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      Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro


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          Dear Editor, COVID-19, caused by the SARS-CoV-2 virus, has now spread worldwide with catastrophic human and economic impacts and currently has infected over 10 million people and killed over 500,000 1 . In an effort to mitigate disease symptoms and impede viral spread, efforts in vaccine development and drug discovery are being conducted at a rapid pace 2 . Recently, we showed that the well-known anticoagulant heparin has exceptional binding affinity to the spike protein (S-protein) of SARS-CoV-2 3 . The S-protein of SARS-CoV-2 bound more tightly to immobilized heparin (K D = ~10−11 M) than the S-proteins of either SARS-CoV (K D = ~10−7 M) or MERS-CoV (K D = ~10-9 M). However, it is not known whether the tight binding of heparin to the SARS-CoV-2 S-protein translates into potent antiviral activity. In the current study, we evaluated the in vitro antiviral properties of heparin and other closely related polysaccharides to assess the relevance of heparin-related GAGs and other sulfated polysaccharides as part of the pharmacopeia of potential therapeutics that target SARS-CoV-2. Vero-CCL81, which expresses both ACE2 and TMPRSS2 4 , were used for viral replication at high titer 5 for use in antiviral assays. Heparin, heparan sulfates, other glycosaminoglycans (GAGs) 3 , and fucoidan and other highly sulfated polysaccharides were screened using surface plasmon resonance (SPR) to measure binding affinity to the SARS-CoV-2 S-protein (Fig. 1a). Briefly, solution competition studies between surface immobilized heparin and other sulfated polysaccharides were evaluated by injecting SARS-CoV-2 S-protein (50 nM) alone or mixed with 1 µM of an indicated polysaccharide in SPR buffer at a flow rate of 30 μL/min. After each run, dissociation and regeneration were performed. For each set of competition experiments, a control experiment (S-protein without polysaccharide) was performed to ensure the surface was fully regenerated. Among the tested polysaccharides, RPI-27 and RPI-28, complex sulfated polysaccharides (fucoidans) extracted from the seaweed Saccharina japonica 6 , chemo-enzymatically synthesized trisulfated (TriS) heparin 7 , and unfractionated USP-heparin itself were able to compete with heparin for S-protein binding. We selected these compounds along with a non-anticoagulant low molecular weight heparin (NACH) 8 for further study (Fig. 1b). The other GAGs including heparan sulfate, the chondroitin sulfates, and keratan sulfate show no competitive binding when compared to the control. Fig. 1 Assessment of antiviral activities of certain sulfated polysaccharides. a Surface plasmon resonance (SPR) experiments were used to screen polysaccharides that outcompete immobilized heparin binding to SARS-CoV-2 S-protein. Data are presented as mean±s.d., n=3 biologically independent samples. A two-sided t-test was performed to test significance against the control (P1<0.0001, P2=0.0003, P3=0.0016, P4=0.0041). b Structural units comprising polysaccharides used for in vitro antiviral studies. c Focus reduction assay images of virus infection on treatment of indicated polysaccharides. At 48h after infection, Vero cells were fixed and probed with SARS-CoV-2 spike primary antibody (1:10000, Sino Bio Inc.) and HRP-conjugated goat rabbit (1:10000, Abcam) secondary antibody. d Vero cells were infected with SARS-CoV-2 at a MOI of 2.5×10−3 at different doses of each polysaccharide for 48h. The viral yield was quantified using a focus reduction assay. Cytotoxicity in Vero cells was measured using a WST-1 assay. The left and right y-axis of the graphs represent mean % inhibition of virus yield and cytotoxicity of the polysaccharides, respectively. Cytotoxicity experiments were performed in duplicate with n=3 biologically independent samples. Focus reduction assay experiments were performed in mean±s.d. (quadruplicate measurements) with n=3 biologically independent samples. e The RBD-ACE2-binding interface is stabilized by an extensive hydrogen bonding network involving sidechains of several residues on both RBD and ACE2. Polar sidechains of N487, Y489, Q493, Q498, and Y505 on the spike protein RBD along with