 1 h of thapsigargin treatment led to a shutdown by 94.3±4.3% (Fig. 2I).

However, in infected cells, the simultaneous or delayed addition of thapsigargin restored (or rescued) protein biosynthesis to approximately 50 % of the level observed in untreated cells (Fig. 2I). These data demonstrate that, although both viral infection and thapsigargin treatment (individually) induce ER stress and cause a translational shut-down, their combination shows no additive harmful effects to the cells. On the contrary, their combination appears to have opposing effects that result in a partial restoration of the cellular metabolic capacity while retaining a profound antiviral effect.

We next assessed if these effects were cell-type or virus-specific. In line with the results described above, the antiviral effects of thapsigargin, the reconstitution of the BiP and IRE1α levels and the lack of additional cytotoxicity in infected cells could be confirmed for diploid MRC-5 embryonic lung fibroblasts infected with HCoV-229E (Fig. 3A-D), as well as for HuH7 cells infected with MERS- CoV and Vero E6 African green monkey kidney epithelial cells infected with SARS-CoV-2 (Fig. 3E- K, Fig. S2A, B). MERS-CoV and SARS-CoV-2 replication were suppressed by thapsigargin with an effective concentration (EC50) (as judged by virus titration) of 4.8 nM and 260 nM, respectively (Fig. 3I, J), while the CC50 for thapsigargin in Vero E6 cells was 17.22 µM (Fig. 3K), resulting in selectivity indices (SI, CC50/EC50) of 66.2 for SARS-CoV-2 and 2229.2 for MERS-CoV, respectively.

To characterize the underlying molecular mechanisms responsible for the observed antiviral effects of thapsigargin, we focused on two highly pathogenic coronaviruses, MERS-CoV and SARS-CoV, for which, to our knowledge, no side-by-side comparison of proteomic changes has been reported to date. The large-scale proteomic study included (i) untreated cells and cells that were (ii) infected with MERS-CoV, (iii) SARS-CoV-2, (iv) treated with thapsigargin, (v and vi) infected with one of these viruses in the presence of thapsigargin. We used label-free quantification of six replicates per sample to determine the expression levels of > 5000 proteins from total cell extracts.

In a systematic approach, we identified differentially expressed proteins (DEPs) based on pairwise comparisons of proteins obtained from untreated cells, virus-infected cells or thapsigargin-treated cells using a p value of -log10 ≥ 1.3 as cut-off. As visualized by Volcano plot representations, MERS-CoV infection suppressed 413 (at 12 h p.i.) and 1171 proteins (at 24 h p.i.), respectively, and increased the levels of 150 proteins (at 12 h p.i.) and 508 proteins (at 24 h p.i.), respectively (Fig. 4A, B), while

SARS-CoV-2 suppressed the expression of 232 proteins at 12 h p.i. and 141 proteins at 24 h p.i. and increased the expression of 184 proteins at 12 h p.i. and 56 proteins at 24 h p.i. (Fig. 4C, D). Thapsigargin treatment alone suppressed / induced large numbers of proteins in HuH7 cells (969 down, 923 up at 12 h; 1711 down, 958 up at 24 h) and in Vero E6 cells (201 down, 179 up at 12 h; 216 down, 149 up at 24 h) (Fig. 4A-D).

As expected, this analysis also identified viral proteins as the most strongly regulated DEPs. A comparison of virus-infected cells with virus-infected cells treated with thapsigargin revealed a complete suppression of all viral proteins and a large number of proteins with increased expression in thapsigargin-treated cells infected with MERS-CoV (676, 12 h p.i.; 1,100, 24  h p.i.; blue groups of proteins) or SARS-CoV-2 (268, 12 h; 326, 24 h; blue groups of proteins) (Fig. 4A-D, right graphs).

Also, similar numbers of proteins were identified with higher expression in virus-infected cells compared to virus-infected cells treated with thapsigargin (Fig. 4A-D, right graphs; red groups of proteins). Together, these data lead us to conclude that thapsigargin causes a profound shift in protein expression in infected cells that likely contributes to the antiviral effects of this compound.

We then devised a bioinformatics strategy to identify patterns of co-regulated or unique pathways and link deregulated protein sets identified in these data to specific (known) biological functions. As shown schematically in Fig. 4E, we sorted the DEPs from each of the four groups shown in Fig. 4A-D into four multiple gene ID lists, annotated the gene IDs to biological pathways and generated hierarchically clustered heatmaps of the top 100 differentially enriched pathway categories for the 12 h

p.i. and 24 h p.i. time points of MERS-CoV- and SARS-CoV-2-infected cells, respectively, versus thapsigargin-treated cells by overrepresentation analysis (ORA) using Metascape software (Zhou et al, 2019). In this analysis, the groups of up- or downregulated proteins were kept separate to preserve information on whether specific DEPs belonging to particular overrepresented pathway terms were regulated in the same or opposite direction.

