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1.
Sci Rep ; 12(1): 20120, 2022 Nov 22.
Article in English | MEDLINE | ID: covidwho-2133636

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/chemistry , Leukocytes, Mononuclear , Antibodies, Viral , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Monoclonal
2.
Structure ; 28(8): 874-878, 2020 08 04.
Article in English | MEDLINE | ID: covidwho-2132441

ABSTRACT

During global pandemics, the spread of information needs to be faster than the spread of the virus in order to ensure the health and safety of human populations worldwide. In our current crisis, the demand for SARS-CoV-2 drugs and vaccines highlights the importance of biological targets and their three-dimensional shape. In particular, structural biology as a field was poised to quickly respond to crises due to previous experience and expertise and because of its early adoption of open access practices.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Viral Proteins/chemistry , COVID-19 , Coronavirus 3C Proteases , Coronavirus RNA-Dependent RNA Polymerase , Cysteine Endopeptidases/chemistry , Databases, Protein , Humans , Models, Molecular , Molecular Biology , Protein Conformation , RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry
3.
Nature ; 593(7857): 136-141, 2021 05.
Article in English | MEDLINE | ID: covidwho-2114170

ABSTRACT

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/immunology , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/isolation & purification , COVID-19/metabolism , COVID-19/virology , Female , HEK293 Cells , Humans , Immune Evasion/genetics , Immune Evasion/immunology , Immunization, Passive , Male , Middle Aged , Models, Molecular , Mutation , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Synthetic/administration & dosage
4.
J Microbiol Biotechnol ; 32(10): 1335-1343, 2022 Oct 28.
Article in English | MEDLINE | ID: covidwho-2115579

ABSTRACT

COVID-19 is an emerging disease that poses a severe threat to global public health. As such, there is an urgent demand for vaccines against SARS-CoV-2, the virus that causes COVID-19. Here, we describe a virus-like nanoparticle candidate vaccine against SARS-CoV-2 produced by an E. coli expression system. The fusion protein of a truncated ORF2-encoded protein of aa 439~608 (p170) from hepatitis E virus CCJD-517 and the receptor-binding domain of the spike protein from SARS-CoV-2 were expressed, purified and characterized. The antigenicity and immunogenicity of p170-RBD were evaluated in vitro and in Kunming mice. Our investigation revealed that p170-RBD self-assembled into approximately 24 nm virus-like particles, which could bind to serum from vaccinated people (p < 0.001) and receptors on cells. Immunization with p170-RBD induced the titer of IgG antibody vaccine increased from 14 days post-immunization and was significantly enhanced after a booster immunization at 28 dpi, ultimately reaching a peak level on 42 dpi with a titer of 4.97 log10. Pseudovirus neutralization tests showed that the candidate vaccine induced a strong neutralizing antibody response in mice. In this research, we demonstrated that p170-RBD possesses strong antigenicity and immunogenicity and could be a potential candidate for use in future SARS-CoV-2 vaccine development.


Subject(s)
COVID-19 , Hepatitis E virus , Viral Vaccines , Animals , Humans , Mice , Antibodies, Neutralizing , Antibodies, Viral , Capsid Proteins/genetics , COVID-19/prevention & control , COVID-19 Vaccines/genetics , Escherichia coli , Mice, Inbred BALB C , Recombinant Proteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Viral Vaccines/genetics
5.
Sci Rep ; 12(1): 19791, 2022 Nov 17.
Article in English | MEDLINE | ID: covidwho-2119245

ABSTRACT

The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Viral , Viral Envelope Proteins , Antiviral Agents/pharmacology , Membrane Glycoproteins/chemistry , SARS-CoV-2 , COVID-19/drug therapy , Antibodies, Monoclonal
6.
Commun Biol ; 5(1): 789, 2022 08 05.
Article in English | MEDLINE | ID: covidwho-2117221

ABSTRACT

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Monoclonal/chemistry , Antibodies, Viral , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/chemistry
7.
Biomolecules ; 12(11)2022 Nov 10.
Article in English | MEDLINE | ID: covidwho-2109922