other residues would be able to bind to heparin and inhibit RBD-ACE2 interaction. Heparin (here an octasaccharide) forms a hydrogen bond network with N448, N450, Q493, and N501 that aids in its occupancy of this binding regions and sterically restrict access to Q498, Y489, and Y505 necessary for ACE2 receptor binding. Standard assays were performed to quantify potential cytotoxicity and antiviral activity. Cytotoxicity determination of the polysaccharides was performed using Vero cells and the standard water-soluble tetrazolium salt-1 (WST-1) assay (Takara Bio Inc., Japan). None of the tested polysaccharides showed toxicity even at the highest concentrations tested. Vero cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 2.5 × 10−3 with varying dosages of polysaccharide to confirm antiviral activity. A focus reduction assay was performed 48 h post infection to determine efficacy. Antiviral activities correlated with the SPR results. The most potent compound tested, RPI-27, is a high molecular weight, branched polysaccharide related to the known compound fucoidan, and had an EC50 of 8.3 ± 4.6 μg/mL, which corresponds to ~83 nM (Fig. 1c, d and Supplementary Table S1). This is substantially more potent than remdesivir having a reported in vitro EC50 value of 770 nM in Vero-E6 cells 9 and 11.4 µM in Vero-CCL81 cells 10 , currently approved for emergency use for severe COVID-19 infections. The smaller RPI-28 has the same basic structure as RPI-27 but a lower molecular weight and, thus, a lower activity (EC50 = 1.2 μM, Supplementary Table S1). Heparin and the TriS-heparin (an intermediate in the bioengineered heparin synthesis pathway 3 ) also have potent antiviral activity with EC50 values of ~2.1 and 5.0 μM, while the lower molecular weight NACH had an approx. EC50 of 55 μM. Similar antiviral activity of heparin has also been demonstrated recently 11 . Heparin and TriS-heparin are similar, with the latter devoid of the relatively small fraction of 3-O-sulfate groups present on heparin (Fig. 1b). Thus, their similar activity is expected. However, the low molecular weight NACH had far lower antiviral activity. Less sulfated GAGs, such as heparan sulfate and various chondroitin sulfates, because of their very low S-protein binding were not tested in the antiviral assay. The high activity of RPI-27 and RPI-28 relative to the other polysaccharides tested may be a result of multivalent interactions between the polysaccharide and viral particle 12 . While heparin, TriS-heparin, and NACH are linear polysaccharides, RPI-27 and RPI-28 are both highly branched (Fig. 1b), possibly conferring added points of interaction in 3-dimensional space. The higher affinity of RPI-27 compared to RPI-28, and hence its more potent antiviral activity, may be due to the far higher molecular weight of the former providing greater opportunity for multipoint binding to the S-protein of SARS-CoV-2. The non-anticoagulant TriS-heparin may be more desirable in some applications than the potent anticoagulant heparin. Our results reveal that specific sulfated polysaccharides bind tightly to the S-protein of SARS-CoV-2 in vitro, which suggests that they can act as decoys to interfere with S-protein binding to the heparan sulfate co-receptor in host tissues 3,11 , inhibiting viral infection. To model this, we constructed a docking model between heparin and the S-protein receptor-binding site (RBD) using the crystal structure of the chimeric RBD-ACE2 complex (PDB ID: 6VW1) 13 (Fig. 1e and detailed docking model described in supplementary information). The RBD’s amino acid residues involved in binding the ACE2 (angiotensin-converting enzyme 2) receptor also participated in heparin binding, suggesting a mechanism of viral entry inhibition by heparin. Moreover, the larger the oligosaccharide model used in docking studies, the tighter the binding. Specifically, the octasaccharide binds tighter than the tetrasaccharide (–7.3 vs. –6.1 kcal/mol). Since these polysaccharides show promising antiviral activity in vitro and low cytotoxicity, we suggest that they may have promising clinical use. Along these lines, SARS-CoV-2 has been found to infect a wide range of tissues that possess sufficient ACE2 levels 14 , including the nose and the gastrointestinal tract 15 . Potential routes of delivery of these