Inspection of the four top 100 clustered heatmaps shows many similarities but also differences in pathways and their enrichment p values in response to virus infection or thapsigargin, all together demonstrating the complexity of the cellular response to CoV infections or chemical stressors (Fig. S3-S4). By condensing this information to the top 5 pathways for up-/or downregulated DEPs we found that many of the most highly enriched categories are related to RNA, DNA, metabolic functions and localization (Fig. S5A).

We then combined the 400 pathway categories and searched this list for identical or unique GO terms in response to MERS-CoV, SARS- CoV-2 or thapsigargin. By filtering 227 pathways (out of 400) with enrichment p values ≤ -log10 3 we found 27 pathway categories shared by both viruses and by thapsigargin, which are mostly related to RNA, folding, stress and localization (Fig. 4F, Fig. S5B). 61 pathway categories unique to thapsigargin almost exclusively represented metabolic and biosynthetic pathways as shown for the top 20 overrepresented pathways containing up- or downregulated DEPs, suggesting that thapsigargin on its own, unlike CoV infection, initiates a broad metabolic response (Fig. 4F, Fig. S5B).

This raised the question of whether the thapsigargin effects were retained in infected cells or, alternatively, drug-sensitive pathway patterns were reprogrammed (or masked) by the virus infection. To address this point, we pooled all pathways enriched under virus+thapsigargin conditions and compared them to virus infection or thapsigargin alone. 53% (132 out 248) pathway terms were shared by these three conditions reflecting multiple stress-related, catabolic and RNA regulatory processes (Fig. 4G, H). 21 pathway terms were unique to the virus+thapsigargin situation. They primarily mapped to specific splicing, signaling (TORC, RHOA, ARF3) and transport/localization pathways (Fig. 4G, H). The 34 categories shared by virus+thapsigargin and thapsigargin conditions but were not detectable in cells infected with virus (only) recapitulate the thapsigargin-regulated metabolic pathways (pyruvate, aldehyde, carbohydrate, amino/nucleotide sugar, amino acid and glutamine

metabolism, TCA cycle, ERAD pathway, N-linked glycosylation) (Fig. 4G, H). For several of these pathways (e.g. ERAD, heat stress, carbohydrate metabolism), some DEPs were induced while others were repressed, indicating remodeling of pathway functions at the protein level (Fig. 4G, H). The 53 pathway terms that were absent in the virus+thapsigargin group of terms (groups 27, 9, 17 of the Venn diagram shown in Fig. 4G) represent a distinct set of terms, mostly related to nucleotide and DNA- related processes, such as DNA repair, DNA unwinding, chromatin silencing (Fig. 4G, H). In summary, the functional analysis of DEPs at the level of differentially enriched pathway categories shows that the antiviral effects of thapsigargin strongly correlate with the activation / suppression of a range of metabolic programs.

The enriched pathway terms provided important overarching information on shared and unique biological processes but not necessarily encompassed identical sets of DEPs as exemplified by the ten pathways shown in Fig. 4I. We therefore refined our analysis to the individual component level to identify proteins with similar regulation between both viruses across both cell types. The proteomes of HuH7 and Vero E6 cells overlap by 57 % (Fig. 5A).

In this group, only 43 identical proteins were found to be deregulated by both MERS-CoV and SARS-CoV-2 (Fig. 5B, left Venn diagrams). However, under thapsigargin+virus conditions, 108 proteins were upregulated and 61 proteins were downregulated, respectively (Fig. 5B, right Venn diagrams). Using the example of the top 50 DEPs, it becomes apparent that the majority of proteins are regulated in the same direction by thapsigargin alone; demonstrating that thapsigargin largely overrides any virus-induced modulation of host processes (Fig. 5C).

In the absence of thapsigargin, the virus infection generally has little or opposite effects on the levels of the 108 proteins, as exemplified by the suppression seen for BiP (HSPA5) or HERPUD1 (Fig. 5C, highlighted in green). The 108 induced factors map to pathways involving COPI-mediated anterograde transport, ER stress, organelle organization and apoptosis (Fig. 5D).

Across their pathway annotations, 59 out of the 108 proteins were reported to strongly interact, thus probably being involved in protein:protein networks that coordinate activities of the enriched pathways (Fig. 5E, left graph). Likewise, the 61 repressed proteins map to specific (though different) pathways, such as fatty acid degradation or viral life cycle (Fig. 5D). 26 components can be allocated to a few small protein interaction networks (Fig. 5E, right graph).

