Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

SummaryThere are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.HighlightsCR3022 neutralises SARS-CoV-2Neutralisation is by destroying the prefusion SPIKE conformationThis antibody may have therapeutic potential alone or with one blocking receptor attachment

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Structural Dissection of Viral Spike-Protein Binding of SARS-CoV-2 and SARS-CoV-1 to the Human Angiotensin-Converting Enzyme 2 (ACE2) as Cellular Receptor

Biomedicines ◽

10.3390/biomedicines9081038 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1038

Author(s):

Deborah Giordano ◽

Luigi De Masi ◽

Maria Antonia Argenio ◽

Angelo Facchiano

Keyword(s):

Angiotensin Converting Enzyme ◽

In Silico Analysis ◽

Host Cells ◽

Spike Protein ◽

Cellular Receptor ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

The World ◽

Silico Analysis

An outbreak by a new severe acute respiratory syndrome betacoronavirus (SARS-CoV-2) has spread CoronaVirus Disease 2019 (COVID-19) all over the world. Immediately, following studies have confirmed the human Angiotensin-Converting Enzyme 2 (ACE2) as a cellular receptor of viral Spike-Protein (Sp) that mediates the CoV-2 invasion into the pulmonary host cells. Here, we compared the molecular interactions of the viral Sp from previous SARS-CoV-1 of 2002 and SARS-CoV-2 with the host ACE2 protein by in silico analysis of the available experimental structures of Sp-ACE2 complexes. The K417 amino acid residue, located in the region of Sp Receptor-Binding Domain (RBD) of the new coronavirus SARS-CoV-2, showed to have a key role for the binding to the ACE2 N-terminal region. The R426 residue of SARS-CoV-1 Sp-RBD also plays a key role, although by interacting with the central region of the ACE2 sequence. Therefore, our study evidenced peculiarities in the interactions of the two Sp-ACE2 complexes. Our outcomes were consistent with previously reported mutagenesis studies on SARS-CoV-1 and support the idea that a new and different RBD was acquired by SARS-CoV-2. These results have interesting implications and suggest further investigations.

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ACE-2-derived Biomimetic Peptides for the Inhibition of Spike Protein of SARS-CoV-2

10.26434/chemrxiv.12335933.v1 ◽

2020 ◽

Author(s):

Saroj Kumar Panda ◽

Parth Sarthi Sen Gupta ◽

Satyaranjan Biswal ◽

Abhik Kumar Ray ◽

Malay Kumar Rana

Keyword(s):

Principal Component ◽

Host Cells ◽

Spike Protein ◽

Peptide Inhibitor ◽

The Novel ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Inhibition Assay ◽

Angiotensin Converting Enzyme 2 ◽

Novel Coronavirus

<p>SARS-CoV-2, a novel coronavirus causing overwhelming death and infection worldwide, has emerged as a pandemic. Compared to its predecessor SARS-CoV, SARS-CoV-2 is more infective for being highly contagious and exhibiting tighter binding with host angiotensin-converting enzyme 2 (hACE-2). The entry of the virus into host cells is mediated by the interaction of its spike protein with hACE-2. Thus, a peptide that has a resemblance to hACE-2 but can overpower the spike protein-hACE-2 interaction will be a potential therapeutic to contain this virus. The non-interacting residues in the receptor-binding domain of hACE-2 have been mutated to generate a library of 136 new peptides. Out of this library, docking and virtual screening discover seven peptides that can exert a stronger interaction with the spike protein than hACE-2. A peptide derived from simultaneous mutation of all the non-interacting residues of hACE-2 yields two-fold stronger interaction than hACE-2 and thus turns out here to be the best peptide-inhibitor of the novel coronavirus. The binding of the spike protein and the best peptide-inhibitor with hACE-2 is explored further by molecular dynamics, free energy, and principal component analysis to demonstrate its efficacy. Further, the inhibition assay study with the best peptide inhibitor is in progress. </p>

