Vitamin B12 inhibits peptidylarginine deiminases and ameliorates rheumatoid arthritis in CAIA mice.

Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.

Natural fucoidans inhibit coronaviruses by targeting viral spike protein and host cell furin.

Fucoidans are a class of long chain sulfated polysaccharides and have multiple biological functions. Herein, four natural fucoidans extracted from Fucus vesiculosus, F. serratus, Laminaria japonica and Undaria pinnatifida, were tested for their HCoV-OC43 inhibition and found to demonstrate EC50 values ranging from 0.15 to 0.61 µg/mL. That from U. pinnatifida exhibited the most potent anti-HCoV-OC43 activity with an EC50 value of 0.15 ± 0.02 µg/mL, a potency largely independent of its sulfate content. Comparison of the gene expression profiles of fucoidan-treated and untreated cells infected with HCoV-OC43 revealed that fucoidan treatment effectively diminished HCoV-OC43 gene expressions associated with induced chemokines, cytokines and viral activities. Further studies using a highly fucoidan-resistant HCoV-OC43 determined that fucoidan inhibited HCoV-OC43 infection via interfering with viral entry and led to the identification of the specific site on the N-terminal region of spike protein, that located adjacent to the host cell receptor binding domain, targeted by the virus. Furthermore, in a SARS-CoV-2 pseudovirus neutralization assay, fucoidan also blocked SARS-CoV-2 entry. In vitro and in vivo, fucoidan decreased SARS-CoV-2 viral loads and inhibited viral infection in Calu-3 or Vero E6 cells and SARS-CoV-2 infected hamsters, respectively. Fucoidan was also found to inhibit furin activity, and reported furin inhibitors were found to inhibit viral infection by wild type HCoV-OC43 or SARS-CoV-2. Accordingly, we conclude that fucoidans inhibit coronaviral infection by targeting viral spike protein and host cell furin to interfere with viral entry.

Correction: Lee et al. The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b. Pharmaceutics 2022, 14, 1511.

In the original publication [...].

The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b.

Ciclesonide is an inhaled corticosteroid used to treat asthma and has been repurposed as a treatment for mildly ill COVID-19 patients, but its precise mechanism of action is unclear. Herein, we report that ciclesonide blocks the coronavirus-induced production of the cytokines IL-6, IL-8, and MCP-1 by increasing IκBα protein levels and significantly decreasing p65 nuclear translocation. Furthermore, we found that the combination of ciclesonide and dbq33b, a potent tylophorine-based coronavirus inhibitor that affects coronavirus-induced NF-κB activation a little, additively and synergistically decreased coronavirus-induced IL-6, IL-8, and MCP-1 cytokine levels, and synergistically inhibited the replication of both HCoV-OC43 and SARS-CoV-2. Collectively, the combination of ciclesonide and dbq33b merits consideration as a treatment for COVID-19 patients who may otherwise be overwhelmed by high viral loads and an NF-κB-mediated cytokine storm.

Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch.

JAK1 depletion or downregulation was previously reported to account for coronavirus inhibition. Here, we found that AG1024, an IR (insulin receptor) and IGF-1R (insulin-like growth factor 1 receptor) inhibitor, diminishes JAK1 protein levels and exerts anti-coronaviral activities with EC50 values of 5.2 ± 0.3 µM against transmissible gastroenteritis coronavirus (TGEV) and 4.3 ± 0.3 µM against human flu coronavirus OC43. However, although the IR and IGF-1R signaling pathways are activated by insulin or IGF-1 in swine testis cells, they are not triggered upon TGEV infection. AG1024, therefore, inhibits coronaviral replication and downregulates JAK1 protein levels independently of IR and IGF-1R. Moreover, JAK1 proteolysis caused by AG1024 was found through activation of upstream Ndfip1/2 and its effector NEDD4-like E3 ligase Itch. In addition, ouabain, which was reported to mediate JAK1 proteolysis causing anti-coronaviral activity by activation of Ndfip1/2 and NEDD4 E3 ligase, additively inhibited anti-coronaviral activity and JAK1 diminishment in combination with AG1024. This study provides novel insights into the pharmacological effects of AG1024 and Itch E3 ligase mediated JAK1 proteolysis and identified Ndfip1/2 as a cognate effector for JAK1 proteolysis via the diversified E3 ligases NEDD4 and NEDD4-like Itch. These findings are expected to provide valued information for the future development of anti-viral agents.

