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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 © Copyright © 2020 Yang, Lee, Hsu, Jan, Lin, Chang, Peng, Yang, Liang, Liao, Chao, Pang, Kao, Huang, Lin, Chang, Niu, Wu, Sytwu, Chen and Lee

Background New therapeutic options to address the ongoing COVID-19 pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for 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, 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., Graphical abstract Image 1

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.

Graphical abstract, Highlights • Natural cardenolides suppress coronaviral activity via downregulating JAK1. • Natural cardenolides downregulate JAK1 in a Na+/K+-ATPase independent manner. • Ouabain (cardenolides) activates Ndfip1/2 and NEDD4 to mediate JAK1 proteolysis. • Ndfip1/2 and NEDD4 mediated JAK1 proteolysis attenuates coronaviral activity., 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.

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.

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., Graphical abstract Image 2, Highlights • Cardenolides were identified as a novel class of anti-TGEV agents. • Cardenolides diminished TGEV replication/viral titers in a dose dependent manner. • Cardenolides blocked TGEV infection induced apoptosis and cytopathic effect. • These cardenolides imparted the same trend of inhibitory activity for Na+/K+-ATPase. • Na+/K+-ATPase was identified as an anti-viral drug target.

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.