Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)–ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low μM range (Kd = 0.207–1.206 μM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, ‘miniproteins’, nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

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Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic

Antibody Therapeutics ◽

10.1093/abt/tbaa025 ◽

2020 ◽

Vol 3 (4) ◽

pp. 246-256

Author(s):

Yaping Sun ◽

Mitchell Ho

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Host Cells ◽

Spike Protein ◽

Inhaled Drugs ◽

Viral Attachment ◽

Global Pandemic ◽

Attachment Sites ◽

Antibody Discovery

Abstract SARS-CoV-2 antibody therapeutics are being evaluated in clinical and preclinical stages. As of 11 October 2020, 13 human monoclonal antibodies targeting the SARS-CoV-2 spike protein have entered clinical trials with three (REGN-COV2, LY3819253/LY-CoV555, and VIR-7831/VIR-7832) in phase 3. On 9 November 2020, the US Food and Drug Administration issued an emergency use authorization for bamlanivimab (LY3819253/LY-CoV555) for the treatment of mild-to-moderate COVID-19. This review outlines the development of neutralizing antibodies against SARS-CoV-2, with a focus on discussing various antibody discovery strategies (animal immunization, phage display and B cell cloning), describing binding epitopes and comparing neutralizing activities. Broad-neutralizing antibodies targeting the spike proteins of SARS-CoV-2 and SARS-CoV might be helpful for treating COVID-19 and future infections. VIR-7831/7832 based on S309 is the only antibody in late clinical development, which can neutralize both SARS-CoV-2 and SARS-CoV although it does not directly block virus receptor binding. Thus far, the only cross-neutralizing antibody that is also a receptor binding blocker is nanobody VHH-72. The feasibility of developing nanobodies as inhaled drugs for treating COVID-19 and other respiratory diseases is an attractive idea that is worth exploring and testing. A cocktail strategy such as REGN-COV2, or engineered multivalent and multispecific molecules, combining two or more antibodies might improve the efficacy and protect against resistance due to virus escape mutants. Besides the receptor-binding domain, other viral antigens such as the S2 subunit of the spike protein and the viral attachment sites such as heparan sulfate proteoglycans that are on the host cells are worth investigating.

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SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2

10.1101/2021.03.16.434488 ◽

2021 ◽

Author(s):

Claire M. Weekley ◽

Damian F. J. Purcell ◽

Michael W. Parker

Keyword(s):

Receptor Binding ◽

Point Mutations ◽

Direct Interaction ◽

Binding Domain ◽

Spike Protein ◽

Genomic Sequencing ◽

Binding Motif ◽

Receptor Binding Domain ◽

Structural Characterisation ◽

Human Receptor

AbstractSince SARS-CoV-2 emerged in 2019, genomic sequencing has identified mutations in the viral RNA including in the receptor-binding domain of the Spike protein. Structural characterisation of the Spike carrying point mutations aids in our understanding of how these mutations impact binding of the protein to its human receptor, ACE2, and to therapeutic antibodies. The Spike G485R mutation has been observed in multiple isolates of the virus and mutation of the adjacent residue E484 to lysine is known to contribute to antigenic escape. Here, we have crystallised the SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2. The crystal structure shows that while the G485 residue does not have a direct interaction with ACE2, its mutation to arginine affects the structure of the loop made by residues 480-488 in the receptor-binding motif, disrupting the interactions of neighbouring residues with ACE2 and with potential implications for antigenic escape from vaccines, antibodies and other biologics directed against SARS-CoV-2 Spike.

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A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

Journal of Fungi ◽

10.3390/jof7070553 ◽

2021 ◽

Vol 7 (7) ◽

pp. 553

Author(s):

Bin Gao ◽

Shunyi Zhu

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Single Point ◽

Binding Domain ◽

Host Cells ◽

Single Point Mutation ◽

Binding Motif ◽

Microsporum Canis ◽

Receptor Binding Domain ◽

Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

Science Translational Medicine ◽

10.1126/scitranslmed.abd6990 ◽

2021 ◽

pp. eabd6990

Author(s):

Sang Il Kim ◽

Jinsung Noh ◽

Sujeong Kim ◽

Younggeun Choi ◽

Duck Kyun Yoo ◽

...

Keyword(s):

B Cells ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants

Journal of Translational Medicine ◽

10.1186/s12967-020-02675-4 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Alice Massacci ◽

Eleonora Sperandio ◽

Lorenzo D’Ambrosio ◽

Mariano Maffei ◽

Fabio Palombo ◽

...

Keyword(s):

Receptor Binding ◽

Consensus Sequence ◽

Cell Epitope ◽

Vaccine Design ◽

Binding Domain ◽

Spike Protein ◽

Antibody Activity ◽

Binding Motif ◽

Receptor Binding Domain ◽

The Impact

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

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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.

10.1101/2021.12.14.472585 ◽

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.

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A hybrid soluble gp130/spike-nanobody fusion protein simultaneously blocks IL-6 trans-signaling and cellular infection with SARS-CoV2

Journal of Virology ◽

10.1128/jvi.01622-21 ◽

2021 ◽

Author(s):

Julia Ettich ◽

Julia Werner ◽

Hendrik T. Weitz ◽

Eva Mueller ◽

Roland Schwarzer ◽

...