ABSTRACT

With its fast-paced mutagenesis, the SARS-CoV-2 Omicron variant has threatened many societies worldwide. Strategies for predicting mutagenesis such as the computational prediction of SARS-CoV-2 structural diversity and its interaction with the human receptor will greatly benefit our understanding of the virus and help develop therapeutics against it. We aim to use protein structure prediction algorithms along with molecular docking to study the effects of various mutations in the Receptor Binding Domain (RBD) of the SARS-CoV-2 and its key interactions with the angiotensin-converting enzyme 2 (ACE-2) receptor. The RBD structures of the naturally occurring variants of SARS-CoV-2 were generated from the WUHAN-Hu-1 using the trRosetta algorithm. Docking (HADDOCK) and binding analysis (PRODIGY) between the predicted RBD sequences and ACE-2 highlighted key interactions at the Receptor-Binding Motif (RBM). Further mutagenesis at conserved residues in the Original, Delta, and Omicron variants (P499S and T500R) demonstrated stronger binding and interactions with the ACE-2 receptor. The predicted T500R mutation underwent some preliminary tests in vitro for its binding and transmissibility in cells; the results correlate with the in-silico analysis. In summary, we suggest conserved residues P499 and T500 as potential mutation sites that could increase the binding affinity and yet do not exist in nature. This work demonstrates the use of the trRosetta algorithm to predict protein structure and future mutations at the RBM of SARS-CoV-2, followed by experimental testing for further efficacy verification. It is important to understand the protein structure and folding to help develop potential therapeutics.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/genetics , Spike Glycoprotein, Coronavirus/chemistry , Molecular Docking Simulation , Peptidyl-Dipeptidase A/chemistry , Receptors, Virus , Protein Binding , Mutation , Protein Folding
8.
Cancer Discov ; 12(4): 958-983, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-2108398

ABSTRACT

Vaccination against coronavirus disease 2019 (COVID-19) relies on the in-depth understanding of protective immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We characterized the polarity and specificity of memory T cells directed against SARS-CoV-2 viral lysates and peptides to determine correlates with spontaneous, virus-elicited, or vaccine-induced protection against COVID-19 in disease-free and cancer-bearing individuals. A disbalance between type 1 and 2 cytokine release was associated with high susceptibility to COVID-19. Individuals susceptible to infection exhibited a specific deficit in the T helper 1/T cytotoxic 1 (Th1/Tc1) peptide repertoire affecting the receptor binding domain of the spike protein (S1-RBD), a hotspot of viral mutations. Current vaccines triggered Th1/Tc1 responses in only a fraction of all subject categories, more effectively against the original sequence of S1-RBD than that from viral variants. We speculate that the next generation of vaccines should elicit Th1/Tc1 T-cell responses against the S1-RBD domain of emerging viral variants. SIGNIFICANCE: This study prospectively analyzed virus-specific T-cell correlates of protection against COVID-19 in healthy and cancer-bearing individuals. A disbalance between Th1/Th2 recall responses conferred susceptibility to COVID-19 in both populations, coinciding with selective defects in Th1 recognition of the receptor binding domain of spike. See related commentary by McGary and Vardhana, p. 892. This article is highlighted in the In This Issue feature, p. 873.


Subject(s)
COVID-19 , Neoplasms , T-Lymphocytes , Antibodies, Neutralizing , COVID-19/immunology , Humans , Neoplasms/complications , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes/immunology
9.
Commun Biol ; 5(1): 1188, 2022 Nov 05.
Article in English | MEDLINE | ID: covidwho-2106511

