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      Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies

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          Abstract

          The recent severe acute respiratory syndrome, known as Coronavirus Disease 2019 (COVID-19) has spread so much rapidly and severely to induce World Health Organization (WHO) to declare a state of emergency over the new coronavirus SARS-CoV-2 pandemic. While several countries have chosen the almost complete lock-down for slowing down SARS-CoV-2 spread, the scientific community is called to respond to the devastating outbreak by identifying new tools for diagnosis and treatment of the dangerous COVID-19. With this aim, we performed an in silico comparative modeling analysis, which allows gaining new insights into the main conformational changes occurring in the SARS-CoV-2 spike protein, at the level of the receptor-binding domain (RBD), along interactions with human cells angiotensin-converting enzyme 2 (ACE2) receptor, that favor human cell invasion. Furthermore, our analysis provides (1) an ideal pipeline to identify already characterized antibodies that might target SARS-CoV-2 spike RBD, aiming to prevent interactions with the human ACE2, and (2) instructions for building new possible neutralizing antibodies, according to chemical/physical space restraints and complementary determining regions (CDR) mutagenesis of the identified existing antibodies. The proposed antibodies show in silico high affinity for SARS-CoV-2 spike RBD and can be used as reference antibodies also for building new high-affinity antibodies against present and future coronaviruses able to invade human cells through interactions of their spike proteins with the human ACE2. More in general, our analysis provides indications for the set-up of the right biological molecular context for investigating spike RBD–ACE2 interactions for the development of new vaccines, diagnostic kits, and other treatments based on the targeting of SARS-CoV-2 spike protein.

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          The online version of this article (10.1007/s00018-020-03580-1) contains supplementary material, which is available to authorized users.

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          Most cited references 102

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          Jalview Version 2—a multiple sequence alignment editor and analysis workbench

          Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org Contact: g.j.barton@dundee.ac.uk
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            COVID-19: consider cytokine storm syndromes and immunosuppression

            As of March 12, 2020, coronavirus disease 2019 (COVID-19) has been confirmed in 125 048 people worldwide, carrying a mortality of approximately 3·7%, 1 compared with a mortality rate of less than 1% from influenza. There is an urgent need for effective treatment. Current focus has been on the development of novel therapeutics, including antivirals and vaccines. Accumulating evidence suggests that a subgroup of patients with severe COVID-19 might have a cytokine storm syndrome. We recommend identification and treatment of hyperinflammation using existing, approved therapies with proven safety profiles to address the immediate need to reduce the rising mortality. Current management of COVID-19 is supportive, and respiratory failure from acute respiratory distress syndrome (ARDS) is the leading cause of mortality. 2 Secondary haemophagocytic lymphohistiocytosis (sHLH) is an under-recognised, hyperinflammatory syndrome characterised by a fulminant and fatal hypercytokinaemia with multiorgan failure. In adults, sHLH is most commonly triggered by viral infections 3 and occurs in 3·7–4·3% of sepsis cases. 4 Cardinal features of sHLH include unremitting fever, cytopenias, and hyperferritinaemia; pulmonary involvement (including ARDS) occurs in approximately 50% of patients. 5 A cytokine profile resembling sHLH is associated with COVID-19 disease severity, characterised by increased interleukin (IL)-2, IL-7, granulocyte-colony stimulating factor, interferon-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α, and tumour necrosis factor-α. 6 Predictors of fatality from a recent retrospective, multicentre study of 150 confirmed COVID-19 cases in Wuhan, China, included elevated ferritin (mean 1297·6 ng/ml in non-survivors vs 614·0 ng/ml in survivors; p 39·4°C 49 Organomegaly None 0 Hepatomegaly or splenomegaly 23 Hepatomegaly and splenomegaly 38 Number of cytopenias * One lineage 0 Two lineages 24 Three lineages 34 Triglycerides (mmol/L) 4·0 mmol/L 64 Fibrinogen (g/L) >2·5 g/L 0 ≤2·5 g/L 30 Ferritin ng/ml 6000 ng/ml 50 Serum aspartate aminotransferase <30 IU/L 0 ≥30 IU/L 19 Haemophagocytosis on bone marrow aspirate No 0 Yes 35 Known immunosuppression † No 0 Yes 18 The Hscore 11 generates a probability for the presence of secondary HLH. HScores greater than 169 are 93% sensitive and 86% specific for HLH. Note that bone marrow haemophagocytosis is not mandatory for a diagnosis of HLH. HScores can be calculated using an online HScore calculator. 11 HLH=haemophagocytic lymphohistiocytosis. * Defined as either haemoglobin concentration of 9·2 g/dL or less (≤5·71 mmol/L), a white blood cell count of 5000 white blood cells per mm3 or less, or platelet count of 110 000 platelets per mm3 or less, or all of these criteria combined. † HIV positive or receiving longterm immunosuppressive therapy (ie, glucocorticoids, cyclosporine, azathioprine).
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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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                Author and article information

                Contributors
                ciro.pierri@uniba.it , ciroleopierri1@gmail.com
                Journal
                Cell Mol Life Sci
                Cell. Mol. Life Sci
                Cellular and Molecular Life Sciences
                Springer International Publishing (Cham )
                1420-682X
                1420-9071
                4 July 2020
                : 1-22
                Affiliations
                [1 ]GRID grid.7644.1, ISNI 0000 0001 0120 3326, Laboratory of Biochemistry, Molecular and Structural Biology, Department of Biosciences, Biotechnologies, Biopharmaceutics, , University of Bari, ; Via E. Orabona, 4, 70125 Bari, Italy
                [2 ]GRID grid.7644.1, ISNI 0000 0001 0120 3326, Department of Soil, Plant and Food Sciences, , University of Bari Aldo Moro, ; Via Amendola 165/A, 70126 Bari, Italy
                [3 ]BROWSer S.r.l. (https://browser-bioinf.com/) c/o Department of Biosciences, Biotechnologies, Biopharmaceutics, University “Aldo Moro” of Bari, Via E. Orabona, 4, 70126 Bari, Italy
                Article
                3580
                10.1007/s00018-020-03580-1
                7334636
                32623480
                © Springer Nature Switzerland AG 2020

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

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