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1.
Int J Mol Sci ; 21(21)2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33158276

RESUMO

Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and the consensus around the molecular origins behind binding preferences of SARS-CoV-2 for binding with the angiotensin-converting enzyme 2 (ACE2) host receptor is yet to be established. In this work, we performed a comprehensive computational investigation in which sequence analysis and modeling of coevolutionary networks are combined with atomistic molecular simulations and comparative binding free energy analysis of the SARS-CoV and SARS-CoV-2 spike protein receptor binding domains with the ACE2 host receptor. Different from other computational studies, we systematically examine the molecular and energetic determinants of the binding mechanisms between SARS-CoV-2 and ACE2 proteins through the lens of coevolution, conformational dynamics, and allosteric interactions that conspire to drive binding interactions and signal transmission. Conformational dynamics analysis revealed the important differences in mobility of the binding interfaces for the SARS-CoV-2 spike protein that are not confined to several binding hotspots, but instead are broadly distributed across many interface residues. Through coevolutionary network analysis and dynamics-based alanine scanning, we established linkages between the binding energy hotspots and potential regulators and carriers of signal communication in the virus-host receptor complexes. The results of this study detailed a binding mechanism in which the energetics of the SARS-CoV-2 association with ACE2 may be determined by cumulative changes of a number of residues distributed across the entire binding interface. The central findings of this study are consistent with structural and biochemical data and highlight drug discovery challenges of inhibiting large and adaptive protein-protein interfaces responsible for virus entry and infection transmission.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Pandemias , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Transdução de Sinais , Internalização do Vírus
2.
PLoS One ; 15(11): e0241168, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33170884

RESUMO

The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called "glue" points) between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer's intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant "glue" point or possible "latch" for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 µ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.


Assuntos
Betacoronavirus/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Mutação Puntual , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Termodinâmica
3.
Nat Commun ; 11(1): 5588, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149112

RESUMO

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/prevenção & controle , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Microscopia Crioeletrônica , Humanos , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Domínios Proteicos/imunologia , Receptores Virais/metabolismo
4.
BMC Pulm Med ; 20(1): 301, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198751

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. METHODS: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. RESULTS: ICU patients showed a marked increase in neutrophils (1.24 × 105 ml- 1, 0.85-2.07), lower lymphocyte (0.97 × 105 ml- 1, 0.024-0.34) and macrophages fractions (0.43 × 105 ml- 1, 0.34-1.62) compared to IMW patients (0.095 × 105 ml- 1, 0.05-0.73; 0.47 × 105 ml- 1, 0.28-1.01 and 2.14 × 105 ml- 1, 1.17-3.01, respectively) (p < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p < 0.1) or antivirals (p < 0.05). CONCLUSIONS: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Infecções por Coronavirus/imunologia , Inflamação/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Leucócitos/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Corticosteroides/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/terapia , Combinação de Medicamentos , Feminino , Humanos , Hidroxicloroquina/uso terapêutico , Unidades de Terapia Intensiva , Interleucina-10/imunologia , Itália , Leucócitos Mononucleares/virologia , Lopinavir/uso terapêutico , Pulmão/citologia , Pulmão/virologia , Linfócitos/imunologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Neutrófilos/imunologia , Pandemias , Pneumonia Viral/terapia , Prognóstico , Estudos Prospectivos , Respiração Artificial/métodos , Ritonavir/uso terapêutico , Glicoproteína da Espícula de Coronavírus/metabolismo , Taxa de Sobrevida , Vírion/metabolismo , Vírion/ultraestrutura
5.
Biol Direct ; 15(1): 21, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33138856

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection spreaded rapidly worldwide, as far as it has become a global pandemic. Therefore, the introduction of serological tests for determination of IgM and IgG antibodies has become the main diagnostic tool, useful for tracking the spread of the virus and for consequently allowing its containment. In our study we compared point of care test (POCT) lateral flow immunoassay (FIA) vs automated chemiluminescent immunoassay (CLIA), in order to assess their specificity and sensibility for COVID-19 antibodies detection. RESULTS: We find that different specificities and sensitivities for IgM and IgG tests. Notably IgM POCT FIA method vs CLIA method (gold standard) has a low sensitivity (0.526), while IgG POCT FIA method vs CLIA method (gold standard) test has a much higher sensitivity (0.937); further, with respect of IgG, FIA and CLIA could arguably provide equivalent information. CONCLUSIONS: FIA method could be helpful in assessing in short time, the possible contagiousness of subjects that for work reasons cannot guarantee "social distancing".