ABSTRACT

SARS-CoV-2 has evolved continuously and accumulated spike mutations with each variant having a different binding for the cellular ACE2 receptor. It is not known whether the interactions between such mutated spikes and ACE2 glycans are conserved among different variant lineages. Here, we focused on three ACE2 glycosylation sites (53, 90 and 322) that are geometrically close to spike binding sites and investigated the effect of their glycosylation pattern on spike affinity. These glycosylation deletions caused distinct site-specific changes in interactions with the spike and acted cooperatively. Of note, the particular interaction profiles were conserved between the SARS-CoV-2 parental virus and the variants of concern (VOCs) Delta and Omicron. Our study provides insights for a better understanding of the importance of ACE2 glycosylation on ACE2/SARS-CoV-2 spike interaction and guidance for further optimization of soluble ACE2 for therapeutic use.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/genetics , Glycosylation , Peptidyl-Dipeptidase A , Protein Binding
10.
Commun Biol ; 5(1): 1170, 2022 Nov 03.
Article in English | MEDLINE | ID: covidwho-2106509

ABSTRACT

The trimeric spike (S) glycoprotein, which protrudes from the SARS-CoV-2 viral envelope, binds to human ACE2, initiated by at least one protomer's receptor binding domain (RBD) switching from a "down" (closed) to an "up" (open) state. Here, we used large-scale molecular dynamics simulations and two-dimensional replica exchange umbrella sampling calculations with more than a thousand windows and an aggregate total of 160 µs of simulation to investigate this transition with and without glycans. We find that the glycosylated spike has a higher barrier to opening and also energetically favors the down state over the up state. Analysis of the S-protein opening pathway reveals that glycans at N165 and N122 interfere with hydrogen bonds between the RBD and the N-terminal domain in the up state, while glycans at N165 and N343 can stabilize both the down and up states. Finally, we estimate how epitope exposure for several known antibodies changes along the opening path. We find that the BD-368-2 antibody's epitope is continuously exposed, explaining its high efficacy.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2 , Peptidyl-Dipeptidase A , Polysaccharides , Epitopes
11.
Sci Rep ; 12(1): 19087, 2022 Nov 09.
Article in English | MEDLINE | ID: covidwho-2106475

ABSTRACT

The World Health Organization categorized SARS-CoV-2 as a variant of concern, having numerous mutations in spike protein, which have been found to evade the effect of antibodies stimulated by the COVID-19 vaccine. The susceptibility to omicron variant by immunization-induced antibodies are direly required for risk evaluation. To avoid the risk of arising viral illness, the construction of a specific vaccine that triggers the production of targeted antibodies to combat infection remains highly imperative. The aim of the present study is to develop a particular vaccine exploiting bioinformatics approaches which can target B- and T-cells epitopes. Through this approach, novel epitopes of the S protein-SARS-CoV-2 were predicted for the development of a multiple epitope vaccine. Multiple epitopes were selected on the basis of toxicity, immunogenicity and antigenicity, and vaccine subunit was constructed having potential immunogenic properties. The epitopes were linked with 3 types of linker EAAAK, AAY and GPGPG for vaccine construction. Subsequently, vaccine structure was docked with the receptor and cloned in a pET-28a (+) vector. The constructed vaccine was ligated in pET-28a (+) vector in E. coli using the SnapGene tool for the expression study and a good immune response was observed. Several computational tools were used to predict and analyze the vaccine constructed by using spike protein sequence of omicrons. The current study identified a Multi-Epitope Vaccine (MEV) as a significant vaccine candidate that could potentially help the global world to combat SARS-CoV-2 infections.


Subject(s)
COVID-19 , Viral Vaccines , Humans , SARS-CoV-2/genetics , COVID-19 Vaccines/genetics , Spike Glycoprotein, Coronavirus/chemistry , COVID-19/prevention & control , Computational Biology , Escherichia coli , Epitopes, B-Lymphocyte , Immunogenicity, Vaccine , Epitopes, T-Lymphocyte
12.
Immunobiology ; 227(6): 152287, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2105123