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Targeting the glycan of receptor binding domain with jacalin as a novel approach to develop a treatment against COVID-19

Royal Society Open Science ◽

10.1098/rsos.200844 ◽

2020 ◽

Vol 7 (9) ◽

pp. 200844

Author(s):

Senthilnathan Rajendaran ◽

Arunchalam Jothi ◽

Veerappan Anbazhagan

Keyword(s):

Angiotensin Converting Enzyme ◽

Receptor Binding ◽

Conformational Changes ◽

In Silico ◽

Binding Domain ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Novel Approach

In silico analysis revealed that a lectin, jacalin from jackfruit seeds, recognizes a glycosylated region of the receptor-binding domain (RBD) of SARS-CoV2. Jacalin binding induces conformational changes in RBD and significantly affects its interaction with human angiotensin-converting enzyme 2. The result may open up exploration of lectin-based strategies against COVID-19.

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Receptor binding and proteolysis do not induce large conformational changes in the SARS-CoV spike

10.1101/292672 ◽

2018 ◽

Cited By ~ 1

Author(s):

Robert N. Kirchdoerfer ◽

Nianshuang Wang ◽

Jesper Pallesen ◽

Daniel Wrapp ◽

Hannah L. Turner ◽

...

Keyword(s):

Conformational Changes ◽

Viral Entry ◽

Cleavage Site ◽

Host Protein ◽

Host Cells ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Protein Receptor ◽

Proteolytic Cleavages ◽

Secondary Cleavage

AbstractSevere acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S undergoes two proteolytic cleavages at S1/S2 and S2’ sites necessary for efficient membrane fusion. Here, we present a cryo-EM analysis of the trimeric SARS-CoV S interactions with ACE2 and of the trypsin-cleaved S. Surprisingly, neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within S or expose the secondary cleavage site, S2’. These observations suggest that S2’ cleavage does not occur in the S prefusion conformation and that additional triggers may be required.

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Spike Glycoprotein-Mediated Entry of SARS Coronaviruses

Viruses ◽

10.3390/v12111289 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1289 ◽

Cited By ~ 1

Author(s):

Lin Wang ◽

Ye Xiang

Keyword(s):

Conformational Changes ◽

Antiviral Agents ◽

Host Cells ◽

Structural Studies ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Public Health Emergencies ◽

Positive Sense ◽

Epidemic Diseases

Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 are enveloped, positive-sense, single-stranded RNA viruses and causes of epidemic diseases that have resulted in public health emergencies worldwide. Angiotensin-converting enzyme 2 (ACE2) is the receptor that allows the entry of these two viruses into host cells, a key step in the life cycle of the pathogens. The characterization of the interactions of ACE2 with the viral spike glycoproteins and structural studies of the ACE2-binding-induced conformational changes in the viral spike glycoproteins have furthered our understanding of the entry processes of these two viruses, and these studies provide useful information that will facilitate the development of antiviral agents and vaccines to control the diseases.

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ACE-2-derived Biomimetic Peptides for the Inhibition of Spike Protein of SARS-CoV-2

10.26434/chemrxiv.12335933 ◽

2020 ◽

Author(s):

Saroj Kumar Panda ◽

Parth Sarthi Sen Gupta ◽

Satyaranjan Biswal ◽

Abhik Kumar Ray ◽

Malay Kumar Rana

Keyword(s):

Principal Component ◽

Host Cells ◽

Spike Protein ◽

Peptide Inhibitor ◽

The Novel ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Inhibition Assay ◽

Angiotensin Converting Enzyme 2 ◽

Novel Coronavirus

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Intermolecular Interaction Analyses on SARS-CoV-2 Spike Protein Receptor Binding Domain and Human Angiotensin-Converting Enzyme 2 Receptor-Blocking Antibody/Peptide Using Fragment Molecular Orbital Calculation