We then validated mass spectrometry data by immunoblotting, confirming the induction of HERPUD1 in thapsigargin-treated cells infected individually with each of the three CoVs (Fig. 5F, G). We also confirmed an additional hit belonging to the enriched pathways GO:0006520 (cellular amino acid metabolic process) and GO:0034976 (response to endoplasmic reticulum stress, as shown in Fig. 4G, H), cystathionine-γ-lyase (CTH), a regulator of glutathione homeostasis and cell survival (Lee et al, 2014), as a further independent example for the fidelity and robustness of the proteomics data (Fig. 5F, G).

The highly inducible HERPUD1 protein has an essential scaffolding function for the organization of components of the core ER-associated protein degradation (ERAD) complex (Leitman et al, 2014; Okuda-Shimizu & Hendershot, 2007). ER quality control (ERQC) and ERAD pathways are critically involved in the qualitative and quantitative control of misfolded or excessively abundant proteins in the ER.

If protein folding in the ER fails, the proteins are retro-translocated through a HERPUD1- dependent ER membrane complex to the cytosol for proteasomal degradation (Behnke et al, 2015). By searching our proteomics data for further ERAD factors we were able to retrieve a total of 34 (for MERS-CoV) and 20 (for SARS-CoV-2) proteins of the canonical ERQC and ERAD pathways for

which a differential expression was observed in virus-infected cells treated with thapsigargin (Fig. 5H). Mapping of these data on the KEGG 04141 pathway suggests that thapsigargin enhances or restores these mechanisms at key nodes of ERQC and ERAD in coronavirus-infected cells (Fig. S6).

We also intersected the 108+59 proteins jointly regulated by thapsigargin in MERS-CoV and SARS- CoV-2 infected cells with data from a recent genome-wide sgRNA screen that reported new ERAD factors required for protein degradation (Leto et al, 2019). This analysis identified 31 additional thapsigargin-regulated factors that may further support antiviral ERAD, including UBA6 and ZNF622, which were recently described either as negative regulators of DNA virus infections or of autophagy, the latter process playing diverse roles during CoV infection (Fig. 5I) (Jia & Bonifacino, 2020; Maier & Britton, 2012; Mun & Punga, 2019).

In conclusion, these data show that thapsigargin forces the (re)expression of a dedicated network of proteins with roles in ER stress, ERQC, ERAD, and a range of metabolic pathways. Collectively, these changes at the protein level confer an “antiviral state” and profoundly suppress CoV replication as summarized schematically in Fig. 5J.

Discussion

In this study, we report a potent inhibitory effect of the chemical thapsigargin on the replication of three human CoVs in three different cell types. Following up on observations that CoVs globally suppress UPR/ER stress factors, we find that thapsigargin counteracts the CoV-induced downregulation of BiP, HERPUD1 (and CTH) and increases IRE1α levels.

In this context, thapsigargin also plays a role in overcoming the coronavirus-induced block of global protein biosynthesis. Proteome-wide data revealed a thapsigargin-mediated reprogramming of metabolic pathways and helped to identify a network of specific thapsigargin-regulated factors, including candidates from the ERQC/ERAD pathways that, most likely, are involved in the destruction of viral proteins.

The positive effects of prolonged thapsigargin treatment on the expression of cellular BiP  and HERPUD1 are well documented (Kokame et al, 2000; Li et al, 1993; Ma & Hendershot, 2004; Sun et al, 2015). Thus, the key finding of our study is that the thapsigargin-mediated induction of ER factors overrides suppressive effects of CoVs on ER functions, as illustrated here for BiP, IRE1α and HERPUD1, but also at the global proteomic scale.

BiP is one of the most abundant cellular proteins (also in our mass spectrometry data) and plays essential roles in development and disease (Wang et al, 2017; Zhu & Lee, 2015). In yeast, the inducible expression of the BiP homologue Karp2 is particularly essential for disposing of toxic proteins and reducing cellular stress (Hsu et al, 2012).

Hence a reduction of BiP levels (as seen during CoV infection) and the contrary effect of thapsigargin-mediated upregulation are likely to have opposing consequences on cell fate upon infection. Similarly, IRE1α is suggested to mediate protective and adaptive responses suitable to alleviate ER stress, e.g. by balancing lipid bilayer stress, an aberrant perturbation of ER membrane structures, which may be expected to occur upon DMV formation in CoV-infected cells (Chen & Brandizzi, 2013; Halbleib et al, 2017; Snijder et al., 2020).

Accordingly, high levels of BiP, HERPUD1 and IRE1α may increase in general the resilience of cells when infected by diverse pathogens. In line with this, our data show that, in cells infected with representative coronaviruses, a protective ER response is initially elicited at the mRNA level (Fig. 1C and (Poppe et al., 2017)). However, the global suppression at the protein level (or the lack of induction) indicates that CoVs have evolved strategies at the posttranscriptional or translational level to escape the protective antiviral activities of BiP, IRE1α and HERPUD1.