non-anticoagulant polysaccharide candidates, including the fucoidans (RPI-27, and RPI-28) and the TriS-heparin, could be through a nasal spray, metered dose inhaler, or oral delivery. This is distinct from remdesivir, which must be delivered intravenously 16 . Indeed, when taken orally, the fucoidans, isolated from edible sulfated seaweed polysaccharides, are considered as “Generally Recognized as Safe” and heparin, an approved drug, is not orally bioavailable. Interestingly, a retrospective clinical study suggests that the administration of anticoagulants, such as heparin, may provide better outcomes for patients hospitalized with COVID-19, including a dramatic reduction in mortality of intubated patients 17 . It is unknown whether this is a result of heparin’s anticoagulation alone, or to some degree is an effect of its anti-SARS-CoV-2 activity. Inhaled heparin has additional benefits such as reducing pulmonary coagulopathy and inflammation without producing systemic bleeding 18 . To this end, we suggest that treatment of fucoidans, nebulized heparin, or possibly TriS-heparin in combination with or without current antiviral therapies, should be assessed first in human primary epithelial cells and then in human patients suffering from COVID-19. Supplementary information Supplementary information, Materials and Table

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          Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

          Dear Editor, In December 2019, a novel pneumonia caused by a previously unknown pathogen emerged in Wuhan, a city of 11 million people in central China. The initial cases were linked to exposures in a seafood market in Wuhan. 1 As of January 27, 2020, the Chinese authorities reported 2835 confirmed cases in mainland China, including 81 deaths. Additionally, 19 confirmed cases were identified in Hong Kong, Macao and Taiwan, and 39 imported cases were identified in Thailand, Japan, South Korea, United States, Vietnam, Singapore, Nepal, France, Australia and Canada. The pathogen was soon identified as a novel coronavirus (2019-nCoV), which is closely related to sever acute respiratory syndrome CoV (SARS-CoV). 2 Currently, there is no specific treatment against the new virus. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. An efficient approach to drug discovery is to test whether the existing antiviral drugs are effective in treating related viral infections. The 2019-nCoV belongs to Betacoronavirus which also contains SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). Several drugs, such as ribavirin, interferon, lopinavir-ritonavir, corticosteroids, have been used in patients with SARS or MERS, although the efficacy of some drugs remains controversial. 3 In this study, we evaluated the antiviral efficiency of five FAD-approved drugs including ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and two well-known broad-spectrum antiviral drugs remdesivir (GS-5734) and favipiravir (T-705) against a clinical isolate of 2019-nCoV in vitro. Standard assays were carried out to measure the effects of these compounds on the cytotoxicity, virus yield and infection rates of 2019-nCoVs. Firstly, the cytotoxicity of the candidate compounds in Vero E6 cells (ATCC-1586) was determined by the CCK8 assay. Then, Vero E6 cells were infected with nCoV-2019BetaCoV/Wuhan/WIV04/2019 2 at a multiplicity of infection (MOI) of 0.05 in the presence of varying concentrations of the test drugs. DMSO was used in the controls. Efficacies were evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR) and confirmed with visualization of virus nucleoprotein (NP) expression through immunofluorescence microscopy at 48 h post infection (p.i.) (cytopathic effect was not obvious at this time point of infection). Among the seven tested drugs, high concentrations of three nucleoside analogs including ribavirin (half-maximal effective concentration (EC50) = 109.50 μM, half-cytotoxic concentration (CC50) > 400 μM, selectivity index (SI) > 3.65), penciclovir (EC50 = 95.96 μM, CC50 > 400 μM, SI > 4.17) and favipiravir (EC50 = 61.88 μM, CC50 > 400 μM, SI > 6.46) were required to reduce the viral infection (Fig. 1a and Supplementary information, Fig. S1). However, favipiravir has been shown to be 100% effective in protecting mice against Ebola virus