Abstract
Since December 2019, the outbreak of a new coronavirus, named 2019-nCoV, has greatly threatened the public health in China and raised great concerns worldwide. No specific treatment for this infection is currently available. We previously reported that teicoplanin, a glycopeptide antibiotic which has routinely been used in the clinic to treat bacterial infection with low toxicity, significantly inhibits the invasion of cells by Ebola virus, SARS-CoV and MERS-CoV, via specifically inhibiting the activity of cathepsin L. Here, we tested the efficacy of teicoplanin against 2019-nCoV virus infection and found that teicoplanin potently prevents the entrance of 2019-nCoV-Spike-pseudoviruses into the cytoplasm, with an IC50 of 1.66 μM. Although the inhibitory effect upon the replication of wildtype viruses ex vivo and in vivo remains to be determined, our preliminary result indicates that the potential antiviral activity of teicoplanin could be applied for the treatment of 2019-nCoV virus infection.
Introduction
The coronaviruses are enveloped, positive sense single-stranded RNA viruses 1,2. The well-known examples, which have emerged as important human pathogens, include severe acute respiratory syndrome CoV (SARS-CoV) in China in 2003 and middle east respiratory syndrome CoV (MERS-CoV) in the Arabian Peninsula since 20123-9. Recently a novel coronavirus outbreaks in December 2019 as the pathogenic agent that causes series of pneumonia cases in Wuhan of China has quickly raised intense attention not only in China but also internationally 10-14. This novel coronavirus, named as 2019 novel coronavirus (2019-nCoV), belongs to the beta-coronavirus according to the sequence released13,15. Evolutionary analyses have shown that the 2019-nCoV shares 79% homology with SARS-CoV and 50% with MERS-CoV 15-17. Given the high infectious rate and the lack of effective treatment for 2019-nCoV, it is quite urgent to develop an efficient antiviral drug for 2019-nCoV.
The spike glycoprotein (S protein) is the leading mediator of viral entry, and the important determinant of host range of coronaviruses 18. The infection of SARS virus is initiated by the attachment of S protein to the receptor ACE219, followed by cleavage with host cell protease TMPRSS2 20-22. The viruses are then transported through the early and late endosomes, subsequently endo/lysosomes, during which host protein extracellular proteases including cathepsin L mediates the further cleavage of S protein in endocytic vesicles23-26. The activated S protein will then activate the fusion between viral and cell membranes and release the genome of SARS-CoV into cytoplasm.
In 2016, our team had found that teicoplanin, a routinely-used clinical glycopeptide antibiotics, significantly inhibited the cellular entry of Ebola virus, SARS-CoV, and MERS-CoV27. Further mechanistic studies showed that teicoplanin blocked virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of glycopeptides as potential inhibitors of cathepsin L-dependent viruses. In this study, we have compared the cleavage site of cathepsin L in 2019-nCoV with that in SARS-CoV and found it is well conserved. Furthermore, we identified that teicoplanin also potently inhibited the entry of 2019-nCoV pseudovirus, which provide a possible strategy to the prophylaxis and treatment for2019-nCoV infection.
Materials and methods
Cell culture and recombinant viruses
The plasmid containing spike (S) gene of 2019-nCoV was purchased from Generay Biotech company (Shanghai, China) and inserted into pcDNA3.1 vector. HIV-1/2019-nCoV-S/pseudoviruses were produced by the co-transfection of pHIV-luciferase, psPAX2, and plasmids expressing different envelope or S proteins into HEK293T cells as previously described 27. Briefly, the pseudoviruses including HIV-luc/2019-nCoV-S, HIV-luc/SARS-CoV-S and HIV-luc/VSV-G were harvested from supernatants after 48 hours post transfection and filtered through a 0.45-μm pore-size filter and stored at -80 °C. HEK293T, A549 and Huh7 cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal calf serum (Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37 °C and 5% CO2.