Together with PERK, all three proteins are key regulators of ERQC/ERAD pathways and ample evidence shows that their expression, regulation and activities are intimately linked (Hetz & Papa, 2018; Karagoz et al., 2019). A recent study reported that PERK activation induces the RPAP2 phosphatase, inactivates IRE1α kinase activity and aborts IRE1α-mediated adaptive functions in response to the chemical stressor Brefeldin A (Chang et al, 2018).

In another report, high BiP levels exerted negative control of IRE1α by direct binding the kinase or by promoting IRE1α degradation (Chang et al., 2018; Sepulveda et al, 2018). Here, we show a different scenario, in which high BiP and IRE1α protein levels coincide with an antiviral state as well as with improved metabolic functions, suggesting unique modes of cross regulation of PERK, BiP and IRE1α in CoV-infected cells exposed to chemical stress.

The regulation of HERPUD1 and several ERAD factors by thapsigargin provides an additional layer of control contributing to the rapid suppression of CoV proteins. While ERAD is generally considered to dispose of unwanted proteins in the ER (Brodsky, 2012; Christianson et al, 2012), a process called “ERAD tuning” has been suggested to segregate ERAD components and thereby positively contribute to replicative organelle formation in RNA virus infections (Byun et al, 2014; Noack et al, 2014). Our data are compatible with a model in which a modulation of ERAD by small molecules may antagonize “ERAD tuning”, thereby preserving normal ERAD-mediated degradation.

Clearly, the mechanistic basis for these effects remains to be identified in additional studies. The proteomic data show that thapsigargin affects multiple pathways beyond the core ER stress response. They also indicate that it will not be trivial to identify the essential targets that mediate thapsigargin’s antiviral effects.

Our data provide a rich resource for further drug target analysis, also in conjunction with the few deep protein sequencing studies available for SARS-CoV-2 (but not MERS-CoV) (Bojkova et al, 2020; Bouhaddou et al, 2020; Grenga et al, 2020; Stukalov et al, 2020). Our study fills an important knowledge gap by providing a direct side-by-side comparison of pharmacologically targeted cells infected with two highly pathogenic human coronaviruses.

In the absence of effective therapeutic and prophylactic strategies (antivirals and vaccines) to combat coronaviruses, and in view of the current SARS-CoV-2 pandemic, we report these observations to invite other laboratories to embark on a broader investigation of this potential therapeutic avenue. Given that thapsigargin concentrations in the lower nanomolar range were shown to abolish CoV replication in cultured cells, even when added later in infection (8 h p.i.), this work identifies thapsigargin as an interesting drug candidate.

The Ca2+ mobilizing and cytotoxic features of plant- derived thapsigargin have been studied for 40 years (Andersen et al, 2015; Patkar et al, 1979). Several analogues have already been designed and efficient and scalable purification or synthesis is now available for application in humans (Chu et al, 2018; Chu et al, 2017; Lopez et al, 2018). Recently, a protease-cleavable prodrug of thapsigargin, mipsagargin, has been evaluated in phase I and II clinical trials for prostate cancer (Doan et al, 2015; Mahalingam et al, 2019; Mahalingam et al, 2016; Patkar et al., 1979).

It is not uncommon to adapt anti-proliferative cytostatic drugs (e.g. azathioprine, cylophosphamid, methotrexate) for the treatment of autoimmune and inflammatory disorders by applying lower doses than those needed for treating cancer (Marder & McCune, 2007).

Similarly, low doses of thapsigargin combined with short term systemic or topical application in the airways might reduce viral load early on or in critically ill patients with a favorable therapeutic index with respect to antiviral versus cytotoxic effects. CoV also activate inflammatory, NF-κB-dependent cytokine and chemokines at the mRNA level (Poppe et al., 2017), some of which (CXCL2, CCL20) escape translational shut-down and are secreted in a cell-type specific manner (Fig. S7). Some of these cytokines may contribute to the cytokine storm observed in some COVID-19 patients (Mehta et al,

2020). While thapsigargin had no effect on IL-8, IL-6, CXCL2 and CCL20 in cell culture (Fig. S7), a single bolus of the compound was shown to efficiently reduce the translation of pro-inflammatory cytokines in preclinical models of sepsis (Wei et al, 2019). Thus, an additional benefit of thapsigargin treatment may arise from dampening overshooting tissue inflammation in COVID-19 patients.

In summary, the study provides several lines of evidence that thapsigargin hits a central mechanism of CoV replication, which may be exploited to develop novel therapeutic strategies. This compound or derivatives with improved specificity, pharmacokinetics and safety profiles may also turn out to be valuable to mitigate the consequences of potential future CoV epidemics more effectively.

reference link: https://doi.org/10.1101/2020.08.26.266304


Provided by University of Nottingham

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