Keyword(s):

Specific Inhibitor ◽

Vero Cells ◽

Organ Damage ◽

Cell Entry ◽

Spike Protein ◽

Antibody Treatment ◽

Glucocorticoid Treatment ◽

Stat3 Phosphorylation ◽

Dual Action ◽

Viral Cell Entry

SARS-CoV2 infection can induce mild to life threatening symptoms. Especially individuals over 60 years of age or with underlying co-morbidities including heart or lung disease, and diabetes or immune compromised patients are at higher risk. Fatal multi-organ damage in COVID19 patients can be attributed to Interleukin (IL-)6 dominated cytokine storm. Consequently, IL-6R monoclonal antibody treatment for severe COVID19 cases has been approved for therapy. High concentrations of soluble IL-6R were found in COVID19 intensive care unit patients suggesting the involvement of IL-6 trans-signaling in disease pathology. Here, in analogy to bispecific antibodies (bsAbs), we developed the first bispecific IL-6 trans-signaling inhibitor c19s130Fc which blocks viral infection and IL-6 trans-signaling. c19s130Fc is a designer protein of the IL-6 trans-signaling inhibitor cs130 fused to a single domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein. c19s130Fc binds with high affinity to IL-6:sIL6R complexes as well as the spike protein of SARS-CoV2 as shown by surface plasmon resonance. Using cell-based assays, we demonstrate that c19s130Fc blocks IL-6 trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells as well as SARS-CoV2 infection and STAT3 phosphorylation in Vero cells. Taken together, c19s130Fc represents a new class of bispecific inhibitors consisting of a soluble cytokine receptor fused to anti-viral nanobodies and principally demonstrates the multi-functionalization of trans-signaling inhibitors. Importance The availability of effective SARS-CoV2 vaccines is a big step forward in managing the pandemic situation. In addition, therapeutic options e.g. monoclonal antibodies to prevent viral cell entry and anti-inflammatory therapies including glucocorticoid treatment are currently developed or in clinical use utilized to treat already infected patients. Here we report a novel dual-specific inhibitor to simultaneously target SARS-Cov2 infection and virus induced hyper-inflammation. This was achieved by fusing an inhibitor of viral cell entry with a molecule blocking IL-6, a key mediator of SARS-CoV2 induced hyper-inflammation. Through this dual action, this molecule may have the potential to efficiently ameliorate symptoms of COVID19 in infected individuals.

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The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour

10.1101/2021.06.07.447341 ◽

2021 ◽

Author(s):

Ana Sofia Oliveira ◽

Deborah Shoemark ◽

Amaurys Avila Ibarra ◽

Andrew D. Davidson ◽

Imre Berger ◽

...

Keyword(s):

Fatty Acid ◽

Binding Site ◽

Acid Site ◽

Host Cells ◽

Spike Protein ◽

Nonequilibrium Molecular Dynamics ◽

Binding Motif ◽

Fatty Acid Binding ◽

Furin Cleavage Site ◽

Fatty Acid Binding Site

The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. The results also show significant differences in behaviour between clinical variants of the spike: e.g. the D614G mutation shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.

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Investigation of the effect of temperature on the structure of SARS-Cov-2 Spike Protein by Molecular Dynamics Simulations

10.1101/2020.06.10.145086 ◽

2020 ◽

Cited By ~ 2

Author(s):

Soumya Lipsa Rath ◽

Kishant Kumar

Keyword(s):

Receptor Binding ◽

Transmembrane Domain ◽

Molecular Level ◽

Spike Protein ◽

Effect Of Temperature ◽

Binding Motif ◽

Structural Protein ◽

Closed Conformation ◽

Different Temperatures ◽

Human Receptor

ABSTRACTStatistical and epidemiological data imply temperature sensitivity of the SARS-CoV-2 coronavirus. However, the molecular level understanding of the virus structure at different temperature is still not clear. Spike protein is the outermost structural protein of the SARS-CoV-2 virus which interacts with the Angiotensin Converting Enzyme 2 (ACE2), a human receptor, and enters the respiratory system. In this study, we performed an all atom molecular dynamics simulation to study the effect of temperature on the structure of the Spike protein. After 200ns of simulation at different temperatures, we came across some interesting phenomena exhibited by the protein. We found that the solvent exposed domain of Spike protein, namely S1, is more mobile than the transmembrane domain, S2. Structural studies implied the presence of several charged residues on the surface of N-terminal Domain of S1 which are optimally oriented at 10-30 °C. Bioinformatics analyses indicated that it is capable of binding to other human receptors and should not be disregarded. Additionally, we found that receptor binding motif (RBM), present on the receptor binding domain (RBD) of S1, begins to close around temperature of 40 °C and attains a completely closed conformation at 50 °C. The closed conformation disables its ability to bind to ACE2, due to the burying of its receptor binding residues. Our results clearly show that there are active and inactive states of the protein at different temperatures. This would not only prove beneficial for understanding the fundamental nature of the virus, but would be also useful in the development of vaccines and therapeutics.Graphical AbstractHighlightsStatistical and epidemiological evidence show that external climatic conditions influence the SARS-CoV infectivity, but we still lack a molecular level understanding of the same.Here, we study the influence of temperature on the structure of the Spike glycoprotein, the outermost structural protein, of the virus which binds to the human receptor ACE2.Results show that the Spike’s S1 domain is very sensitive to external atmospheric conditions compared to the S2 transmembrane domain.The N-terminal domain comprises of several solvent exposed charged residues that are capable of binding to human proteins. The region is specifically stable at temperatures ranging around 10-30° C.The Receptor Binding Motif adopts a closed conformation at 40°C and completely closes at higher temperatures making it unsuitable of binding to human receptors

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SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

10.1101/2020.03.21.001933 ◽

2020 ◽

Cited By ~ 4

Author(s):

Vinicio Armijos-Jaramillo ◽

Justin Yeager ◽

Claire Muslin ◽

Yunierkis Perez-Castillo

Keyword(s):

Receptor Binding ◽

Evolutionary Dynamics ◽

Hot Spot ◽

Critical Role ◽

Human Cells ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Salt Bridges

AbstractThe emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.

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