IC50 determination
IC50 determination was carried out by luciferase assay from the infected cells in the presence of various concentrations (2-fold dilutions) of the teicoplanin. The IC50 curve was determined by using software from GraphPad (San Diego).
siRNA transfection, RNA isolation, and RT-PCR
Sequences of siRNA against cathepsin Lor TMPRSS2 were predesigned by Ribobio Company (Guangzhou, China). A549 cells were seeded in 105 cells per ml in each well of 12-well-microtiter plates and transiently transfected with siRNA (10–20 pmol/well) using lipofectinRNAimax reagent according to the manufacturer’s instructions in serum-free medium with suitable scrambled siRNA control. Twenty-four hours later, total RNAs from the transfected cells were extracted for knocking out efficiency detection by qRT-PCR.
Sequence data collection and alignment
The genome sequences of SARS-CoV and 2019-nCoV were collected from the GenBank database (https://www.ncbi.nlm.nih.gov/nuccore/). The sequences of SARS-CoV circulating in 2003 contain 6 strains (accession number: AY278488, AY545918, AY545917, AY394977, AY394978, AY394979) . The sequences of 2019-nCoV include 9 from Wuhan (MN908947, NC_045512, MN988668, MN988669, MN996527, MN996528, MN996529, MN996530, MN996531), 2 from Shenzhen (MN975262, MN938384) and 5 from USA (MN985325, MN988713, MN994467, MN994468, MN997409). The S gene sequences were obtained from the genome of SARS-CoV and 2019-nCoV according the annotation in the GenBank database. The sequence datasets were aligned using the ClustalW program implemented in MEGA X software28. Consensus sequences were created using the BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) based on the multiple alignment of SARS-CoV and 2019-nCoV, respectively. The amino acid sequence logos generated by using WebLogo29.
Statistics
Statistical analysis was performed with GraphPad Prism 7. For data with a normal distribution, we used a Student’s t test. For multiple comparisons a one-way or two-way ANOVA (for parametric data) followed by Bonferroni’s correction (only two groups were compared) were used. P values < 0.05 were considered statistically significant.
Results
Cathepsin L is required for the cell entry of 2019-nCoV
The proteolytic processing of the SARS-CoV S protein is essential for the virus entry and fusion. Many cleavage sites of the relevant exogenous protease including cathepsin L and TMPRSS2 involved in the proteolytic processing of the SARS-CoV S protein have been experimentally validated 20,30,31. Because cathepsin L has been identified as a target of teicoplanin, it is important to identify whether the cleavage site of cathepsin L exists on the 2019-nCOV S protein. After alignment, we found that the cleavage site of cathepsin L is well conserved between the SARS-CoV and 2019-nCoV S protein (Figure 1A), suggesting that cathepsin L could participant in the 2019-nCoV entry and fusion26,31,32. Moreover, the cleavage site of cathepsin L was consistent among the epidemic strains of 2019-nCoV retrieved from the GenBank database, including 9 from Wuhan, 2 from Shenzhen, and 5 from USA (Figure 1B). As such, we hypothesized that cathepsin L could be an important target for the entry of 2019-nCoV. Accordingly, we infected the cells with SARS-CoV-, or 2019-nCoV -S-pseudotyped HIV-1 viruses after siRNA-mediated knockdown of the expression of cathepsin L and TMPRSS2 and found that both cathepsin L and TMPRSS2 depletion impaired the cell entry of pseudoviruses (Figure 1C), indicating that both cathepsin L and TMPRSS2 are required for 2019-nCoV cell entry.
Teicoplanin specifically inhibits the entry of 2019-nCoV
Based on our previous reported, we generated the HIV-luc/2019-nCoV-S pseudoviruses (Figure 2A)27. The resulting viruses were then used to infect A549 cells in the presence of teicoplanin. To exclude the possibility that the drug inhibited the early events of HIV-1 life cycle, HIV-luc/VSV-G pseudoviruses bearing vesicular stomatitis virus (VSV) glycoproteins were set up for a negative control group while pseudoviruses bearing the SARS-CoV-S were used a positive control27. Here, we identified that teicoplanin acted specifically as a 2019-nCoV entry inhibitor in a dose-dependent manner (Figure 2B). It demonstrated an IC50 of 1.66 uM for its inhibitory effect on HIV-luc/2019-nCoV-S pseudoviruses (Figure. 2C).
