key: cord-0745557-fz97xoga
authors: Shaban, Mohammed Samer; Mayr-Buro, Christin; Meier-Soelch, Johanna; Albert, Benadict Vincent; Schmitz, M. Lienhard; Ziebuhr, John; Kracht, Michael
title: Thapsigargin: key to new host-directed coronavirus antivirals?
date: 2022-04-18
journal: Trends Pharmacol Sci
DOI: 10.1016/j.tips.2022.04.004
sha: f2d573a80b22aa7a6aabd4b40c391ebbaa3740c0
doc_id: 745557
cord_uid: fz97xoga

Despite the great success of vaccines that protect against RNA virus infections and the development and clinical use of a limited number of RNA virus-specific drugs, there is still an urgent need for new classes of antiviral drugs against circulating or emerging RNA viruses. To date, it has proven difficult to efficiently suppress RNA virus replication by targeting host cell functions, and there are no approved drugs of this type. This opinion paper discusses the recent discovery of a pronounced and sustained antiviral activity of the plant-derived natural compound thapsigargin against enveloped RNA viruses such as SARS-CoV-2, MERS-CoV, and influenza A virus. Based on its mechanisms of action, thapsigargin represents a new prototype of compounds with multimodal host-directed antiviral activity.

membrane spherules (DMSs). New 3D ultra-structural studies of SARS-CoV-2-infected cells provide strong evidence that viral RNA synthesis occurs inside ER-derived DMVs, which are considered as the major replicative organelles (RO) [8] [9] [10] [11] . In the course of infection, newly translated structural proteins translocate from ER membranes to the ER-to-Golgi intermediate compartment (ERGIC) . Guided by interactions of newly formed nucleocapsids with other structural proteins, virus particles are formed by budding into the lumen of secretory vesicular compartments and released from the cell through the secretory exocytotic pathway [7] . This brief summary shows that key steps of the CoV replication cycle involve interactions with a large variety of cytoplasmic membranes, most prominently ER membranes [12] [13] [14] . Although the molecular details of how viral and cellular factors (inter)act to trigger the profound membrane rearrangements in CoV-infected cells are not completely understood, the available information provides a solid basis to suggest that these processes may be attractive targets for antiviral therapies.

In a recent study aimed at identifying commonalities and differences between mechanisms involved in canonical ER stress and CoV-mediated ER stress response, respectively, Shaban et al. used thapsigargin as a reference compound, as done before by many other groups working in this field. The study revealed that thapsigargin strongly inhibits the replication of human CoV 229E (HCoV-229E), Middle-East respiratory syndrome CoV (MERS-CoV), severe acute respiratory syndrome CoV 2 (SARS-CoV-2) and influenza A virus (IAV, strain KAN-1) in cell culture [15] . Altogether, thapsigargin was found to interfere with coronavirus replication at multiple levels, including viral RNA synthesis and genome replication, protein translation and production of infectious virus progeny in different cell types [15] .

The antiviral effect was observed with a half-maximal effective concentration (EC 50 ) in the lower nM range, well below the (known) half-maximal cytotoxic concentrations (CC 50 ) of thapsigargin [15] . The inhibitory effect was readily detectable, even when the compound was added as late as eight hours post infection (p.i.). Moreover, the effect appeared to be remarkably long-lasting, with a single drug application being sufficient to suppress viral replication for up to three days [15] . Shortly after the Shaban et al. manuscript was published as a pre-print [16] , Al-Beltagi et al. reported that thapsigargin has similar inhibitory effects on HCoV-OC43, respiratory syncytial virus (RSV), SARS-CoV-2 and various IAV H1N1 strains [17] . Both groups independently confirmed the suppression of SARS-CoV-2 replication in normal human differentiated bronchial epithial (NHBE) cells grown at an air-liquid interface [15] or in undifferentiated NHBE cells [17, 18] , which were used as ex-vivo models mimicking the natural site of infection in humans. In a follow-up study, Al-Beltagi et al. showed that thapsigargin also suppresses the SARS-CoV-2 alpha, beta, and delta variants of concern [19] . The selectivity indices (CC 50 / EC 50 ) reported in these studies ranged from > 70 (for SARS-CoV-2) to > 900 (for IAV, RSV, MERS-CoV, HCoV-229E and HCoV-OC43) [15, 17, 18] . Taken together, these studies strongly support the idea that thapsigargin may be developed into a broadspectrum anti-CoV drug for application in the upper respiratory tract and warrants further pre-clinical and clinical studies.

