scholarly journals The inhibitory effect of a Corona virus spike protein fragment with ACE2

2020 ◽  
Author(s):  
E. K. Peter ◽  
A. Schug
Keyword(s):  
Viral Entry ◽  
Md Simulations ◽  
Inhibitory Effect ◽  
Human Cells ◽  
Spike Protein ◽  
Receptor Protein ◽  
Binding Motif ◽  
Protein Fragment ◽  
S Protein ◽  
Binding Interface

ABSTRACTIn this paper, we investigate the molecular assembly processes of a Coronavirus Spike protein fragment, the hexapeptide YKYRYL on the ACE2 receptor and its inhibitory effect on the aggregation and activation of the CoV-2 spike receptor protein at the same receptor protein. In agreement with an experimental study, we find a high affinity of the hexapeptide to the binding interface between the spike receptor protein and ACE2, which we investigate using 20 independent equilibrium MD simulations over a total of 1 μs and a 200 ns enhanced MD simulation. We then evaluate the effect of the hexapeptide on the aggregation process of the spike receptor protein to ACE2 in long-time enhanced MD simulations. In that set of simulations, we find that the spike receptor protein does not bind to ACE2 with the binding motif shown in experiments, but it rotates due to an electrostatic repulsion and forms a hydrophobic interface with ACE2. Surprisingly, we observe that the hexapeptide binds to the spike receptor domain, which has the effect that this protein only weakly attaches to ACE2, so that the activation of the spike protein receptor might be inhibited in this case. Our results indicate that the hexapeptide might be a possible treatment option which prevents the viral activation through the inhibition of the interaction between ACE2 and the spike receptor protein.SIGNIFICANCEA novel coronavirus, CoV-19 and a later phenotype CoV-2 were identified as primary cause for a severe acute respiratory syndrome (SARS CoV-2). The spike (S) protein of CoV-2 is one target for the development of a vaccine to prevent the viral entry into human cells. The inhibition of the direct interaction between ACE2 and the S-protein could provides a suitable strategy to prevent the membrane fusion of CoV-2 and the viral entry into human cells. Using MD simulations, we investigate the assembly process of a Coronavirus Spike protein fragment, the hexapeptide YKYRYL on the ACE2 receptor and its inhibitzory effect on the aggregation and activation of the CoV-2 spike receptor protein at the same receptor protein.

scholarly journals The effect of salt on the dynamics of CoV-2 RBD at ACE2

2020 ◽  
Author(s):  
E. K. Peter ◽  
A. Schug
Keyword(s):  
Viral Entry ◽  
Membrane Surface ◽  
Direct Interaction ◽  
Human Cells ◽  
Spike Protein ◽  
Terminal Part ◽  
Binding Interface ◽  
Strong Binding ◽  
Protein Receptor

ABSTRACTIn this article, we investigate the effect of electrolytes on the stability of the complex between the coronavirus type 2 spike protein receptor domain (CoV-2 RBD) and ACE2, which plays an important role in the activation cascade at the viral entry of CoV-2 into human cells. At the cellular surface, electrolytes play an important role, especially in the interaction of proteins near the membrane surface. Additionally, the binding interface of the CoV-2 RBD - ACE2 complex is highly hydrophilic. We simulated the CoV-2 RBD - ACE2 complex at varying salt concentrations over the concentration range from 0.03 M to 0.3 M of calcium and sodium chloride over an individual simulation length of 750 ns in 9 independent simulations (6.75 µs total). We observe that the CoV-2 RBD - ACE2 complex is stabilized independent of the salt concentration. We identify a strong negative electrostatic potential at the N-terminal part of CoV-2 RBD and we find that CoV-2 RBD binds even stronger at higher salt concentrations. We observe that the dynamics of the N-terminal part of CoV-2 RBD stabilize the protein complex leading to strong collective motions and a stable interface between CoV-2 RBD and ACE2. We state that the sequence of CoV-2 RBD might be optimized for a strong binding to ACE2 at varying salt concentrations at the cellular surface, which acts as a key component in the activation of CoV-2 for its viral entry.SIGNIFICANCEA novel coronavirus, coronavirus type 2 (CoV-2), was identified as primary cause for a worldwide pandemic of the severe acute respiratory syndrome (SARS CoV-2). The CoV-2 spike protein is a major target for the development of a vaccine and potential strategies to inhibit the viral entry into human cells. At the cellular surface, CoV-2 activation involves the direct interaction between ACE2 and CoV-2 RBD. At the cellular surface, electrolytes play an important role, especially in the interaction of proteins near the membrane surface. We thus investigate the effect of ion conditions on the interaction of the CoV-2 RBD - ACE2 complex and find stabilizing effects. We speculate that CoV-2 RBD is optimized for strong binding to ACE2 at varying salt concentrations.