Remdesivir and Cyclosporine Synergistically Inhibit the Human Coronaviruses OC43 and SARS-CoV-2.

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Repurposing old drugs as antiviral agents for coronaviruses.

BACKGROUND: New therapeutic options to address the ongoing coronavirus disease 2019 (COVID-19) pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Due to the commercial unavailability of SARS-CoV-2 drugs for treating COVID-19, we screened approximately 250 existing drugs or pharmacologically active compounds for their inhibitory activities against feline infectious peritonitis coronavirus (FIPV) and human coronavirus OC43 (HCoV-OC43), a human coronavirus in the same genus (Betacoronavirus) as SARS-CoV-2. METHODS: FIPV was proliferated in feline Fcwf-4 cells and HCoV-OC43 in human HCT-8 cells. Viral proliferation was assayed by visualization of cytopathic effects on the infected Fcwf-4 cells and immunofluorescent assay for detection of the nucleocapsid proteins of HCoV-OC43 in the HCT-8 cells. The concentrations (EC50) of each drug necessary to diminish viral activity to 50% of that for the untreated controls were determined. The viabilities of Fcwf-4 and HCT-8 cells were measured by crystal violet staining and MTS/PMS assay, respectively. RESULTS: Fifteen out of the 252 drugs or pharmacologically active compounds screened were found to be active against both FIPV and HCoV-OC43, with EC50 values ranging from 11 nM to 75 µM. They are all old drugs as follows, anisomycin, antimycin A, atovaquone, chloroquine, conivaptan, emetine, gemcitabine, homoharringtonine, niclosamide, nitazoxanide, oligomycin, salinomycin, tilorone, valinomycin, and vismodegib. CONCLUSION: All of the old drugs identified as having activity against FIPV and HCoV-OC43 have seen clinical use in their respective indications and are associated with known dosing schedules and adverse effect or toxicity profiles in humans. Those, when later confirmed to have an anti-viral effect on SARS-CoV-2, should be considered for immediate uses in COVID-19 patients.

Natural cardenolides suppress coronaviral replication by downregulating JAK1 via a Na+/K+-ATPase independent proteolysis.

An unprecedented biological function of natural cardenolides independent of their membrane target Na+/K+-ATPase is disclosed. Previously, we reported that cardenolides impart anti-transmissible gastroenteritis coronavirus (anti-TGEV) activity through the targeting of Na+/K+-ATPase and its associated PI3K_PDK1_RSK2 signaling. Swine testis cells with Na+/K+-ATPase α1 knocked down exhibited decreased susceptibility to TGEV infectivity and attenuated PI3K_PDK1_RSK2 signaling. Herein, we further explored a Na+/K+-ATPase-independent signaling axis induced by natural cardenolides that also afforded significant anti-coronaviral activity for porcine TGEV and human HCoV-OC43. Using pharmacological inhibition and gene silencing techniques, we found that this anti-TGEV or anti-HCoV-OC43 activity was caused by JAK1 proteolysis and mediated through upstream activation of Ndfip1/2 and its effector NEDD4. This study provides novel insights into the pharmacological effects of natural cardenolides, and is expected to inform their future development as antiviral agents.

Artificial intelligence approach fighting COVID-19 with repurposing drugs.

Background: The ongoing COVID-19 pandemic has caused more than 193,825 deaths during the past few months. A quick-to-be-identified cure for the disease will be a therapeutic medicine that has prior use experiences in patients in order to resolve the current pandemic situation before it could become worsening. Artificial intelligence (AI) technology is hereby applied to identify the marketed drugs with potential for treating COVID-19. Methods: An AI platform was established to identify potential old drugs with anti-coronavirus activities by using two different learning databases; one consisted of the compounds reported or proven active against SARS-CoV, SARS-CoV-2, human immunodeficiency virus, influenza virus, and the other one containing the known 3C-like protease inhibitors. All AI predicted drugs were then tested for activities against a feline coronavirus in in vitro cell-based assay. These assay results were feedbacks to the AI system for relearning and thus to generate a modified AI model to search for old drugs again. Results: After a few runs of AI learning and prediction processes, the AI system identified 80 marketed drugs with potential. Among them, 8 drugs (bedaquiline, brequinar, celecoxib, clofazimine, conivaptan, gemcitabine, tolcapone, and vismodegib) showed in vitro activities against the proliferation of a feline infectious peritonitis (FIP) virus in Fcwf-4 cells. In addition, 5 other drugs (boceprevir, chloroquine, homoharringtonine, tilorone, and salinomycin) were also found active during the exercises of AI approaches. Conclusion: Having taken advantages of AI, we identified old drugs with activities against FIP coronavirus. Further studies are underway to demonstrate their activities against SARS-CoV-2 in vitro and in vivo at clinically achievable concentrations and doses. With prior use experiences in patients, these old drugs if proven active against SARS-CoV-2 can readily be applied for fighting COVID-19 pandemic.

Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Inhibition of SARS-CoV-2 by Highly Potent Broad-Spectrum Anti-Coronaviral Tylophorine-Based Derivatives.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7-8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2-3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6-7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5-14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

Tylophorine-based compounds are therapeutic in rheumatoid arthritis by targeting the caprin-1 ribonucleoprotein complex and inhibiting expression of associated c-Myc and HIF-1α.

Interruption of the Warburg effect - the observation that un-stimulated macrophages reprogram their core metabolism from oxidative phosphorylation toward aerobic glycolysis to become pro-inflammatory M1 macrophages upon stimulation - is an emerging strategy for the treatment of cancer and anti-inflammatory diseases such as rheumatoid arthritis. We studied this process with view to the discovery of novel therapeutics, and found that tylophorine-based compounds targeted a ribonucleoprotein complex containing caprin-1 and mRNAs of c-Myc and HIF-1α in LPS/IFN-γ stimulated Raw264.7 cells, diminished the protein levels of c-Myc and HIF-1α, and consequently downregulated their targeted genes that are associated with the Warburg effect, as well as the pro-inflammatory iNOS and COX2. The tylophorine-based compound DBQ 33b significantly meliorated the severity and incidence of type II collagen-monoclonal antibody-induced rheumatoid arthritis and diminished gene expressions of c-Myc, HIF-1α, iNOS, COX2, TNFα, and IL-17A in vivo. Moreover, pharmacological inhibition of either c-Myc or HIF-1α exhibited similar effects as the tylophorine-based compound DBQ 33b, even though inhibition of c-Myc reversed the induction of iNOS and COX2 in LPS/IFN-γ stimulated Raw264.7 cells to a lesser degree. Therefore, simultaneous inhibition of both c-Myc and HIF-1α is efficacious for anti-inflammation in vitro and in vivo and merits further study.

The cardenolides ouabain and reevesioside A promote FGF2 secretion and subsequent FGFR1 phosphorylation via converged ERK1/2 activation.

Na+/K+-ATPase α1 was reported to directly interact with and recruit FGF2 (fibroblast growth factor 2), a vital cell signaling protein implicated in angiogenesis, to the inner plasma membrane for subsequent secretion. Cardenolides, a class of cardiac glycosides, were reported to downregulate FGF2 secretion upon binding to Na+/K+-ATPase α1 in a cell system with ectopically expressed FGF2 and Na+/K+-ATPase α1. Herein, we disclose that the cardenolides ouabain and reevesioside A significantly enhance the secretion/release of FGF2 and the phosphorylation of FGFR1 (fibroblast growth factor receptor 1) in a time- and dose-dependent manner, in A549 carcinoma cells. A pharmacological approach was used to elucidate the pertinent upstream effectors. Only the ERK1/2 inhibitor U0126 but not the other inhibitors examined (including those inhibiting the unconventional secretion of FGF2) was able to reduce ouabain-induced FGF2 secretion and FGFR1 activation. ERK1/2 phosphorylation was increased upon ouabain treatment, a process found to be mediated through upstream effectors including ouabain-induced phosphorylated EGFR and a reduced MKP1 protein level. Therefore, at least two independent lines of upstream effectors are able to mediate ouabain-induced ERK1/2 phosphorylation and the subsequent FGF2 secretion and FGFR1 activation. These finding constitute unprecedent insights into the regulation of FGF2 secretion by cardenolides.

Correction: Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget.