In the following sections we discuss recent observations that, mechanistically, thapsigargin's profound antiviral activity is linked to the simulatenous interference with several interconnected cellular processes and pathways. The differential effects of PERK inhibitors and thapsigargin on CoV replication J o u r n a l P r e -p r o o f Journal Pre-proof Shaban et al. went on to perform a detailed molecular study to obtain more insight into how thapsigargin inhibits viral replication. Both thapsigargin and CoV infection activate the PERK protein kinase, a major ER stress sensor that phosphorylates eukaryotic translation initiation factor (eIF) 2α to block protein translation [20] . A highly selective PERK inhibitor, GSK2656157, was shown to suppress viral replication of MERS-CoV and HCoV-229E by approximately one order of magnitude but failed to abrogate the more than 100-fold suppression of replication resulting from treatment with thapsigargin [15] . These data show that PERK has a role in the replication of (at least some) CoVs. In contrast, thapsigargin appears to act downstream of PERK and was shown to block the replication of all human CoVs investigated so far, as discussed above. It thus appears possible that PERK inhibitors might be further developed into strain-specific CoV antivirals, while thapsigargin appears to have a much more potent and broad-spectrum antiviral activity.

In previous transcriptome studies unrelated to viral infections, thapsigargin was reported to cause multiple changes in cellular mRNA expression, thereby significantly extending the list of changes that are typically involved in physiological adaptive UPR responses induced by an excess of foldingcompetent or folding-defective model protein substrates targeted to the ER [21, 22] . These data suggested that additional mechanisms contribute to the phenotypes observed for thapsigargin-treated cells. Also, it was unknown to what extent thapsigargin-induced mRNA changes correlate with changes of the proteome, in particular under conditions of the profound global shutdown of translation that is typically observed in RNA virus-infected cells. Along with a strong activation of PERK, CoV infection was found to reduce cellular protein biosynthesis by about 90%. Thapsigargin treatment of infected cells was observed to improve translation rates significantly. Together with data from cell viability assays, these results suggest that the compound improves, but does not completely restore, the overall metabolic status of infected cells [15] . To address these points, Shaban et al. performed comparative proteomic and bioinformatics studies of cells infected with MERS-CoV-and SARS-CoV-2, respectively. Enrichment analyses revealed a thapsigargin-mediated activation of a broad spectrum of metabolic pathways which, probably, contribute to the protective response mounted in infected cells that were treated with this compond [15] . To further corroborate the significance of these observations, it will be important to define the enzymes and their metabolites that contribute to the thapsigargin-mediated metabolic changes of infected host cells and to answer the question of whether metabolic reprogramming can be exploited as a general (cell-intrinsic) strategy to suppress RNA virus replication.

At the level of individual components, Shaban et al. identified a set of proteins that are specifically induced (120 proteins) or downregulated (63 proteins) in thapsigargin-treated and simultaneously infected cells, as shown in Fig. 7 of their publication [15] . Many of the upregulated factors are annotated as being involved in the regulation of intracellular membrane compartments (ER, Golgi apparatus-associated vesicle biogenesis and transport, endocytosis) and membrane-associated processes such as ER stress and ER-associated degradation (ERAD, Box1) [15] ). As discussed above, membrane rearrangement and fusion events are major hallmarks of the coronavirus replication cycle and are critically involved in virus entry, replication and budding [8] [9] [10] . Based on the proteomic data, it is tempting to suggest that thapsigargin prevents, or activates, unknown membrane-associated processes that counteract efficient viral replication. To elucidate the underlying mechanisms, it will be J o u r n a l P r e -p r o o f Journal Pre-proof important to study individually the (enzymatic) activities, interactomes (i.e. protein:protein interactions and networks), localizations and functions of each of the specific factors identified by proteomics. Ideally, this approach should also include information from the plethora of proteomic, interactomic and CRISPR/Cas9-based single guide (sg) RNA genome-wide screens that have been performed recently for SARS-CoV-2 and other coronaviruses [23] [24] [25] [26] [27] [28] [29] . We firmly believe that such studies would provide new avenues for antiviral approaches that target very specifically the profound structural and functional changes of intracellular membrane compartments in +RNA virus-infected cells.