Compared with SARS-CoV2 wild type's spike protein, the SARS-CoV2 omicron's receptor binding motif has adopted a more SARS-CoV1 and/or bat/civet-like structure.

2021 ◽  
Author(s):  
Michael O. Glocker ◽  
Kwabena F. M. Opuni ◽  
Hans-Juergen Thiesen
Keyword(s):  
Free Energy ◽  
Receptor Binding ◽  
Free Energy Calculations ◽  
Viral Fitness ◽  
Spike Protein ◽  
Receptor Protein ◽  
Binding Motif ◽  
Energy Calculations ◽  
Selection Processes ◽  
Protein Receptor

Our study focuses on free energy calculations of SARS-Cov2 spike protein receptor binding motives (RBMs) from wild type and variants-of-concern with particular emphasis on currently emerging SARS- CoV2 omicron variants of concern (VOC). Our computational free energy analysis underlines the occurrence of positive selection processes that specify omicron host adaption and bring changes on the molecular level into context with clinically relevant observations. Our free energy calculations studies regarding the interaction of omicron's RBM with human ACE2 shows weaker binding to ACE2 than alpha's, delta's, or wild type's RBM. Thus, less virus is predicted to be generated in time per infected cell. Our mutant analyses predict with focus on omicron variants a reduced spike-protein binding to ACE2--receptor protein possibly enhancing viral fitness / transmissibility and resulting in a delayed induction of danger signals as trade-off. Finally, more virus is produced but less per cell accompanied with delayed Covid-19 immunogenicity and pathogenicity. Regarding the latter, more virus is assumed to be required to initiate inflammatory immune responses.

scholarly journals Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Yutaka Terada ◽  
Kengo Kawachi ◽  
...  
Keyword(s):  
Middle East ◽  
Viral Entry ◽  
Cathepsin L ◽  
Inhibitory Effect ◽  
Middle East Respiratory Syndrome ◽  
Cell Entry ◽  
Target Cells ◽  
Furin Cleavage ◽  
S Protein ◽  
Entry Assay

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

scholarly journals Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

2020 ◽  
Vol 117 (45) ◽  
pp. 28046-28055 ◽  
Author(s):  
Anum Glasgow ◽  
Jeff Glasgow ◽  
Daniel Limonta ◽  
Paige Solomon ◽  
Irene Lui ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Surface Display ◽  
Random Mutagenesis ◽  
Yeast Surface Display ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Protein ◽  
Yeast Surface

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

Abstract 12928: Vascular Complications in Covid-19 and Role of Zinc in Viral Inhibition

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Donghui Zhu ◽  
Zhen Zhao
Keyword(s):  
Viral Entry ◽  
Vascular Complications ◽  
Functional Change ◽  
Cell Entry ◽  
Host Cells ◽  
Binding Motif ◽  
Vascular Cells ◽  
Viral Inhibition ◽  
S Protein

Although COVID-19 is associated with severe respiratory dysfunctions, conspicuous vascular complications and neurological manifestations have been reported worldwide. Of note, two distinctive features have been noticed in severe patients, progressive increase of inflammation and an unusual trend of hypercoagulation. Interestingly, evidence is mounting that healthy blood vessels protect children from serious effects of COVID-19, such as stroke. These findings suggest vascular complications play a key role in the progress of COVID-19, warranting an investigation to its pathophysiology and treatment strategy related to vascular cells. Cell entry of this SARS-CoV-2 virus depends on binding of the viral spike (S) proteins to cellular receptor ACE2, which could be a key target for blocking the viral entry into host cells. ACE2 is a zinc (Zn) binding metallopeptidase while Zn possesses distinct antiviral properties against many human viruses including coronaviruses. Although the mechanistic studies are lacking, Zn appears to inhibit viral protease and polymerase enzymatic processes, and physical processes such as virus attachment, cell entry, and uncoating. In fact, our data showed that ACE2 has multiple affinity binding sites for Zn. Excess bindings of ionic Zn to ACE2 led to its conformational or functional change, therefore, interfering with its ability to metabolize its substrate as well as inhibiting its binding to S protein. Computational modeling also revealed that one critical Zn binding motif is located in ACE2’s binding domain to S protein, and docking affinity of S protein to ACE2 was significantly reduced after Zn binding to this specific site. Moreover, cell and animal studies using pseudo-virus bearing CoV-2-S protein validated that significantly lower infection of vascular cells in the presence of Zn was observed. Thus, targeting vascular complications in COVID-19 may offer strong benefits including the potential therapeutic role of Zn.

