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 [...].

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.

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.

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.

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.

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.

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.