There are multiple connections between ERQC, ERAD, the ubiquitin proteasome system (UPS) and autophagy pathways as outlined in Box 1. ERAD is an UPR-activated degradative mechanism that serves to retro-translocate misfolded proteins from the lumen or membrane of the ER into the cytosol, whereupon they get ubiquitinylated and proteasomally hydrolyzed [30] . The regulation of ERAD and the related process called ER-phagy by viruses is incompletely understood [31, 32] . The proteomic analyses mentioned above revealed that thapsigargin induces a strong upregulation of several ERAD factors, including homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (HERPUD1), a central scaffold for ERAD complexes assembling in the ER membrane [33] ( Figure 1, key figure) . Thapsigargin also induced proteins involved in ubiquitinylation and ubiquitinlike posttranslational modifications. Ubiquitinylation of target proteins proceeds in three steps through ubiquitin-activating (E1), -conjugating (E2) and -ligating (E3) enzymes [34] . Shaban et al. observed an upregulation of ubiquilins (UBQLN1 and 4), the E2 enzyme ubiquitin-conjugating enzyme E2 G1 (UBE2G1), and the E1 enzyme ubiquitin-like modifier-activating enzyme 6 (UBA6) in cells infected with HCoV-229E, MERS-CoV, and SARS-CoV-2, respectively [15] . UBA6 and ubiquitin-activating enzyme E1 (UBE1, also called ubiquitin-like modifier-activating enzyme 1 (UBA1)) are the two main E1 enzymes that re-direct proteins to the proteasome [35] . UBA6 can also activate the ubiquitin-like protein (UBL) FAT10 (also called ubiquitin D, UBD) and transfer FAT10 to its substrate proteins for rapid proteasomal destruction [35, 36] . FAT10 is upregulated by the pro-inflammatory mediators interferon (IFN)γ, tumor necrosis factor (TNF)α or lipopolysaccharide (LPS) and has been found to modify ubiquitin-binding protein p62 / sequestosome-1 (p62 / SQSTM1), connecting this pathway to autophagy and suggesting a broader role of FAT10 in immune functions [37] . These results suggest a role for specific components of the UPS in viral protein turnover downstream of ERAD / autophagy. Therefore, it is an intriguing hypothesis that the thapsigargin-mediated autophagic block described below re-directs CoV proteins to an increased ERAD and thereby contributes to their rapid clearence and, as a result, suppression of viral replication (Figure 1 ).