ABSTRACT

BACKGROUND: Epitope selection is the key to peptide vaccines development. Bioinformatics tools can efficiently improve the screening of antigenic epitopes and help to choose the right ones. OBJECTIVE: To predict, synthesize and testify peptide epitopes at spike protein, assess the effect of mutations on epitope humoral immunity, thus provide clues for the design and development of epitope peptide vaccines against SARS-CoV-2. METHODS: Bioinformatics servers and immunological tools were used to identify the helper T lymphocyte, cytotoxic T lymphocyte, and linear B lymphocyte epitopes on the S protein of SARS-CoV-2. Physicochemical properties of candidate epitopes were analyzed using IEDB, VaxiJen, and AllerTOP online software. Three candidate epitopes were synthesized and their antigenic responses were evaluated by binding antibody detection. RESULTS: A total of 20 antigenic, non-toxic and non-allergenic candidate epitopes were identified from 1502 epitopes, including 6 helper T-cell epitopes, 13 cytotoxic T-cell epitopes, and 1 linear B cell epitope. After immunization with antigen containing candidate epitopes S206-221, S403-425, and S1157-1170 in rabbits, the binding titers of serum antibody to the corresponding peptide, S protein, receptor-binding domain protein were (415044, 2582, 209.3), (852819, 45238, 457767) and (357897, 10528, 13.79), respectively. The binding titers to Omicron S protein were 642, 12,878 and 7750, respectively, showing that N211L, DEL212 and K417N mutations cause the reduction of the antibody binding activity. CONCLUSIONS: Bioinformatic methods are effective in peptide epitopes design. Certain mutations of the Omicron would lead to the loss of antibody affinity to Omicron S protein.


Subject(s)
COVID-19 , Viral Vaccines , Animals , Humans , Rabbits , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Computational Biology/methods , Epitopes, T-Lymphocyte/genetics , COVID-19 Vaccines/genetics , Immunity, Humoral , Epitopes, B-Lymphocyte/genetics , Vaccines, Subunit , Peptides
13.
Mol Divers ; 26(6): 3143-3155, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2104017

ABSTRACT

Oxidative stress, which occurs when an organism is exposed to an adverse stimulus that results in a misbalance of antioxidant and pro-oxidants species, is the common denominator of diseases considered as a risk factor for SARS-CoV-2 lethality. Indeed, reactive oxygen species caused by oxidative stress have been related to many virus pathogenicity. In this work, simulations have been performed on the receptor binding domain of SARS-CoV-2 spike glycoprotein to study what residues are more susceptible to be attacked by ·OH, which is one of the most reactive radicals associated to oxidative stress. The results indicate that isoleucine (ILE) probably plays a crucial role in modification processes driven by radicals. Accordingly, QM/MM-MD simulations have been conducted to study both the ·OH-mediated hydrogen abstraction of ILE residues and the induced modification of the resulting ILE radical through hydroxylation or nitrosylation reactions. All in all, in silico studies show the importance of the chemical environment triggered by oxidative stress on the modifications of the virus, which is expected to help for foreseeing the identification or development of antioxidants as therapeutic drugs.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Binding Sites , Molecular Dynamics Simulation , Protein Binding , Oxidative Stress
14.
Biomolecules ; 12(11)2022 Oct 31.
Article in English | MEDLINE | ID: covidwho-2099330

ABSTRACT

After the SARS-CoV-2 Wuhan variant that gave rise to the pandemic, other variants named Delta, Omicron, and Omicron-2 sequentially became prevalent, with mutations spread around the viral genome, including on the spike (S) protein; in order to understand the resultant in gains in infectivity, we interrogated in silico both the equilibrium binding and the binding pathway of the virus' receptor-binding domain (RBD) to the angiotensin-converting enzyme 2 (ACE2) receptor. We interrogated the molecular recognition between the RBD of different variants and ACE2 through supervised molecular dynamics (SuMD) and classic molecular dynamics (MD) simulations to address the effect of mutations on the possible S protein binding pathways. Our results indicate that compensation between binding pathway efficiency and stability of the complex exists for the Omicron BA.1 receptor binding domain, while Omicron BA.2's mutations putatively improved the dynamic recognition of the ACE2 receptor, suggesting an evolutionary advantage over the previous strains.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Humans , Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Protein Binding , Peptidyl-Dipeptidase A/chemistry , COVID-19/genetics , Receptors, Virus/genetics , Mutation
15.
Chem Commun (Camb) ; 58(93): 12939-12942, 2022 Nov 22.
Article in English | MEDLINE | ID: covidwho-2096844