Teicoplanin and dalbavancin repress the entry of 2019-nCoV in different cell types
Considering that ACE2 is a major receptor of 2019-nCoV, we have further analyzed the expression of ACE2 in different cell types from the GEO Profiles database and it was important to examine whether teicoplanin could repress the entry of 2019-nCoV viruses into different types of cells. The data showed that teicoplanin also effectively repressed virus entrance into HEK293T cells (Figure. 3A), and Huh7 cells (Figure 3B). As we have previously demonstrated that several glycopeptide antibiotics, including teicoplanin and dalbavancin, exhibited specific inhibitory effects on cathepsin L, we therefore hypothesized that teicoplanin and its homologs could also inhibit the entry of 2019-nCoV. The data illustrated that the dalbavancin also repressed the entry of 2019-nCoV in a dose-dependent manner (Figure 3C). However, vancomycin, another routinely-used antibiotics, did not inhibit the 2019-nCoV entry (Figure. 3D). There results were consistent with the previous inhibitory effect of several glycopeptide antibiotics on SARS-CoV and MERS-CoV27.
Discussion
Host cell entry is the first step of viral life cycle and is an ideal drug target for viral infection. In this study, we identified that teicoplanin could inhibit the entry of HIV-1-2019-nCoV-S pseudoviruses with the IC50 value of 1.66 uM. During the invasion phase, 2019-nCoV first binds to the receptor ACE2 on the surface of host cells. The interaction between RBD domain of S protein and ACE2 triggers conformational changes within S protein, which render the S protein susceptible to activation by host cell protease TMPRSS220,30,33. Subsequently, the 2019-nCoV virus enters the early endosome of the cell through endocytosis or macropinocytosis. During the early endosome maturation process, the endosome gradually acidifies, which has an important impact on the entry of virus into cells. The inhibitory effect of chloroquine on 2019-nCoV support this hypothesis17,34. The cysteine proteases cathepsin L in the endosome can further cleave the S protein, and activate the membrane fusion. It has been proposed that sequential cleavages of SARS-CoV S protein by TMPRSS2 and cathepsin L are necessary for fully exposure of fusion peptide at S2 region to the late endosome/lysosome membrane35. Our work indicates that both TMPRSS2 and cathepsin L are required for 2019-nCoV entrance, and teicoplanin potently prevents the 2019-nCoV S protein activation by directly inhibiting the enzymatic activity of cathepsin L (Figure 4)27.
Teicoplanin is a glycopeptide antibiotic, which is mainly used for serious infections caused by Gram-positive bacteria such as staphylococcus aureus and streptococcus 36-38. As a routinely-used clinical antibiotics, teicoplanin is well known for its much low toxic and side effects, long half-life in blood plasma, convenient administration, and high safety when used in combination with other antibiotics. The recommended plasma concentration of teicoplanin for clinical use to inhibit Gram-positive bacteria is 15 mg / L, or 8.78 μM, and the commonly used dose is 400 mg / day. Here we found the IC50 inhibition of 2019-nCOV was only 1.66 uM, which is much lower than the routine clinical drug concentration. Therefore, the routinely-used dose (400 mg / day) could be considered for patients with 2019-nCOV infection. If the effect is not significant, dose could be optimized because of its low toxicity. The doses such as 800 mg / day or 1200 mg / day could be considered to improve the drug efficiency. Given that the principle of antiviral therapy is to prevent virus infection and amplification at a stage as early as possible, it is reasonable to recommend the use of teicoplanin for 2019-nCoV in the early stage. Alternatively, it could substitute vancomycin or other antibiotics to treat the co-infection with Gram-positive bacteria at a proper time. As such, teicoplanin could function as a dual inhibitor for both the 2019-nCOV infection and co-infection with Gram-positive bacteria.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by National Key Research and Development Program of China (2020YFC0841400), the National Special Research Program of China for Important Infectious Diseases (2018ZX10302103, 2017ZX10202102-003), the Important Key Program of Natural Science Foundation of China (81730060), and the Joint-Innovation Program in Healthcare for Special Scientific Research Projects of Guangzhou (201803040002) to H.Z. This work was also supported by the Pearl River S&T Nova Program of Guangzhou (201806010118) and the National Natural Science Foundation of China (81971918) to T.P.