Another factor that is consistently found to be regulated by thapsigargin is p62 / SQSTM1 (Box 1 and Figure 1) , a multi-functional signaling protein and cargo receptor for selective autophagy clients [38] . The potential role of (macro)autophagy in CoV replication is under active debate, with evidence for both pro-and antiviral functions, depending on the virus strains or model systems used (reviewed in [39, 40] ). As shown in the study by Shaban et al., HCoV-229E, MERS-CoV or SARS-CoV-2 infections downregulate the level of p62 / SQSTM1, albeit to a different extent. All three viruses have comparably limited effects on LC3B-II, which is the lipidated (i.e. phosphatidylethanolamineconjugated) form of autophagy-related protein LC3 / microtubule-associated proteins 1A / 1B light chain 3B (Atg8/LC3B), an ubiquitin-like molecule or modifier (ULM) that is essential for autophagosome formation early in the autophagy pathway [41] . Autophagy is a highly dynamic process that proceeds through several steps until the respective membrane-engulfed cargo is degraded by fusion of autophagosomes with lysosomes [42] . The activity of this pathway is commonly assessed by determining the turnover of autophagy proteins, which is generally referred to as the autophagic flux [43] . Application of bafilomycin A 1 , an inhibitor of the lysosomal enzyme V-ATPase, to CoVinfected cells resulted in (i) a strong increase in the p62 (and, to lesser extent, LC3B-II) protein levels and (ii) a 10-fold reduction of viral titers for some of the CoVs included in this study [15] . These results are in line with (more variable) antiviral effects observed for several other lysosomal inhibitors including (hydroxy)chloroquine [44] . Taken together, the available data indicate that human CoVs stimulate selective autophagy at some point during viral replication but, clearly, more evidence is needed to corroborate this hypothesis. In the presence of thapsigargin, strongly increased levels of p62 / SQSTM1 and LC3B were observed in virus-infected cells by a bafilomycin A 1 -independent mechanism. Also, thapsigargin strongly inhibited the autophagic flux in these cells [15] . These results are best reconciled with the work by Ganley et al. who showed that, independently from ER stress signaling and the endosomal pathway, thapsigargin blocks the fusion of autophagosomes with lysosomes late in the autophagy pathway by an unknown mechanism [45, 46] . It is therefore possible that such a mechanism prevents (irreversibly) the correct fusion of lysosomes with intracellular vesicles throughout the CoV replication cycle and, possibly, also interferes with membrane fusion events required for the formation of replicative organelles early in the infection cycle [7] .

The best understood mechanism of thapsigargin's action is related to intracellular calcium homeostasis [47] . Thapsigargin was first described as a modulator of the Ca 2+ -dependent histamine release from mast cells [48] [49] [50] . Functional and structural evidence suggested that thapsigargin increases intracellular Ca 2+ by specific and potent inhibition of the sarco-/endoplasmic reticulum (ER) Ca 2+ -ATPase (SERCA) (Box 2) [51] [52] [53] . The depletion of ER Ca 2+ and prolonged activation of the UPR are considered to cause the massive cell death seen with thapsigargin or its analogues in various cell types [54] . Furthermore, a recent report suggested that overexpression of SERCA may counteract the antiviral effects of thapsigargin against two RNA viruses infecting sheep and goat (Peste des petits ruminants virus, PPRV) or birds (Newcastle disease virus, NDV) [55] . In Drosophila, the SERCA Ca 2+ pump is important for membrane fusion events of lysosomes [56] , suggesting that the block of autophagy and RNA virus replication caused by thapsigargin may be related, at least in part, to changes in subcellular Ca 2+ gradients that support vesicle fusion events. This hypothesis may be an attractive avenue for further investigations into the antiviral effects of thapsigargin.

