ferentially expressed snoRNAs is needed in much more detail no matter whether they mediate the host antiviral response or the virus life cycle. Furthermore, GO evaluation showed that therapy with cinobufagin, telocinobufagin, or bufalin through MERS-CoV infection upregulated the genes involved inside the regulation of ion channel activity, and downregulated receptor and receptor ligands like cytokines. Cardiotonic steroids reportedly inhibit the Na+ /K+ -ATPase pumps and the inhibition from the ATP1A1 subunit of Na+ /K+ -ATPase pumps by bufalin inhibits MERS-CoV infection at an early stage [5]. As a result of the inhibition of Na+ /K+ -ATPase pumps by cinobufagin, telocinobufagin, or bufalin, the host cells could upregulate the regulation of ion channel activity to compensate for the intracellular ion concentrations and retain homeostasis. In contrast, MERS-CoV infection induced the production of cytokines which include interferon and activated receptors in Calu-3 cells. However, ligand production and activation induced by MERS-CoV infection were downregulated by cinobufagin, telocinobufagin, or bufalin treatment. In addition, the toxicity of digitoxin, bufalin, cinobufagin, telocinobufagin, bufotalin, and cinobufotalin was AChE Molecular Weight compared using 5-day repeated dose toxicity studies in mice. Despite the fact that the intraperitoneal HDAC6 Molecular Weight administration of 2 mg/kg/day of these compounds resulted in one hundred survival, administration of bufalin, cinobufagin, or digitoxin at 10 mg/kg/day resulted in one hundred death at 1, two, and four days immediately after administration, respectively; administration of telocinobufagin, bufotalin, and cinobufotalin at 10 mg/kg/day resulted in 100 survival. These data recommend that bufalin had the highest anti-coronaviral activity as well as the strongest toxicity. For that reason, cinobufagin and telocinobufagin had been chosen for their higher anti-coronavirus activity and low toxicity plus the pharmacokinetic properties of these compounds were additional examined. These data recommend that telocinobufagin had improved microsomal stability and reduce CYP inhibition than cinobufagin, though these compounds inhibited hERG channels by about 20 , and the PPB rates have been 80 . Investigation in the pharmacokinetic properties showed that the oral bioavailability of telocinobufagin was much better than that of cinobufagin, suggesting that telocinobufagin was additional promising among the cardiotonic steroids for being created as an anti-coronaviral drug. 5. Conclusions Within this study, the anti-coronaviral activity in the cardiotonic steroids, digitoxin, bufalin, cinobufagin, telocinobufagin, bufotalin, cinobufotalin, and resibufogenin against MERSCoV, SARS-CoV, and SARS-COV-2 was examined and compared. The proof of idea (POC) of cardiotonic steroids was performed only in vitro. Hence, in vivo POC as well as the therapeutic target study in detail must be executed in additional research. Investigations in to the efficacy of antiviral activity, 5-day repeated dose toxicity, and pharmacokinetic properties suggested that telocinobufagin was one of the most promising therapeutic candidate among the tested cardiotonic steroids for use against emerging coronaviruses including COVID-19.Pharmaceutics 2021, 13,12 ofAuthor Contributions: Conceptualization, Y.-H.J. and S.K. (Sunoh Kwon); methodology, Y.-H.J. and S.K. (Sunoh Kwon); validation, Y.-H.J., S.J., J.L., S.K. (Seungtaek Kim), M.S.J., C.M.P., J.H.S., H.R.K. and S.K. (Sunoh Kwon); formal evaluation, Y.-H.J. and S.K. (Sunoh Kwon); investigation, Y.-H.J., S.J., J.L., M.S.J., C.M.P.