ABSTRACT

Here we show using mass photometry how proline substitutions, commonly used for SARS-CoV-2 spike stabilisation in vaccine design, directly affects ACE2 receptor interactions via dynamics of open and closed states. Conformational changes and ACE2 binding were influenced by spike variant and temperature, but independent of site-specific N-glycosylation.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Angiotensin-Converting Enzyme 2 , Spike Glycoprotein, Coronavirus/chemistry , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Protein Binding , Photometry , Molecular Dynamics Simulation , Binding Sites
16.
ACS Infect Dis ; 8(11): 2259-2270, 2022 Nov 11.
Article in English | MEDLINE | ID: covidwho-2096630

ABSTRACT

SARS-CoV-2, a coronavirus strain that started a worldwide pandemic in early 2020, attaches to human cells by binding its spike (S) glycoprotein to a host receptor protein angiotensin-converting enzyme 2 (ACE2). Blocking the interaction between the S protein and ACE2 has emerged as an important strategy for preventing viral infection. We systematically developed and optimized an AlphaLISA assay to investigate binding events between ACE2 and the ectodomain of the SARS-CoV-2 S protein (S-614G: residues 1-1208 with a D614G mutation). Using S-614G permits discovering potential allosteric inhibitors that stabilize the S protein in a conformation that impedes its access to ACE2. Over 30,000 small molecules were screened in a high-throughput format for activity against S-614G and ACE2 binding using the AlphaLISA assay. A viral entry assay was used to validate hits using lentiviral particles pseudotyped with the full-length S protein of the Wuhan-1 strain. Two compounds identified in the screen, oleic acid and suramin, blocked the attachment of S-614G to ACE2 and S protein-driven cell entry into Calu-3 and ACE2-overexpressing HEK293T cells. Oleic acid inhibits S-614G binding to ACE2 far more potently than to the receptor-binding domain (RBD, residues 319-541 of SARS-CoV-2 S), potentially indicating a noncompetitive mechanism. The results indicate that using the full-length ectodomain of the S protein can be important for identifying allosteric inhibitors of ACE2 binding. The approach reported here represents a rapidly adaptable format for discovering receptor-binding inhibitors to S-proteins of future coronavirus strains.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Oleic Acid , HEK293 Cells , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism
17.
J Agric Food Chem ; 70(45): 14403-14413, 2022 Nov 16.
Article in English | MEDLINE | ID: covidwho-2096615

ABSTRACT

COVID-19 is initiated by binding the SARS-CoV-2 spike protein to angiotensin-converting enzyme 2 (ACE2) on host cells. Food factors capable of suppressing the binding between the SARS-CoV-2 spike protein and ACE2 or reducing the ACE2 availability through ACE2 inhibitions may potentially reduce the risk of SARS-CoV-2 infection and COVID-19. In this study, the chemical compositions of clove water and ethanol extracts were investigated, along with their potentials in suppressing SARS-CoV-2 spike protein-ACE2 binding, reducing ACE2 availability, and scavenging free radicals. Thirty-four compounds were tentatively identified in the clove water and ethanol extracts, with six reported in clove for the first time. Clove water and ethanol extracts dose-dependently suppressed SARS-CoV-2 spike protein binding to ACE2 and inhibited ACE2 activity. The water extract had stronger inhibitory effects than the ethanol extract on a dry weight basis. The clove water extract also had more potent free radical scavenging activities against DPPH• and ABTS•+ (536.9 and 3525.06 µmol TE/g, respectively) than the ethanol extract (58.44 and 2298.01 µmol TE/g, respectively). In contrast, the ethanol extract had greater total phenolic content (TPC) and relative HO• scavenging capacity (HOSC) values (180.03 mg GAE/g and 2181.08 µmol TE/g, respectively) than the water extract (120.12 mg GAE/g and 1483.02 µmol TE/g, respectively). The present study demonstrated the potential of clove in reducing the risk of SARS-CoV-2 infection and COVID-19 development.