Taken together, these observations suggest that thapsigargin hits, in a unique manner, a combination of cellular processes that are essential for CoV replication (Figure 2A) . It remains to be studied if these processes can be targeted even more specifically using a combination of pathway-specific drugs, such as combinations of PERK and lysosomal inhibitors, with the aim to reduce potential side effects (Figure 2A) . The pharmacodynamics of thapsigargin in the context of Ca 2+ depletion from the ER and induction of cell death have been studied for four decades, primarily with the aim to use this drug in cancer therapy [6, 49] . The synthesis and production of thapsigargin and derivatives thereof is well established and formulations are available for application in humans [57] [58] [59] . A major obstacle for its potential use in antiviral therapy relates to the toxicity expected to occur in vivo. In this context, two studies have recently been performed in animal models. Oral doses of thapsigargin applied to mice 12 h before or 12 h after infection with IAV significantly improved the survival rates [17, 18] . Starting one day after a lethal challenge with IAV, a daily oral application of thapsigargin for 5 days was found to prevent infection-related death of mice infected with this virus [17] . In another study, thapsigargin was applied J o u r n a l P r e -p r o o f Journal Pre-proof 15 h before a lethal dose of LPS or cecal ligation and puncture (CLP)-induced sepsis. Thapsigargin was shown to improve, in a dose-dependent manner, the survial rate of two mouse strains after seven days [60] . Although these preclinical data on a short-term application of the drug are encouraging, more in vivo data are required to thoroughly assess the pharmacokinetics, efficacy and toxicity of thapsigargin using animal models of infection and inflammation. This includes the application of thapsigargin in suitable SARS-CoV-2 animal models [61] . While systemic thapsigargin-mediated toxicity in humans may be appropriately addressed by using very low, single-bolus doses or specific topical applications in the upper respiratory tract (Figure 2B ), it may also be possible to design suitable pro-drugs in which thapsigargin is coupled to peptides or other moieties to limit potential toxic side effects (Figure 2C, D) [62] . Thus, for example, mipsagargin, a protease-cleavable inactive prodrug of thapsigargin, has been applied in phase I and II clinical trials for prostate or liver cancer therapy with acceptable toxicity [63] [64] [65] [66] . By using a similar approach, it may be feasible to develop a cell-permeable thapsigargin derivative that is specifically cleaved by viral (or specific cellular) proteases, thereby restricting the release of the active drug to virus-infected cells (Figure 2C, D) . An additional clinical benefit of thapsigargin may arise from the compound-mediated suppression of inflammatory cytokines at the post-transcriptional level by interfering with their translation or secretion [67] . These effects have been observed in CoV-infected cultured cells and in the LPS / CLP animal models [15, 60] . The suppression of inflammation-mediated lung injury by low doses of the anti-inflammatory glucocorticoid dexamethasone reduces COVID-19 mortality [68] . In analogy, it will be important to find out if low dose thapsigargin will not only suppress CoV replication but also tissue-specific or systemic imunopathologies relevant for COVID-19 disease.

Here, we reviewed the available evidence that supports a potent antiviral effect of thapsigargin against CoVs, which is linked to an improvement (but not complete rescue) of the cellular metabolic state and the survival of CoV-infected cells treated with this natural compound. Also, there is initial evidence that thapsigargin inhibits the replication of the flavivirus tick-borne encephalitis virus (TBEV), albeit by a different mechanism that involves the UPR-dependent priming of an interferon response [69] . Athough many questions remain to be answered regarding virus-specific effects and the underlying antiviral actions of this compound at the molecular level ('see Outstanding Questions'), particlularly with respect to changes of intracellular membrane structures and functions in infected cells, we think it is worth pursuing further (pre-clinical and clinical) studies on thapsigargin and its derivatives to (i) establish topical, low-dosage and short-term therapeutic applications, (ii) restrict toxic effects to virusinfected cells and (iii) address the question of whether or not RNA viruses evolve drug-resistent variants that escape these multimodal antiviral mechanisms. Figure 1 . Hypothetical antiviral mechanism of thapsigargin involving ERAD. The scheme shows the organisation of the ER membrane-associated ERAD complex as recently reviewed [30] . ERAD is intimately linked to the major proteasomal and autophagy-related protein degradation pathways [70] [71] [72] (see also Box 1) . Red colors highlight the proteins BiP, HERPUD1, UBA6, LC3 and p62 / SQSTM1 that, by mass spectrometry and immunoblotting, have consistently been found to be induced by thapsigargin in CoV-infected cells. Question marks indicate potential functional links between these factors. Because thapsigargin-treated infected cells show a strong reduction of coronavirus protein levels, it is tempting to suggest that thapsigargin enhances ERAD activity in favour of autophagy (which is blocked by the compound late in the autophagy pathway) and, thereby, causes disposal of viral proteins primarily through the protasome pathway as part of its antiviral mechanism. BiP, endoplasmic reticulum chaperone BiP (GRP-78, HSPA5); DERL1, Derlin-1; SEL1L, protein sel-1 homolog 1; HERPUD1, homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like J o u r n a l P r e -p r o o f (A) The scheme shows the different levels of cellular processes that have been shown to be activated or suppressed by thapsigargin in cells infected with various coronaviruses. Also included are the effects of drugs blocking the lysosome (bafilomycin A 1 ) or PERK kinase (PERKi, GSK2656157 or GSK2606414). At present, the role of thapsigargin-mediated calcium depletion in coronavirus replication is unclear. Adapted from Reference [15] . (B) Possible routes and modes of application of thapsigargin in humans. While short-term oral administration was effective in mice, the bioavailability and pharmacokinetics of thapsigargin for the use as antiviral therapy in humans are currently unclear. (C) Structure of thapsigargin. (D) Potential protease-cleavable prodrugs in which thapsigargin is coupled to a peptide (in green) that will be cleaved off by cell type-specific proteases, by (inducible) proteases present only in infected cells or by coronavirus proteases in order to minimize side effects by restricting the active drug primarily or exclusively to infected cells.

