Antiviral activity of digoxin and ouabain against SARS-CoV-2 infection and its implication for COVID-19

The current coronavirus (COVID-19) pandemic is exacerbated by the absence of effective therapeutic agents. Notably, patients with COVID-19 and comorbidities such as hypertension and cardiac diseases have a higher mortality rate. An efficient strategy in response to this issue is repurposing drugs with antiviral activity for therapeutic effect. Digoxin (DIG) and ouabain (OUA) are FDA drugs for heart diseases that have antiviral activity against several coronaviruses. Thus, we aimed to assess antiviral activity of DIG and OUA against SARS-CoV-2 infection. The half-maximal inhibitory concentrations (IC50) of DIG and OUA were determined at a nanomolar concentration. Progeny virus titers of single-dose treatment of DIG, OUA and remdesivir were approximately 103-, 104- and 103-fold lower (> 99% inhibition), respectively, than that of non-treated control or chloroquine at 48 h post-infection (hpi). Furthermore, therapeutic treatment with DIG and OUA inhibited over 99% of SARS-CoV-2 replication, leading to viral inhibition at the post entry stage of the viral life cycle. Collectively, these results suggest that DIG and OUA may be an alternative treatment for COVID-19, with potential additional therapeutic effects for patients with cardiovascular disease.

Notably, these agents have been used to treat various heart diseases and were mainly identified to bind to the transmembrane protein sodium/potassium ATPase (Na + /K + -ATPase) and inhibit ion-exchange, leading to increased intracellular Ca ++ concentration and heart muscle contraction [25][26][27] . Therefore, in this study, we evaluated the antiviral activity of DIG and OUA based on viral growth kinetics and inhibition at different stages of viral infection, and compared it to that of vehicle (DMSO), chloroquine (CHQ) and REM to identify a suitable and potent antiviral agent to treat COVID-19 patients with cardiac diseases.
After treating the virus-infected cells with drugs, viral N mRNA expression, viral copy number, and progeny virus titer were measured at 8, 24 and 48 h (the duration of the complete virus replication was assessed via growth kinetics) ( Fig. 2A-C). Viral N mRNA expression was almost fully suppressed (> 99%) by all drugs at 8 and 24 hpi. However, the expression of viral N mRNA was significantly restored in CHQ-treated cells at 48 hpi ( Fig. 2A). Viral copy numbers were also markedly reduced at 48 h in the DIG-, OUA-, and REM-treated cell culture supernatants (Fig. 2B). Progeny virus titer in the culture medium of DIG, OUA and REM treatment, as measured using plaque assays, revealed a 10 3 -and 10 4 -fold reduction, respectively, and an inhibition ratio of > 99% for the drugs compared to those for the carrier (DMSO, 1.80 × 10 6 plaque forming units (pfu)/mL). Moreover, the virus titer was considerably reduced in the cells treated with DIG (1.83 × 10 3 pfu/mL), OUA (1.80 × 10 2 pfu/mL) and REM (7.0 × 10 3 pfu/mL), but not CHQ (2.45 × 10 6 pfu/mL) at 48 hpi ( Fig. 2C, Table 1 and Supplementary Fig. S2).

Determination of the inhibition step in the SARS-CoV-2 life cycle by drug treatment.
To determine which step of the virus life-cycle is inhibited by drug treatment, DIG, OUA, CHQ and REM were administered at the different time points of treatment: prophylactic (1 h prior to infection and maintenance for 24 h), entry (0 h of infection and maintenance for 2 h), and therapeutic (2 h following infection and maintenance for 24 h). All drugs demonstrated high efficacy upon prophylactic administration. Viral copy number, mRNA expression, and viral N protein expression were lower in prophylactic-treated cells than in non-treated cells by approximately 99% (Fig. 3). In entry-treated cells, CHQ and OUA treatment significantly inhibited viral RNA and protein levels to approximately 60% and 30% of those of DMSO, respectively, whereas DIG and REM treatment did not effectively inhibit virus propagation ( Fig. 3A-C). In comparison, OUA, DIG, and REM, but not CHQ, treatment markedly reduced viral replication in the therapeutic-treated cells ( Fig. 3A-C).