challenge, although its EC50 value in Vero E6 cells was as high as 67 μM, 4 suggesting further in vivo studies are recommended to evaluate this antiviral nucleoside. Nafamostat, a potent inhibitor of MERS-CoV, which prevents membrane fusion, was inhibitive against the 2019-nCoV infection (EC50 = 22.50 μM, CC50 > 100 μM, SI > 4.44). Nitazoxanide, a commercial antiprotozoal agent with an antiviral potential against a broad range of viruses including human and animal coronaviruses, inhibited the 2019-nCoV at a low-micromolar concentration (EC50 = 2.12 μM; CC50 > 35.53 μM; SI > 16.76). Further in vivo evaluation of this drug against 2019-nCoV infection is recommended. Notably, two compounds remdesivir (EC50 = 0.77 μM; CC50 > 100 μM; SI > 129.87) and chloroquine (EC50 = 1.13 μM; CC50 > 100 μM, SI > 88.50) potently blocked virus infection at low-micromolar concentration and showed high SI (Fig. 1a, b). Fig. 1 The antiviral activities of the test drugs against 2019-nCoV in vitro. a Vero E6 cells were infected with 2019-nCoV at an MOI of 0.05 in the treatment of different doses of the indicated antivirals for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Cytotoxicity of these drugs to Vero E6 cells was measured by CCK-8 assays. The left and right Y-axis of the graphs represent mean % inhibition of virus yield and cytotoxicity of the drugs, respectively. The experiments were done in triplicates. b Immunofluorescence microscopy of virus infection upon treatment of remdesivir and chloroquine. Virus infection and drug treatment were performed as mentioned above. At 48 h p.i., the infected cells were fixed, and then probed with rabbit sera against the NP of a bat SARS-related CoV 2 as the primary antibody and Alexa 488-labeled goat anti-rabbit IgG (1:500; Abcam) as the secondary antibody, respectively. The nuclei were stained with Hoechst dye. Bars, 100 μm. c and d Time-of-addition experiment of remdesivir and chloroquine. For “Full-time” treatment, Vero E6 cells were pre-treated with the drugs for 1 h, and virus was then added to allow attachment for 2 h. Afterwards, the virus–drug mixture was removed, and the cells were cultured with drug-containing medium until the end of the experiment. For “Entry” treatment, the drugs were added to the cells for 1 h before viral attachment, and at 2 h p.i., the virus–drug mixture was replaced with fresh culture medium and maintained till the end of the experiment. For “Post-entry” experiment, drugs were added at 2 h p.i., and maintained until the end of the experiment. For all the experimental groups, cells were infected with 2019-nCoV at an MOI of 0.05, and virus yield in the infected cell supernatants was quantified by qRT-PCR c and NP expression in infected cells was analyzed by Western blot d at 14 h p.i. Remdesivir has been recently recognized as a promising antiviral drug against a wide array of RNA viruses (including SARS/MERS-CoV 5 ) infection in cultured cells, mice and nonhuman primate (NHP) models. It is currently under clinical development for the treatment of Ebola virus infection. 6 Remdesivir is an adenosine analogue, which incorporates into nascent viral RNA chains and results in pre-mature termination. 7 Our time-of-addition assay showed remdesivir functioned at a stage post virus entry (Fig. 1c, d), which is in agreement with its putative anti-viral mechanism as a nucleotide analogue. Warren et al. showed that in NHP model, intravenous administration of 10 mg/kg dose of remdesivir resulted in concomitant persistent levels of its active form in the blood (10 μM) and conferred 100% protection against Ebola virus infection. 7 Our data showed that EC90 value of remdesivir against 2019-nCoV in Vero E6 cells was 1.76 μM, suggesting its working concentration is likely to be achieved in NHP. Our preliminary data (Supplementary information, Fig. S2) showed that remdesivir also inhibited virus infection efficiently in a human cell line (human liver cancer Huh-7 cells), which is sensitive to 2019-nCoV. 2 Chloroquine, a widely-used anti-malarial and autoimmune disease drug, has recently been reported as a potential broad-spectrum antiviral drug. 8,9 Chloroquine is known to block virus infection by increasing endosomal pH required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors of SARS-CoV. 10 Our time-of-addition assay demonstrated that chloroquine functioned at both entry, and at post-entry stages of the 2019-nCoV infection in Vero E6 cells (Fig. 1c, d). Besides its antiviral activity, chloroquine has an immune-modulating activity, which may synergistically enhance its antiviral effect in vivo. Chloroquine is widely distributed in the whole body, including lung, after oral administration. The EC90 value of chloroquine against the 2019-nCoV in Vero E6 cells was 6.90 μM, which can be clinically achievable as demonstrated in the plasma of rheumatoid arthritis patients who received 500 mg administration. 