Outstanding Questions  Are SERCA and SERCA-dependent processes such as Ca 2+ -signaling, Ca 2+ -dependent regulation of cellular factors or enzymes and subcellular changes in Ca 2+ concentrations relevant for thapsigargin-mediated antiviral activities? What are the roles of local Ca 2+ -sensing or effector mechanisms inside organelles and at their interfaces?  How is cell morphology affected by thapsigargin early upon viral replication and, related to this, can thapsigargin lead to the resolution of already established ROs? Can these questions be solved by 3D high resolution imaging, including advanced electron microscopy?  Present evidence suggests that thapsigargin antiviral activity crucially relies on the inhibition / activation of several cellular processes, but it is unclear if some of them are more important than others. Does thapsigargin hit a process or a specific molecular event that is common to all enveloped viruses, even though they have highly divergent life cycles such CoV or IAV? Can these issues be addressed by meta-analyses of functional genome-wide genetic screens performed in various infection models?  Several hACE2-transgenic mice and also mouse-adapted SARS-CoV-strains are now available.

Will thapsigargin suppress the replication of SARS-CoV-2 in animal models of COVID-19 disease? What are the longer-term consequences?  Multiple chemical analogues of thapsigargin have been synthesized recently and can be studied in RNA virus infection models with respect to replication versus cell survival. Can they be used to separate thapsigargin-mediated cytotoxicity from its antiviral effects?  Can thapsigargin analogues be designed in a way that they are cleaved, after internalization, by cell-specific or CoV main proteases?  Can thapsigargin be specifically targeted to sites of infection in vivo? For example, can (inactive) peptide-thapsigargin conjugates be targeted to cell surface molecules only expressed on CoVinfected cells?

J o u r n a l P r e -p r o o f

Mechanisms, regulation and functions of the unfolded protein response

Pharmacological targeting of endoplasmic reticulum stress in disease

The Unfolded Protein Response: Detecting and Responding to Fluctuations in the Protein-Folding Capacity of the Endoplasmic Reticulum

The unfolded protein response in immunity and inflammation

Thapsigargine and thapsigargicine, two new histamine liberators from Thapsia garganica L

Thapsigargin--from Thapsia L. to mipsagargin

Coronavirus biology and replication: implications for SARS-CoV-2

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells

Modification of intracellular membrane structures for virus replication

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

The interplay between emerging human coronavirus infections and autophagy

Multi-level inhibition of coronavirus replication by chemical ER stress

Inhibiting coronavirus replication in cultured cells by chemical ER stress

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication

Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

Fine-tuning PERK signaling to control cell fate under stress

Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides

Three branches to rule them all? UPR signalling in response to chemically versus misfolded proteins-induced ER stress

Crisp(r) New Perspective on SARS-CoV-2 Biology

A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2

Mapping the SARS-CoV-2-Host Protein-Protein Interactome by Affinity Purification Mass Spectrometry and Proximity-Dependent Biotin Labeling: A Rational and Straightforward Route to Discover Host-Directed Anti-SARS-CoV-2 Therapeutics