Discussion
SARS-CoV-2 infection causes not only multiple organ failure but also higher mortality rate in patients with underlying cardiac diseases 4,28,29 . Notably, the angiotensin-converting enzyme 2 (ACE2) receptor, which serves as a functional receptor for coronaviruses, is systemically distributed in multiple organs and is especially highly expressed in the heart and lungs 29 . Therefore, these organs may be directly attacked by SARS-CoV-2 or indirectly damaged by elevated levels of proinflammatory cytokines [30][31][32][33][34][35] .
The drugs DIG and OUA have been used to treat heart conditions of patients for over 10 decades, and thus their clinical dosage regimen, bioavailability, pharmacokinetic profile information, and safety are well known 27 . Hence, these drugs may exert multiple benefits in patients with COVID-19 in terms of antiviral and symptom management and safety. Although the IC 50 in an in vitro study is a poor guide for clinically relevant concentrations of DIG and OUA, previous studies of cancer therapy with DIG using human cells reported IC 50 ranges Scientific RepoRtS | (2020) 10:16200 | https://doi.org/10.1038/s41598-020-72879-7 www.nature.com/scientificreports/ from 0.02 to 0.34 μM, and the correspond plasma concentrations were safe and in the acceptable range from 0.8 to 2.6 nM (1 ng/mL = 1.28 nmol/L) in patients with cardiac diseases 27,39 . Moreover, the serum levels of patients taking oral doses of 0.25 mg/day (3.4 to 5.1 μg/kg/day) DIG are in the range from 1 to 2.6 nM 40,41 . Therefore, the IC 50 of DIG and OUA described in this study may be helpful for further pre-clinical and clinical studies. In this study, single dose of DIG and OUA treatment consistently showed superior antiviral activity against human isolate BetaCoV/Korea/KCDC03/2020 infection, as evident from the evaluation of viral mRNA expression, copy number (released virions in cell supernatant), and progeny virus titer up to 48 hpi in vitro. The progeny virus titer at 48 hpi in the DIG and OUA treatment groups was comparable to that in REM and reduced more than 10 3 -10 4 -fold compared to either the non-treated vehicle (DMSO) or CHQ groups, indicating that DIG and OUA have effective antiviral activity with stability up to 48 hpi (this time point represents the peak viral titer on growth kinetics in Vero cells, corresponding to maximal SARS-CoV-2 replication) 36 . Moreover, DIG and OUA treatment significantly inhibited over 99% of viral mRNA expression, which is more effective than REM (> 60%) and CHQ (> 30%) at 48 hpi. These results suggest that DIG and OUA could be an alternative treatment against SARS-CoV-2 infection.
Notably, DIG and OUA significantly inhibited viral mRNA expression, copy number, and viral protein expression when administered at the post-entry stage, although DIG did not show effective antiviral activity at the host entry stage of the virus cycle. Clinically, these results are very important for therapeutics, as a large numbers of patients are asymptomatic at the initial stage of SARS-CoV-2 infection 37,38 . Moreover, the results indicate that the inhibition mechanism of SARS-CoV-2 by DIG may be similar to that of respiratory syncytial virus (RSV), wherein inhibition occurs at the step of viral RNA synthesis 18 . However, OUA may have another inhibition mechanism, as OUA treatment at the entry stage inhibited approximately 30% of viral mRNA and protein expression. This suggests that an OUA may have an alternative antiviral mechanism related to blocking Src-mediated endocytosis in the entry step of coronaviruses 42 . Interestingly, a recent study reported that digitoxin, a CG,  www.nature.com/scientificreports/ suppresses proinflammatory cytokines in influenza A virus-infected cotton rat lung 43 , which suggests that DIG and OUA may have an additional therapeutic role against COVID-19 with hypercytokinemia. Taken together, we demonstrated a more effective antiviral activity of DIG and OUA against SARS-CoV-2 infection in vitro than previously approved antiviral agents such as chloroquine and remdesivir. We propose that these agents may be used as therapeutic options for patients with COVID-19 and comorbid cardiac diseases. progeny titer were assessed using qRT-PCR and plaque assays. Viral mRNA expression was normalized to GAPDH expression. DMSO was set to 1, and the remaining values are represented as a relative value. Viral copy number was calculated using a standard curve. Values are presented as mean ± SD (n = 3). Statistically significantly differences between DMSO and drug treatment are represented as *P < 0.05, **P < 0.01 and ***P < 0.001 determined using the two-way ANOVA with Bonferroni post-tests (each column compared to control). ns not significant. cloning and linearity determination of the RnA reference. The PCR products with SARS-CoV-2 N primers were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA) and in vitro transcribed using the RiboMAX Large Scale RNA Production System-T7 (Promega). The linearity of the RNA reference template was evaluated with a tenfold serial dilution of in vitro transcribed N RNA of SARS-CoV-2 (10 4 -10 10 copies). The copy number of RNA was calculated using: [X g/μL RNA/(transcript length in nucleotides -340)] × 6.022 × 10 23 = Y molecules/μL 45 . The linear range was determined using a standard curve generated with diluted reference RNAs and the best-fit line to the raw data was established by linear regression analysis with 95% confidence intervals using GraphPad Prism (version 5.01; La Jolla, CA, USA).