11 Chloroquine is a cheap and a safe drug that has been used for more than 70 years and, therefore, it is potentially clinically applicable against the 2019-nCoV. Our findings reveal that remdesivir and chloroquine are highly effective in the control of 2019-nCoV infection in vitro. Since these compounds have been used in human patients with a safety track record and shown to be effective against various ailments, we suggest that they should be assessed in human patients suffering from the novel coronavirus disease. Supplementary information Supplementary information, Materials and Figures
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            Structural basis of receptor recognition by SARS-CoV-2

            Summary A novel SARS-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans 1,2 . A key to tackling this epidemic is to understand the virus’s receptor recognition mechanism, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor - human ACE2 (hACE2) 3,4 . Here we determined the crystal structure of SARS-CoV-2 receptor-binding domain (RBD) (engineered to facilitate crystallization) in complex of hACE2. Compared with SARS-CoV RBD, a hACE2-binding ridge in SARS-CoV-2 RBD takes a more compact conformation; moreover, several residue changes in SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD/hACE2 interface. These structural features of SARS-CoV-2 RBD enhance its hACE2-binding affinity. Additionally, we showed that RaTG13, a bat coronavirus closely related to SARS-CoV-2, also uses hACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in hACE2 recognition shed light on potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies targeting receptor recognition by SARS-CoV-2.
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              SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

              We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.

                Author and article information

                Cell Discov
                Cell Discov
                Cell Discovery
                Springer Singapore (Singapore )
                24 July 2020
                24 July 2020
                : 6
                : 50
                [1 ]GRID grid.33647.35, ISNI 0000 0001 2160 9198, Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, , Rensselaer Polytechnic Institute, ; Troy, NY USA
                [2 ]GRID grid.33647.35, ISNI 0000 0001 2160 9198, Department of Chemistry and Chemical Biology, , Rensselaer Polytechnic Institute, ; Troy, NY USA
                [3 ]GRID grid.249967.7, ISNI 0000 0004 0636 3099, National Primate Research Center, , Korea Research Institute of Bioscience and Biotechnology, ; Cheongju, Chungcheongbuk Republic of Korea
                [4 ]GRID grid.469325.f, ISNI 0000 0004 1761 325X, College of Biotechnology and Bioengineering, , Zhejiang University of Technology, ; Hangzhou, 310014 China
                [5 ]GRID grid.33647.35, ISNI 0000 0001 2160 9198, Department of Biological Sciences, , Rensselaer Polytechnic Institute, ; Troy, NY USA
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                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                : 23 May 2020
                : 10 July 2020
                Funded by: Global Research Laboratory Program of the Korean National Research Foundation.
                Funded by: FundRef https://doi.org/10.13039/501100003715, Korea Research Institute of Bioscience and Biotechnology (KRIBB);
                Award ID: KGM4572013
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                mechanisms of disease,glycobiology
                mechanisms of disease, glycobiology


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