Structure and function analysis of a potent human neutralizing antibody CA521LALA against SARS-CoV-2

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.

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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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Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2

Science ◽

10.1126/science.abc0870 ◽

2020 ◽

Vol 369 (6508) ◽

pp. 1261-1265 ◽

Cited By ~ 45

Author(s):

Kui K. Chan ◽

Danielle Dorosky ◽

Preeti Sharma ◽

Shawn A. Abbasi ◽

John M. Dye ◽

...

Keyword(s):

Host Cells ◽

Viral Escape ◽

Spike Protein ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

High Affinity ◽

Decoy Receptors ◽

Catalytically Active ◽

Interaction Surface

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90–glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.

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In vitro measurements of protein–protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

10.1101/2020.12.20.422820 ◽

2020 ◽

Author(s):

Sebastian Fiedler ◽

Monika A. Piziorska ◽

Viola Denninger ◽

Alexey S. Morgunov ◽

Alison Ilsley ◽

...

Keyword(s):

Protein Interactions ◽

Vaccine Development ◽

Serum Antibody ◽

Neutralizing Antibody ◽

Binding Energies ◽

Host Cells ◽

Spike Protein ◽

Antibody Affinity ◽

Plasma Therapy

AbstractThe humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2–spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the binding energies of the binary interactions between the ACE2 receptor and the spike protein, and the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential areas of application ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.

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Targeting SARS-CoV-2 Spike Protein of COVID-19 with Naturally Occurring Phytochemicals: An in Silco Study for Drug Development

10.26434/chemrxiv.12094203.v1 ◽

2020 ◽

Cited By ~ 6

Author(s):

Jitendra Subhash Rane ◽

Aroni Chatterjee ◽

Abhijeet Kumar ◽

Shashikant Ray

Keyword(s):

Receptor Binding ◽

Docking Study ◽

Binding Energies ◽

Host Cells ◽

Spike Protein ◽

Molecular Docking Study ◽

Angiotensin Converting Enzyme 2 ◽

Naturally Occurring

Spike glycoprotein found on the surface of SARS-CoV-2 (SARS-CoV-2S) is a class I fusion protein which helps the virus in its initial attachment with human Angiotensin converting enzyme 2 (ACE2) receptor and its consecutive fusion with the host cells. The attachment is mediated by the S1 subunit of the protein via its receptor binding domain. Upon binding with the receptor the protein changes its conformation from a pre-fusion to a post-fusion form. The membrane fusion and internalization of the virus is brought about by the S2 domain of the spike protein. From ancient times people have relied on naturally occurring substances like phytochemicals to fight against diseases and infection. Among these phytochemicals, flavonoids and non-flavonoids have been found to be the active source of different anti-microbial agents. Recently, studies have shown that these phytochemicals have essential anti-viral activities. We performed a molecular docking study using 10 potential naturally occurring flavonoids/non-flavonoids against the SARS-CoV-2 spike protein and compared their affinity with the FDA approved drug hydroxychloroquine (HCQ). Interestingly, the docking analysis suggested that C-terminal of S1 domain and S2 domain of the spike protein are important for binding with these compounds. Kamferol, curcumin, pterostilbene, and HCQ interact with the C-terminal of S1 domain with binding energies of -7.4, -7.1, -6.7 and -5.6 Kcal/mol, respectively. Fisetin, quercetin, isorhamnetin, genistein, luteolin, resveratrol and apigenin on the other hand, interact with the S2 domain of spike protein with the binding energies of -8.5, -8.5, -8.3, -8.2, -8.2, -7.9, -7.7 Kcal/mol, respectively. Our study suggested that, these flavonoid and non-flavonoid moieties have significantly high binding affinity for the two main important domains of the spike protein which is responsible for the attachment and internalization of the virus in the host cell and their binding affinities are much higher compared to that of HCQ. In addition, ADME (absorption, distribution, metabolism and excretion) analysis also suggested that these compounds consist of drug likeness property which may help for further explore as anti-SARS-CoV-2 agents. Further, in vitro and in vivo study of these compounds will provide a clear path for the development of novel compounds that would most likely prevent the receptor binding or internalization of the SARS-CoV-2 spike protein and therefore could be used as drugs for COVID-19 therapy.