scholarly journals Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Hannah Kleine-Weber ◽  
Mahmoud Tarek Elzayat ◽  
Lingshu Wang ◽  
Barney S. Graham ◽  
Marcel A. Müller ◽  
...  
Keyword(s):  
Middle East ◽  
Protein Binding ◽  
Receptor Binding ◽  
Viral Entry ◽  
Middle East Respiratory Syndrome ◽  
Spike Protein ◽  
Target Cells ◽  
S Protein ◽  
Hospital Outbreak ◽  
The Republic

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4. IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

scholarly journals Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

2021 ◽  
Vol 22 (4) ◽  
pp. 1913
Author(s):  
Yu Jung Kim ◽  
Min Ho Lee ◽  
Se-Ra Lee ◽  
Hyo-Young Chung ◽  
Kwangmin Kim ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Phage Display ◽  
Viral Entry ◽  
Phage Display Library ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Receptor Protein ◽  
In Vitro Assays ◽  
Global Threat

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19–663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs’ activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08–1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.

scholarly journals Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction

2020 ◽  
Author(s):  
Xiaoling Qiang ◽  
Shu Zhu ◽  
Jianhua Li ◽  
Ping Wang ◽  
Kevin J. Tracey ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Viral Entry ◽  
Protein Targeting ◽  
Inflammatory Responses ◽  
Human Monocyte ◽  
Spike Protein ◽  
Binding Motif ◽  
Gm Csf ◽  
Future Assessment

AbstractA severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected more than 25.6 million and killed 852,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibody responses. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human monocyte and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited “cytokine storm”, and provided a potentially useful criteria for future assessment of innate immune-modulating properties of various SARS-CoV-2 vaccines.One Sentence SummaryRBM-binding Antibodies Inhibit GM-CSF Induction.

scholarly journals Binding mechanism of SARS-CoV-2 spike protein with human ACE2 receptor

2020 ◽  
Author(s):  
Rajendra P Koirala ◽  
Bidhya Thapa ◽  
Shyam P Khanal ◽  
Jhulan Powrel ◽  
Rajendra P Adhikari ◽  
...  
Keyword(s):  
Free Energy ◽  
Contact Surface ◽  
Hydrophobic Interactions ◽  
Binding Free Energy ◽  
Umbrella Sampling ◽  
Spike Protein ◽  
Receptor Protein ◽  
Virus Protein ◽  
Binding Mechanism ◽  
Binding Interface

Abstract SARS-CoV-2 virus interacts via C-terminal domain of spike protein to human cell receptor protein hACE2. Amino acid residues residing at the interface play vital role in binding of SARS-CoV-2 CTD to hACE2. The detailed atomic level inves- tigation of interactions at binding interface of SARS-CoV-2 CTD/hACE2 provides indispensable information on better understanding of location for drug target. In the present work, we have studied the dynamical behaviour of the complex by ana- lyzing the molecular dynamics (MD) trajectories. The major interacting residues of SARS-CoV-2 CTD and hACE2 have been identified by analyzing the non-bonded interactions such as hydrogen bondings, salt bridges, hydrophobic interactions, van der Waals interactions etc. Umbrella sampling method has been used to estimate the binding free energy for in-depth understanding of binding mechanism between virus protein and host receptor. The binding free energy difference, key residues at the interface, important atomic interactions and contact surface areas have been compared with the molecular complex of SARS-CoV and hACE2. Relatively larger contact surface area, more non-bonded interactions as well as greater binding free energy provide the evidence for favorable binding of SARS-CoV-2 with hACE2 receptor than SARS-CoV.

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