Subject(s)
COVID-19 , Syzygium , Humans , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Syzygium/metabolism , SARS-CoV-2 , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Binding Sites , Free Radicals , Water , Ethanol
18.
J Comput Aided Mol Des ; 36(11): 797-804, 2022 11.
Article in English | MEDLINE | ID: covidwho-2094694

ABSTRACT

Evaluation of the intramolecular stability of proteins plays a key role in the comprehension of their biological behavior and mechanism of action. Small structural alterations such as mutations induced by single nucleotide polymorphism can impact biological activity and pharmacological modulation. Covid-19 mutations, that affect viral replication and the susceptibility to antibody neutralization, and the action of antiviral drugs, are just one example. In this work, the intramolecular stability of mutated proteins, like Spike glycoprotein and its complexes with the human target, is evaluated through hydropathic intramolecular energy scoring originally conceived by Abraham and Kellogg based on the "Extension of the fragment method to calculate amino acid zwitterion and side-chain partition coefficients" by Abraham and Leo in Proteins: Struct. Funct. Genet. 1987, 2:130 - 52. HINT is proposed as a fast and reliable tool for the stability evaluation of any mutated system. This work has been written in honor of Prof. Donald J. Abraham (1936-2021).


Subject(s)
Oncogene Proteins , Spike Glycoprotein, Coronavirus , Humans , Oncogene Proteins/chemistry , Spike Glycoprotein, Coronavirus/chemistry
19.
Protein Sci ; 31(11): e4447, 2022 11.
Article in English | MEDLINE | ID: covidwho-2094239

ABSTRACT

SARS-CoV-2 infects cells by attachment to its receptor-the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation-, and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affects the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism and capacity to evade immune responses during evolution.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/chemistry , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Binding
20.
Am J Physiol Heart Circ Physiol ; 323(6): H1176-H1193, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2088958

ABSTRACT

Patients with diabetes infected with COVID-19 have greater mortality than those without comorbidities, but the underlying mechanisms remain unknown. This study aims to identify the mechanistic interactions between diabetes and severe COVID-19. Microparticles (MPs), the cell membrane-derived vesicles released on cell activation, are largely increased in patients with diabetes. To date, many mechanisms have been postulated for increased severity of COVID-19 in patients with underlying conditions, but the contributions of excessive MPs in patients with diabetes have been overlooked. This study characterizes plasma MPs from normal human subjects and patients with type 2 diabetes in terms of amount, cell origins, surface adhesive properties, ACE2 expression, spike protein binding capacity, and their roles in SARS-CoV-2 infection. Results showed that over 90% of plasma MPs express ACE2 that binds the spike protein of SARS-CoV-2. MPs in patients with diabetes increase 13-fold in quantity and 11-fold in adhesiveness when compared with normal subjects. Perfusion of human plasma with pseudo-typed SARS-CoV-2 virus or spike protein-bound MPs into human endothelial cell-formed microvessels-on-a chip demonstrated that MPs from patients with diabetes, not normal subjects, interact with endothelium and carry SARS-CoV-2 into cells through endocytosis, providing additional virus entry pathways and enhanced infection. Results also showed a large percentage of platelet-derived tissue factor-bearing MPs in diabetic plasma, which could contribute to thrombotic complications with SARS-CoV-2 infection. This study reveals a dual role of diabetic MPs in promoting SARS-CoV-2 entry and propagating vascular inflammation. These findings provide novel mechanistic insight into the high prevalence of COVID-19 in patients with diabetes and their propensity to develop severe vascular complications.NEW & NOTEWORTHY This study provides the first evidence that over 90% of human plasma microparticles express ACE2 that binds SARS-CoV-2 S protein with high affinity. Thus, the highly elevated adhesive circulating microparticles identified in patients with diabetes not only have greater SARS-CoV-2 binding capacity but also enable additional viral entry through virus-bound microparticle-endothelium interactions and enhanced infection. These findings reveal a novel mechanistic insight into the adverse outcomes of COVID-19 in patients with diabetes.


Subject(s)
COVID-19 , Diabetes Mellitus, Type 2 , Humans , Angiotensin-Converting Enzyme 2 , COVID-19/complications , Diabetes Mellitus, Type 2/complications , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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