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Targeting SARS-CoV-2 Spike Protein of COVID-19 with Naturally Occurring Phytochemicals: An in Silco Study for Drug Development

10.26434/chemrxiv.12094203 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jitendra Subhash Rane ◽

Aroni Chatterjee ◽

Abhijeet Kumar ◽

Shashikant Ray

Keyword(s):

Receptor Binding ◽

Docking Study ◽

Binding Energies ◽

Host Cells ◽

Spike Protein ◽

Molecular Docking Study ◽

Angiotensin Converting Enzyme 2 ◽

Naturally Occurring

Spike glycoprotein found on the surface of SARS-CoV-2 (SARS-CoV-2S) is a class I fusion protein which helps the virus in its initial attachment with human Angiotensin converting enzyme 2 (ACE2) receptor and its consecutive fusion with the host cells. The attachment is mediated by the S1 subunit of the protein via its receptor binding domain. Upon binding with the receptor the protein changes its conformation from a pre-fusion to a post-fusion form. The membrane fusion and internalization of the virus is brought about by the S2 domain of the spike protein. From ancient times people have relied on naturally occurring substances like phytochemicals to fight against diseases and infection. Among these phytochemicals, flavonoids and non-flavonoids have been found to be the active source of different anti-microbial agents. Recently, studies have shown that these phytochemicals have essential anti-viral activities. We performed a molecular docking study using 10 potential naturally occurring flavonoids/non-flavonoids against the SARS-CoV-2 spike protein and compared their affinity with the FDA approved drug hydroxychloroquine (HCQ). Interestingly, the docking analysis suggested that C-terminal of S1 domain and S2 domain of the spike protein are important for binding with these compounds. Kamferol, curcumin, pterostilbene, and HCQ interact with the C-terminal of S1 domain with binding energies of -7.4, -7.1, -6.7 and -5.6 Kcal/mol, respectively. Fisetin, quercetin, isorhamnetin, genistein, luteolin, resveratrol and apigenin on the other hand, interact with the S2 domain of spike protein with the binding energies of -8.5, -8.5, -8.3, -8.2, -8.2, -7.9, -7.7 Kcal/mol, respectively. Our study suggested that, these flavonoid and non-flavonoid moieties have significantly high binding affinity for the two main important domains of the spike protein which is responsible for the attachment and internalization of the virus in the host cell and their binding affinities are much higher compared to that of HCQ. In addition, ADME (absorption, distribution, metabolism and excretion) analysis also suggested that these compounds consist of drug likeness property which may help for further explore as anti-SARS-CoV-2 agents. Further, in vitro and in vivo study of these compounds will provide a clear path for the development of novel compounds that would most likely prevent the receptor binding or internalization of the SARS-CoV-2 spike protein and therefore could be used as drugs for COVID-19 therapy.

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Molecular Mechanism of the N501Y Mutation for Enhanced Binding between SARS-CoV-2’s Spike Protein and Human ACE2 Receptor

10.1101/2021.01.04.425316 ◽

2021 ◽

Author(s):

Binquan Luan ◽

Haoran Wang ◽

Tien Huynh

Keyword(s):

Molecular Mechanism ◽

Hydrophobic Interactions ◽

Neutralizing Antibody ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Global Pandemic ◽