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV

A SARS-CoV-2 -host proximity interactome

CRISPR genetic screens to discover host-virus interactions

Disposing of misfolded ER proteins: A troubled substrate's way out of the ER

ER-phagy: Eating the Factory

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways

Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD

The spatial and temporal organization of ubiquitin networks

Activating the ubiquitin family: UBA6 challenges the field

UBA6 and Its Bispecific Pathways for Ubiquitin and FAT10

The ubiquitin-like modifier FAT10 interferes with SUMO activation

Regulation of selective autophagy: the p62/SQSTM1 paradigm

Coronavirus interactions with the cellular autophagy machinery

Coronavirus Interplay With Lipid Rafts and Autophagy Unveils Promising Therapeutic Targets

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Mechanism and medical implications of mammalian autophagy

Monitoring and Measuring Autophagy

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest

Intracellular Ca(2+) Sensing: Its Role in Calcium Homeostasis and Signaling

The ability of thapsigargin and thapsigargicin to activate cells involved in the inflammatory response

On the mechanism of histamine release induced by thapsigargin from Thapsia garganica L

From Plant to Patient: Thapsigargin, a Tool for Understanding Natural Product Chemistry, Total Syntheses, Biosynthesis, Taxonomy, ATPases, Cell Death, and Drug Development

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Inhibition of the sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase by thapsigargin analogs induces cell death via ER Ca(2+) depletion and the unfolded protein response

Inhibitor of Sarco/Endoplasmic Reticulum Calcium-ATPase Impairs Multiple Steps of Paramyxovirus Replication

Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification

Divergent synthesis of thapsigargin analogs

Scalable Synthesis of (-)-Thapsigargin

Use of a temporary immersion bioreactor system for the sustainable production of thapsigargin in shoot cultures of Thapsia garganica

Pharmacological preconditioning with the cellular stress inducer thapsigargin protects against experimental sepsis

Animal models for SARS-CoV-2

Targeting Strategies for Tissue-Specific Drug Delivery

Targeting thapsigargin towards tumors

A Phase II, Multicenter, Single-Arm Study of Mipsagargin (G-202) as a Second-Line Therapy Following Sorafenib for Adult Patients with Progressive Advanced Hepatocellular Carcinoma. Cancers (Basel)

Mipsagargin, a novel thapsigargin-based PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics

The Crosstalk of Endoplasmic Reticulum (ER) Stress Pathways with NF-kappaB: Complex Mechanisms Relevant for Cancer, Inflammation and Infection

After 62 years of regulating immunity, dexamethasone meets COVID-19

Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response

Herp depletion protects from protein aggregation by up-regulating autophagy

Emerging Principles of Selective ER Autophagy

ER-associated degradation in health and disease -from substrate to organism

Defining human ERAD networks through an integrative mapping strategy

ER-Phagy: Quality Control and Turnover of Endoplasmic Reticulum

Implications for Disease. Trends Biochem Sci

Mechanisms governing autophagosome biogenesis

Linking Biochemical and Structural States of SERCA: Achievements, Challenges, and New Opportunities

Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

E3 ubiquitin-protein ligase synoviolin; LC3, autophagy-related protein LC3, microtubule-associated proteins 1A / 1B light chain 3A/B; SQSTM1, sequestosome-1, ubiquitin-binding protein p62

UBA6, ubiquitin-like modifier-activating enzyme 6; VCP, transitional endoplasmic reticulum ATPase, 5S Mg (2+) -ATPase p97 subunit

M.K. wrote the initial paper draft and prepared the figures. M.S.S., C.M-B., J.M-S., B.V.A., J.Z. and M.L.S. helped to finalize the paper. All authors approved the submitted version of the paper.

