As a neuraminidase inhibitor, oseltamivir was approved by the Food and Drug Admission (FDA) in 1999¹. Since then, it has played an essential role in treating against influenza A and influenza B and is becoming more widespread. The atypical pneumonia caused by severe acute respiratory syndrome coronavirus (SARS-CoV) that broke out in Guangzhou, China, in 2003, linked oseltamivir to coronavirus. Zhang et al. found that the active site of the Spike (S) 1 Protein of SARS is similar to that of neuraminidase, suggesting that neuraminidase inhibitors may be useful to treat SARS-CoV². However, despite this similarity, no clinical data suggest that oseltamivir is effective in treating SARS-CoV. With the epidemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, oseltamivir has once again become a hot topic. A study from Thailand reported that a 71-year-old patient with severe COVID-19 on January 29, 2020 underwent a 48-hour treatment with lopinavir/ritonavir combined with oseltamivir, which improved the patient’s condition, and the throat swab test became negative³. Although a single case report does not prove the effectiveness of oseltamivir for COVID-19, it once again linked oseltamivir to the treatment of a coronavirus-induced disease. As frontline healthcare workers fighting against COVID-19, we were interested in the treatment regimen of this case and found most patients with COVID-19 who were symptomatic have used oseltamivir. We believed that it is of practical significance to further study the effect of oseltamivir on COVID-19. Because of its significant effectiveness on influenza and being sold over the counter, oseltamivir is not only stocked in common households but also a common antiviral drug in primary hospitals. Therefore, if oseltamivir is effective for COVID-19 the treatment could be family-oriented. If, instead, the treatment effectiveness is not significant, the use of oseltamivir should be stopped, which will avoid delaying other treatments and multiple oseltamivir adverse reactions, such as nausea, vomiting, epilepsy, elevated liver enzymes, and arrhythmias^4,5. Therefore, the study of the antiviral effect of oseltamivir against SARS-CoV-2 could have a positive effect on the treatment of COVID-19.

SARS-CoV-2 belongs to the β-genus of the coronavirus family and is the seventh coronavirus found to infect humans. Therefore, it has the structural features of coronaviruses, such as structural proteins, including S protein and Nucleoprotein (NC), and also non-structural proteins, including the main key enzymes 3C-like protease (3CLpro), papain-like protease (PLpro), and RNA-directed RNA polymerase (Pol/RdRp). These main structural proteins and key enzymes of SARS-CoV-2 are important targets for inhibiting SARS-CoV-2 infection^6–9. Since the SARS outbreak in 2003, the S protein, NC, 3CLpro, PLpro, and Pol/RdRp of the coronavirus known to infect humans have been extensively studied due to their potential as therapeutic targets^10–15.

Oseltamivir is a competitive inhibitor of neuraminidase designed by computer-assisted technology. In this context, the effective binding of oseltamivir to the active site of key proteins of SARS-CoV-2 is particularly important. Therefore, here we: 1. analyzed whether S protein, NC protein, 3CLpro, PLpro, and Pol/RdR have active centers similar to neuraminidase; 2. explored whether oseltamivir can form effective docking with key protein active centers of the virus using molecular simulation; 3. accessed the antiviral effect of oseltamivir against SARS-CoV-2 in vitro; 4. used case review to analyze whether oseltamivir is a factor to improve the conditions of patients with COVID-19.

The obtainment of the viral protein structure

The structures of the S protein (PDB ID: 6VSB) and 3CL pro (PDB ID: 6LU7) of SARS-CoV-2 were obtained from the RCSB Protein Data Bank (http://www.rcsb.org). Then the structures of NC, PLPro, and Pol/RdRp were built through the method of homology modeling. We first downloaded the protein sequences of NC (YP_009724397.2), nsp3 (YP_009725299.1), and Pol/RdRp (YP_009725307.1) from the GenePept protein sequence database and used Swiss-model (www.swissmodel.org) program for homology modeling. The models were constructed successfully and shown in Fig1A-C. Among them, the NC of SARS-CoV-2 contains 419 amino acid residues. The search of a homology template using the Swissmodel found that the 35–174 residues of NC have an identity of 92.37% with the N-terminal RNA-binding domain (NRBD) of NC from SARS-CoV(PDB ID: 1ssk), which is the active region for the binding of NC of SARS-CoV with RNA. Inhibiting NRBD activity can affect coronavirus RNA transcription and replication, and virion assembly¹⁶. Thus, it is one of the targets for inhibiting coronaviruses. Therefore, NRBD is the main activity center of NC, and this model can be used for the active site of NC from SARS-CoV-2 for further research.