The Journal of Physical Chemistry Letters ◽

10.1021/acs.jpclett.1c00663 ◽

2021 ◽

Vol 12 (16) ◽

pp. 4059-4066

Author(s):

Kazuki Watanabe ◽

Chiduru Watanabe ◽

Teruki Honma ◽

Yu-Shi Tian ◽

Yusuke Kawashima ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Receptor Binding ◽

Molecular Orbital Calculation ◽

Spike Protein ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Blocking Antibody ◽

Angiotensin Converting Enzyme 2 ◽

Receptor Blocking ◽

Protein Receptor

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480 Preliminary evaluation of a novel coronavirus vaccine (CORVax) using electroporation of plasmid DNA encoding a stabilized prefusion SARS-CoV-2 spike protein alone or with transfection of plasmid IL-12

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0480 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A514-A514

Author(s):

Shawn Jensen ◽

Christopher Twitty ◽

Christopher Paustian ◽

Madelein Laws ◽

Glenna McDonnell ◽

...

Keyword(s):

Immune Responses ◽

Receptor Binding ◽

Binding Domain ◽

Tumor Rejection ◽

Spike Protein ◽

Preliminary Evaluation ◽

List Type ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Viral Neutralization

BackgroundSARS-CoV-2 (CoV2) has precipitated a global pandemic and the effectiveness of standard vaccine strategies to induce potent and persistent immunity to CoV2 is in question, particularly for the elderly. This problem is not dissimilar to what we have struggled with in our quest to induce immunity to cancer antigens, where vaccine-induced anti-cancer immune responses can be weak. Here, we describe a novel vaccine approach which leverages electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax). As IL-12 has been shown to augment the efficacy of immunotherapy in aged mice,1 we have initiated studies to evaluate if plasmid IL-12 (TAVO™) can similarly augment anti-CoV2 immune responses in young mice and have planned studies in aged animals.MethodsA prefusion stabilized CoV2 spike plasmid expression vector was constructed, a master cell bank generated and clinical-grade plasmid manufactured. C57BL/6 and BALB/c were vaccinated via intramuscular (IM) and/or intradermal (ID) injection followed immediately by EP of plasmids encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12 on days 1 and 14 or 21. Mice were followed for >120 days to assess safety. Splenocytes and serum were harvested at different time points to interrogate virus-specific cellular responses as well anti-spike IgG1/IgG2 antibody titers. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble hACE2R binding to immobilized CoV2 spike.ResultsPreliminary data shows that EP of CORVax alone or combined with IL-12 was safe. EP of CORVax was able to elicit anti-Spike IgG antibodies (IC50 = 1/2112), as well as IgG antibodies targeting the receptor binding domain of the Spike protein (IC50 = 1/965) approximately 40 days after the booster vaccination. In 2 of 2 experiments, CORVax combined with IL-12 significantly (P<0.0001) increased the sVNT titers at 2 months, but this benefit was lost by 3 months.ConclusionsEarly preclinical data shows that EP of CORVax can induce IgG responses to CoV2 Spike and the receptor binding domain (RBD) as well as apparent viral neutralizing activity. The addition of IL-12, at least transiently, increased sVNT titer. We plan to investigate alternate vaccine boosting strategies while extending these studies into aged animals and initiate a clinical trial in the near future.ReferencesRuby CE, Weinberg AD. OX40-Enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol2009;182:1481–9. https://doi.org/10.4049/jimmunol.182.3.1481.