The authors declare no competing interests.J o u r n a l P r e -p r o o f Glossary Air-Liquid Interface: Cell culture systems by which the apical site of a cell layer is exposed to air. Atg8/LC3: The autophagy-related protein LC3 also called microtubule-associated proteins 1A / 1B light chain 3A / B, a major autophagy regulator. Autophagy flux: The turnover of components degraded by the autophagy pathway. BiP: Endoplasmic reticulum chaperone BiP (GRP-78, HSPA5), a major ER factor supporting protein folding. Cecal Ligation and Puncture (CLP)-induced Sepsis: A procedure for modeling sepsis in vivo, in which the puncture of the cecum, which is full of bacteria, results in polymicrobial peritonitis, bacteremia, septic shock, multi-organ dysfunction and death. Coronaviruses (CoV): A group of RNA viruses with very large, mRNA-like, +ssRNA genomes. Efficacy: maximum effect or therapeutic response achievable from a pharmaceutical drug. Endoplasmic Reticulum (ER): An interconnected, intracellular network of flattened, membraneenclosed sacs involved in multiple processes including protein synthesis, folding and transport. Endosomal Pathway: A membrane transport pathway derived from the trans Golgi network to allow transport from the plasma membrane to the lysosome or back to the cell membrane. Ubiquitin: A small (8.6 kDa) regulatory protein that is covalently attached to a substrate by ubiquitinylation to regulate its decay or activity. Unfolded protein response (UPR): A transcriptional response program that induces multiple genes / corresponding proteins to compensate ER stress. Viral Replication/Transcription Complexes (vRTC): several non-structural CoV proteins (nsps) that replicate the CoV genome inside of RO.

One third of mammalian proteins are synthesized in the ER whose maturation is overseen by the ER quality control (ERQC) system. ERQC employs lectin chaperones or the chaperone BiP (in concert with ER-localized co-factors) that together with protein disulfide or peptidylprolyl isomerases catalyze protein folding reactions. Accurately folded and assembled proteins are released from chaperones, exit the ER and are transported to the Golgi via COPII vesicles to their final destinations. If ERQC fails, proteins will be disposed of by two degradative systems, ER-associated degradation (ERAD) or ERphagy. ERAD encompasses the identification of misfolded proteins, their retro-translocation to the cytosol and the degradation by the ubiquitin proteasome system (UPS). ERAD clients first bind to Sel1L which interacts directly with the E3 ligase HRD1. By oligomerization of its transmembrane domains, HRD1 forms the central, funnel like structure of the retro-translocon. Upon insertion into the HRD1 channel, the client starts to cross the ER membrane, reaches the cytosol and becomes (poly)ubiquitinylated by HRD1. At this point, the AAA-ATPase, p97, is recruited to the ER membrane via its association with VIMP, recognizes the poly-ubiquitin chains on the ERAD client and provides the final energy to extract it fully from the ER membrane. The ERAD client is then delivered to the 26S proteasome and degraded. ERAD involves many additional (UPR-regulated) proteins as assessed by large scale screens [73] . ER-phagy, a (macro)autophagy-related process, is used to remove larger proteins, protein aggregates, damaged ER and to reduce ER size after stress-induced expansion. During autophagy, multiple autophagy-related (ATG) proteins cooperate to elongate and bend pieces of membranes to engulf cytoplasmic components or organelles. The resulting structure, the autophagosome, is decorated with phosphatidylethanolamine (PE)-conjugated, lipidated LC3 (LC3-II) at the inner side of the lipid bilayer membrane. In case of selective autophagy, substrates often carry ubiquitin chains to tether them to specific autophagy receptors (e.g. p62 / SQSTM1). Mammalian ERphagy is promoted by six ER-resident receptors (FAM134B, RTN3L, CCPG1, SEC62, TEX264, and ATL3) that possess LC3-interacting regions (LIR). LIR-domains help to recruit autophagy substrates to the interior of the autophagosome precursor, called isolation membrane or phagophore. To complete autophagy, the pore in the phagophore closes and the outer autophagosomal membrane fuses with the lysosome resulting in disintegration of the inner autophagosomal membrane and degradation of the sequestered materials (for excellent reviews on these topics see [30, [74] [75] [76] ).J o u r n a l P r e -p r o o f