Alignment of the structure of viral proteins and influenza A neuraminidase

To explore whether oseltamivir is effective for SARS-CoV-2, we first analyzed whether there was structural similarity between the structure of viral protein and influenza A neuraminidase (PDB ID: 3Ti6), especially whether there was a similarity in the active center. TM-align program (https://zhanglab.ccmb.med.umich.edu/TM-align/) was used for structural alignment¹⁷. The alignment of the structure of influenza B neuraminidase (PDB ID: 4CPY) with 3Ti6 was selected as a positive control. TM-score was used to analyze the structural similarity; 0.0 < TM-score < 0.30 was defined as “random structural similarity”, and 0.5 < TM-score < 1.00 was defined as “in about the same fold”. The active site of influenza A and B neuraminidase were determined based on the location of ligand in crystal structure (Fig. 2A)^18,19. By comparing 4CPY with 3Ti6, the TM-score of the two was found to be 0.94460, which proved that the two were structurally homologous. Subsequently, we aligned the structures of S protein, NRBD, 3CL pro, PLpro, and Pol/RdRp with 3Ti6; the TM-scores were 0.30077, 0.19254, 0.28766, 0.30666, and 0.34047, respectively (Fig. 2G). The results suggested that there was only a structural similarity between the protein structure of the above SARS-CoV-2 and 3Ti6. The active center of the S protein was mainly the RBD domain (Fig. 2B green region) and S2 subunit HR1 domain (Fig. 2B blue region), which were also two important targets for studying the inhibition of S protein^20,21. We found that the similar structure of 3Ti6 and S protein is located in the N-terminal domain (NTD) region of S protein. As the active center of NC, we found that NRBD did not match the active center of 3Ti6 (Fig2C). The active center of 3CLpro was determined based on that it is homologous to the 3CLpro from SARS-CoV. Previous studies have confirmed the active center of 3CLpro of SARS-CoV. Thus, this region was used as the active center of 3CLpro of SARS-CoV-2¹². By alignment, we found that the active centers of the two proteins were similar (Fig. 2D). Subsequently, we also determined the active center of the PLpro and Pol/RdRp based on the homology of the proteins of SARS-CoV-2 and those of SARS-CoV. And we found that the active center of PLpro did not match the active center from 3Ti6 through the alignment of structure (Fig. 2E)²². In studies focused on the Pol/RdRp of SARS-CoV, it has found that Thr680, Asn691, and Asp623 can bind nucleotides and promote viral gene transcription, and Val557 also plays an important role in the replication of the virus¹⁹. Thus, the region where the above residues are located was determined as the active center of SARS-CoV-2 (Fig. 2F). It was found that the similar structure between Pol/RdRp and neuraminidase was not located in the active center. The above results suggested that there was a theoretical possibility of effective binding of oseltamivir with the active center of 3CLpro because it was found to be similar to the active center from the neuraminidase.

Molecular Docking Studies for predicting the antiviral effect of oseltamivir

To pursue our hypothesis, we used molecular docking to analyze whether oseltamivir carboxylic acid could stably bind to the active center of the viral protein structure. We used ChemBioDraw Ultra 17.0 software to draw the structure of oseltamivir carboxylic acid and N3 inhibitor, then converted them into three-dimensional (3D) structure using ChemBio3D Ultra 17.0 software. Then the structure of agents was preprocessing by MMFF94 force field. AutoDock Tools 1.5.6 software was used to remove water molecules and ligands in the protein, and then add hydrogen atoms, combine non-polar hydrogen atoms, add charges, and converted protein and drugs into the format of PDBQT. The GridBox module was used to generate a 3D ensemble around the active site, and the grid points in the ensemble were calculated for energy score evaluation. AutoDock Vina was used for molecular docking²³. Exhaustiveness was set at 20 and other parameters were used default value. Finally, we selected the conformation with the highest docking score to analyze the result using Free Maestro 11.9. The docking conformations were described in Fig3A-E. The results showed that the docking energies of oseltamivir carboxylic acid with NRBD, 3CLpro, PLpro, and Pol/RdRp were −4.7 kcal/mol, −7.0 kcal/mol, −5.8 kcal/mol, and −5.4 kcal/mol, respectively (Fig3F). The lower the binding energy, the more suitable the binding of the inhibitor molecule to the receptor. In terms of binding energy, oseltamivir carboxylic acid was more favorable to bind to the active site of 3CLpro effectively. However, we concluded that the ability of oseltamivir to inhibit 3CLpro is not strong compared with N3 inhibitor, which exhibited strong inhibition for 3CLpro of SARS-CoV-2 in vitro and its docking energy was −9.5 kcal/mol²⁴, which was lower than that of oseltamivir carboxylic acid.

In vitro study for accessing the antiviral effect of oseltamivir

To further verify our hypothesis, we evaluated the antiviral efficiency of oseltamivir against SARS-CoV-2 in vitro as described in the previous work²⁵. Briefly, Vero E6 cells infected with the SARS-CoV-2 were cultured with the different doses of the oseltamivir carboxylic acid. Cell culture supernatant was collected after incubated with agent for 24h and viral RNA was extracted. Quantitative real-time RT-PCR was performed to evaluate the quantification of viral copy numbers in the cell supernatant. Fig4A showed that treatment of oseltamivir did not inhibit SARS-CoV-2 replication, with half-inhibitory concentration (IC₅₀) value above 100 μM. Chloroquine was selected as a positive control and can inhibit SARS-CoV-2 replication (Fig4B). Therefore, we concluded that oseltamivir is ineffective against SARS-CoV-2.