Dynamics Simulations ◽

Antibody Cocktail

AbstractCoronavirus disease 2019 (COVID-19) has been an ongoing global pandemic for over a year. Recently, an emergent SARS-CoV-2 variant (B.1.1.7) with an unusually large number of mutations had become highly contagious and wide-spreading in United Kingdom. From genome analysis, the N501Y mutation within the receptor binding domain (RBD) of the SARS-CoV-2’s spike protein might have enhanced the viral protein’s binding with the human angiotensin converting enzyme 2 (hACE2). The latter is the prelude for the virus’ entry into host cells. So far, the molecular mechanism of this enhanced binding is still elusive, which prevents us from assessing its effects on existing therapeutic antibodies. Using all atom molecular dynamics simulations, we demonstrated that Y501 in mutated RBD can be well coordinated by Y41 and K353 in hACE2 through hydrophobic interactions, increasing the overall binding affinity between RBD and hACE2 by about 0.81 kcal/mol. We further explored how the N501Y mutation might affect the binding between a neutralizing antibody (CB6) and RBD. We expect that our work can help researchers design proper measures responding to this urgent virus mutation, such as adding a modified/new neutralizing antibody specifically targeting at this variant in the therapeutic antibody cocktail.Abstract Figure

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Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2

10.1101/2020.11.13.381533 ◽

2020 ◽

Author(s):

Sarah A. Clark ◽

Lars E. Clark ◽

Junhua Pan ◽

Adrian Coscia ◽

Lindsay G.A. McKay ◽

...

Keyword(s):

Antibody Response ◽

Molecular Basis ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Antiviral Effect ◽

Host Cells ◽

Human Antibody ◽

Angiotensin Converting Enzyme 2 ◽

Antibody Heavy Chain ◽

Major Target

AbstractThe SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

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Investigation of Interaction Between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

10.21203/rs.3.rs-456888/v1 ◽

2021 ◽

Author(s):

Yuning Shang ◽

Feixiang Chen ◽

Shasha Li ◽

Lijuan Song ◽

Yunzhen Gao ◽

...

Keyword(s):

Viral Entry ◽

Living Cells ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Wild Type ◽

Protein Variant ◽

In Vitro System ◽

Angiotensin Converting Enzyme 2

Abstract Background: The Interaction between severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) spike protein with Angiotensin converting enzyme 2 (ACE2) on the host cells is a crucial step for the viral entry and infection. Therefore, investigating the molecular mechanism underlying the interaction is of great importance for the prevention of the infection of SARS-CoV-2. In this study, we aimed to establish a virus-free in vitro system to study the interaction between the spike protein and host cells of SARS-CoV-2.Results: Our results show that ACE2-overexpressing HEK293T cells are captured by immobilized spike protein, and the cell capturing process can be inhibited by the receptor binding domain of the spike protein or antibodies against S protein. Furthermore, spike protein variant with D614G mutant show a higher cell capturing ability than wild type spike protein. In addition, the captured cells can be eluted as living cells for further investigation.Conclusions: This study provides a new in vitro system for investigating the interaction between SARS-CoV-2 and host cells and purifying ACE2-expressing cells.

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The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2 in vitro

PLoS ONE ◽

10.1371/journal.pone.0257705 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257705

Author(s):

Riho Tateyama-Makino ◽

Mari Abe-Yutori ◽

Taku Iwamoto ◽

Kota Tsutsumi ◽

Motonori Tsuji ◽

...

Keyword(s):

Protease Activity ◽

Sodium Dodecyl ◽

Host Cells ◽

Spike Protein ◽

Inhibitory Effects ◽

Inhibitor Binding ◽

Angiotensin Converting Enzyme 2 ◽

Docking Simulations ◽

Gingival Mucosa

SARS-CoV-2 enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the tongue and gingival mucosa, the oral cavity is a potential entry point for SARS-CoV-2. This study evaluated the inhibitory effects of general ingredients of toothpastes and mouthwashes on the spike protein-ACE2 interaction and the TMPRSS2 protease activity using an in vitro assay. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to inhibitor-binding site of ACE2. Furthermore, tranexamic acid exerted inhibitory effects on TMPRSS2 protease activity. Our findings suggest that these toothpaste and mouthwash ingredients could help prevent SARS-CoV-2 infection.

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