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Angiotensin Converting Enzyme 2 (ACE2) Expression in the Visual System

10.21203/rs.3.rs-296818/v1 ◽

2021 ◽

Author(s):

James M. Hill ◽

Christian Clement ◽

L. Arceneaux ◽

Walter Lukiw

Keyword(s):

Signal Processing ◽

Angiotensin Converting Enzyme ◽

Occipital Lobe ◽

Cell Types ◽

Receptor Expression ◽

Host Cells ◽

Visual Signal ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

The Brain

Abstract Background: Multiple lines of evidence currently indicate that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)gains entry into human host cells via a high-affinity interaction with the angiotensin-converting enzyme 2 (ACE2) transmembrane receptor. Research has further shown the widespread expression of the ACE2 receptor on the surface of many different immune, non-immune and neural host cell types, and that SARS-CoV-2 has there markable capability to attack many different types of human-host cells simultaneously. One principal neuroanatomical region for highACE2 expression patterns occurs in the brainstem, an area of the brain containing regulatory centers for respiration, and this may in part explain the predisposition of many COVID-19 patients to respiratory distress. Early studies also indicated extensive ACE2 expression in the whole eye and the brain’s visual circuitry. In this study we analyzed ACE2 receptor expression at the mRNA and protein level in multiple cell types involved in human vision, including cell types of the external eye and several deep brain regions known to be involved in the processing of visual signals.Methods: ACE2 mRNA and protein analysis; multiple eye and brain cells and tissues; gamma32P-adenosine tri-phosphate ([γ-32P]dATP) radiolabeled probes; Northern analysis; ELISA.Results: The four main findings were: (i)that many different optical and neural cell types of the human visual system provide receptors essential for SARS-CoV-2 invasion; (ii)the remarkable ubiquity of ACE2 presence in cells of the eye and anatomical regions of the brain involved in visual signal processing; (iii)that ACE2 receptor expression in different ocular cell types and visual processing centers of the brain provide multiple compartments for SARS-CoV-2 infiltration; and (iv)a gradient of increasing ACE2 expression from the anterior surface of the eye to the visual signal processing areas of the occipital lobe and the primary visual neocortex.Conclusion: A gradient of ACE2 expression from the eye surface to the occipital lobe provide the SARS-CoV-2 virus a novel pathway from the outer eye into deeper anatomical regions of the brain involved in vision. These findings may explain, in part, the many recently reported neuro-ophthalmic manifestations of SARS-CoV-2infection in COVID-19 affected patients.

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Virtual screening as therapeutic strategy of COVID-19 targeting angiotensin-converting enzyme 2

Frontiers in Molecular Immunology ◽

10.25082/fmi.2021.01.002 ◽

2021 ◽

Vol 2 (1) ◽

pp. 16-27

Author(s):

Zahra Sharifinia ◽

◽

Samira Asadi ◽

Mahyar Irani ◽

Abdollah Allahverdi ◽

...

Keyword(s):

Virtual Screening ◽

Angiotensin Converting Enzyme ◽

Host Cells ◽

Spike Protein ◽

Potential Drug ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Approved Drugs ◽

Fda Approved Drugs

Objective: The receptor-binding domain (RBD) of the S1 domain of the SARS-CoV- 2 Spike protein performs a key role in the interaction with Angiotensin-converting enzyme 2 (ACE2), leading to both subsequent S2 domain-mediated membrane fusion and incorporation of viral RNA in host cells. Methods: In this study, we investigated the inhibitor’s targeted compounds through existing human ACE2 drugs to use as a future viral invasion. 54 FDA approved drugs were selected to assess their binding affinity to the ACE2 receptor. The structurebased methods via computational ones have been used for virtual screening of the best drugs from the drug database. Key Findings: The ligands “Cinacalcet” and “Levomefolic acid” highaffinity scores can be a potential drug preventing Spike protein of SARS-CoV-2 and human ACE2 interaction. Levomefolic acid from vitamin B family was proved to be a potential drug as a spike protein inhibitor in previous clinical and computational studies. Besides that, in this study, the capability of Levomefolic acid to avoid ACE2 and Spike protein of SARS-CoV-2 interaction is indicated. Therefore, it is worth to consider this drug for more in vitro investigations as ACE2 and Spike protein inhibition candidate. Conclusion: The two Cinacalcet and Levomefolic acid are the two ligands that have highest energy binding for human ACE2 blocking among 54 FDA approved drugs.

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