Quantitative real-time PCR (qRT-PCR).
For the quantification of viral copy number, total RNA was isolated from cell supernatants using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized from 1 μg of total RNA using SuperScript IV (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. qRT-PCR was performed using Power SYBR Green PCR master mix (Applied Bio-  Figure S3; full image of the blots). Viral mRNA expression was normalized to GAPDH levels and represented as relative values. Values are presented as mean ± SD (n = 3). Anti-GAPDH blots were used as loading controls. Viral NP protein and GAPDH protein were blotted in the same gel. Statistically significantly differences between DMSO and drug treatment are represented as **P < 0.01 and ***P < 0.001 determined using the two-way ANOVA with Bonferroni post-tests. ns not significant. www.nature.com/scientificreports/ systems, Foster City, CA, USA) and an Applied Biosystems QuantStudio3 (Applied Biosystems) following the manufacturer's protocol. For measurement of viral mRNA, total RNA was isolated using TRIzol (Ambion, Leicestershire, UK) reagent. Subsequently, cDNA was synthesized from 1 μg of total RNA using a SuperScript IV (Invitrogen). qRT-PCR was performed using Power SYBR Green PCR master mix (Applied Biosystems) and Applied Biosystems QuantS-tudio3 (Applied Biosystems) as follows: denaturation at 95 ℃ for 5 min, followed by 40 cycles of 95 ℃ for 30 s and 60 ℃ for 30 s. The sequences of the GAPDH primers used were: 5′-GAA CGG GAA GCT TGT CAT CAA TGG-3′ and 5′-TGT GGT CAT GAG TCC TTC CAC GAT-3′. The sequences of the N primers used were: 5′-GGG AGC CTT GAA TAC ACC AAA A-3′ and 5′-TGT AGC ACG ATT GCA GCA TTG-3′ 46 .
Plaque assay. Monolayers of Vero cells were prepared in 12-well plates. The cells were infected with tenfold serial dilutions of supernatant from treated cells and incubated at 37 °C for 1 h. The medium was removed and cells were washed with PBS. Each well was overlaid with MEM/agarose (Gibco) and maintained at room temperature until the overlay turned solid. The plates were incubated at 37 °C for 3 days. The cells were then fixed with 2% paraformaldehyde (Thermo Scientific) and stained with 1% crystal violet (Sigma-Aldrich) overnight. Subsequently, cell viability was measured using the PrestoBlue Cell Viability reagent (Invitrogen), and viral RNA was isolated from cell supernatants and cDNA was synthesized. qRT-PCR was performed using Power SYBR Green PCR master mix (Applied Biosystems) and Applied Biosystems QuantStudio3. The copy number was calculated based on the reference RNA template and the IC 50 value was calculated using GraphPad Prism.
Drug treatment. Vero cells were pre-treated with DIG (150 nM), OUA (100 nM), CHQ (10 μM) and REM (10 μM) for 1 h, and then the virus was applied for 1 h to allow infection. The drug-virus mixture was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in the presence of fresh medium containing the optimal concentrations of the drugs, and the cells and supernatant were collected at 8, 24 and 48 hpi for quantification of viral mRNA, copy number, and progeny virus titer.
For the prophylactic condition, Vero cells were pre-treated with the drugs in infection medium for 1 h and the virus was applied for 2 h to allow infection. Subsequently, the cells were washed twice with PBS and incubated for 24 h in the presence of the drugs in fresh infection medium.
For the entry condition, the cells were treated with the drugs for the infection period (2 h), followed by removal of the drug-virus mixture and washing of the cells. Subsequently, the cells were incubated in infection medium without the drugs for 24 h.
For the therapeutic condition, following viral infection without the drugs for 2 h, the virus was discarded and the cells were washed. Then, drugs in fresh infection medium were added to the cells for 24 h. Statistical analysis. The statistical significance of DMSO control and drug treatments were assessed by one-way ANOVA with Dunnett's multiple comparison test. The statistical comparison of the viral copy number, mRNA expression, and progeny virus titer was performed using two-way ANOVA with Bonferroni post-tests. Data plotting and statistical analysis were performed using GraphPad Prism. A P value < 0.05 was considered statistically significant.

Data availability
The data that support the findings of this study are available from the corresponding author on reasonable request.