[This corrects the article DOI: 10.18632/oncotarget.3236.].

The cardenolide ouabain suppresses coronaviral replication via augmenting a Na+/K+-ATPase-dependent PI3K_PDK1 axis signaling.

Cardenolides are plant-derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti-viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti-transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na+/K+-ATPase. Herein, we further explore the potential signaling cascades associated with this anti-TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV-induced production of IL-6 in a dose dependent manner, with 50% inhibitory concentrations of 37 nM and 23 nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV-infected ST cells, and ouabain imparted a degree of anti-TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na+/K+-ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti-viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti-viral applications for the cardenolides in the future.

Targeting Coronaviral Replication and Cellular JAK2 Mediated Dominant NF-κB Activation for Comprehensive and Ultimate Inhibition of Coronaviral Activity.

Tylophorine-based compounds exert broad spectral, potent inhibition of coronaviruses. NF-κB activation is a common pro-inflammatory response of host cells to viral infection. The aims of this study were to (i) find an effective combination treatment for coronaviral infections through targeting of the virus per se and cellular NF-κB activity; and (ii) to study the underling mechanisms. We found that tylophorine-based compounds target the TGEV viral RNA and effectively inhibit TGEV replication. NF-κB inhibition also leads to anti-TGEV replication. NF-κB activation induced by TGEV infection was found to be associated with two convergent pathways, IKK-2_IκBα/p65 and JAK2 mediated p65 phosphorylation, in swine testicular cells. JAK2 inhibition either by CYT387 (a JAK family inhibitor) or by silencing JAK2-expression revealed a dominant JAK2 mediated p65 phosphorylation pathway for NF-κB activation and resulted in NF-κB inhibition, which overrode the IκBα regulation via the IKK-2. Finally, tylophorine-based compounds work cooperatively with CYT387 to impart comprehensive anti-TGEV activities. The combination treatment, wherein a tylophorine compound targets TGEV and a JAK2 inhibitor blocks the alternative dominant NF-κB activation mediated by JAK2, is more effective and comprehensive than either one alone and constitutes a feasible approach for the treatment of SARS-CoV or MERS-CoV.

Design, Synthesis, and Evaluation of Thiazolidine-2,4-dione Derivatives as a Novel Class of Glutaminase Inhibitors.

Humans have two glutaminase genes, GLS (GLS1) and GLS2, each of which has two alternative transcripts: the kidney isoform (KGA) and glutaminase C (GAC) for GLS, and the liver isoform (LGA) and glutaminase B (GAB) for GLS2. Initial hit compound (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (2), a thiazolidine-2,4-dione, was obtained from a high throughput screening of 40 000 compounds against KGA. Subsequently, a series of thiazolidine-2,4-dione derivatives was synthesized. Most of these were found to inhibit KGA and GAC with comparable activities, were less potent inhibitors of GAB, and were moderately selective for GLS1 over GLS2. The relationships between chemical structure, activity, and selectivity were investigated. The lead compounds obtained were found to (1) offer in vitro cellular activities for inhibiting cell growth, clonogenicity, and cellular glutamate production, (2) exhibit high concentrations of exposure in plasma by a pharmacokinetic study, and (3) reduce the tumor size of xenografted human pancreatic AsPC-1 carcinoma cells in mice.

Identification of anti-viral activity of the cardenolides, Na+/K+-ATPase inhibitors, against porcine transmissible gastroenteritis virus.

A series of naturally occurring cardenolides that exhibit potent anti-transmissible gastroenteritis virus (TGEV) activity in swine testicular (ST) cells has been identified. In an immunofluorescence assay, these cardenolides were found to diminish the expressions of TGEV nucleocapsid and spike protein, which was used as an indication for viral replication; block TGEV infection induced apoptosis and cytopathic effects; and impart the same trend of inhibitory activity against Na+/K+-ATPase as for anti-TGEV activity. The viral titer inhibition was found to take place in a dose-dependent manner. Knocking down expression of Na+/K+-ATPase, the cellular receptor of cardenolides, in ST cells was found to significantly impair the susceptibility of ST cells to TGEV infectivity. Thus, we have identified Na+/K+-ATPase as an anti-viral drug target and its antagonists, cardenolides, a novel class of anti- TGEV agents.