Assuntos
Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Testes Sorológicos , Sequência de Aminoácidos , Feminino , Humanos , Imunoensaio , Imunoglobulina M/metabolismo , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Pandemias , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
J Mol Model ; 26(12): 338, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33175236

RESUMO

A novel coronavirus (SARS-CoV-2) identified in Wuhan state of China in 2019 is the causative agent of deadly disease COVID-19. It has spread across the globe (more than 210 countries) within a short period. Coronaviruses pose serious health threats to both humans and animals. A recent publication reported an experimental 3D complex structure of the S protein of SARS-CoV-2 showed that the ectodomain of the SARS-CoV-2 S protein binds to the peptidase domain (PD) of human ACE2 with a dissociation constant (Kd) of ~ 15 nM. In this study, we focused on inhibitors for ACE2: S protein complex using virtual screening and inhibition studies through molecular docking for over 200,000 natural compounds. Toxicity analysis was also performed for the best hits, and the final complex structures for four complexes were subjected to 400 ns molecular dynamics simulations for stability testing. We found two natural origin inhibitors for the S protein: human ACE2 complex (Andrographolide and Pterostilbene) which displayed better inhibition potential for ACE2 receptor and its binding with the S protein of SARS-CoV-2. Comparative studies were also performed to test and verify that these two drug candidates are also better than hydroxychloroquine which is known to inhibit this complex. However, we needed better potential drug candidates to overcome the side effects of hydroxychloroquine. Supplementary experimental studies need to be carried forward to corroborate the viability of these two new inhibitors for ACE2: S protein complex so as to curb down COVID-19.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/epidemiologia , Peptídeo Hidrolases/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/epidemiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Peptídeo Hidrolases/genética , Peptidil Dipeptidase A/genética , Pneumonia Viral/virologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/genética
7.
Molecules ; 25(20)2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053830

RESUMO

Docking of over 160 aminothiourea derivatives at the SARS-CoV-2 S-protein-human ACE2 receptor interface, whose structure became available recently, has been evaluated for its complex stabilizing potency and subsequently subjected to quantitative structure-activity relationship (QSAR) analysis. The structural variety of the studied compounds, that include 3 different forms of the N-N-C(S)-N skeleton and combinations of 13 different substituents alongside the extensive length of the interface, resulted in the failure of the QSAR analysis, since different molecules were binding to different parts of the interface. Subsequently, absorption, distribution, metabolism, and excretion (ADME) analysis on all studied compounds, followed by a toxicity analysis using statistical models for selected compounds, was carried out to evaluate their potential use as lead compounds for drug design. Combined, these studies highlighted two molecules among the studied compounds, i.e., 5-(pyrrol-2-yl)-2-(2-methoxyphenylamino)-1,3,4-thiadiazole and 1-(cyclopentanoyl)-4-(3-iodophenyl)-thiosemicarbazide, as the best candidates for the development of future drugs.


Assuntos
Antivirais/farmacologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Semicarbazidas/química , Glicoproteína da Espícula de Coronavírus/química , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/virologia , Humanos , Modelos Estatísticos , Estrutura Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
Med Hypotheses ; 143: 110197, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33017906

RESUMO

Coronavirus disease 2019 (COVID-19) may have a metabolic origin given strong links with risk factors such as lipids and glucose and co-morbidities such as obesity and type 2 diabetes mellitus. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein mediates viral cellular entry via the ACE2 receptor. The cytoplasmic tail of this spike protein is heavily palmitoylated. Emerging studies suggest that SARS-CoV-2 alters lipid metabolism in the lung epithelial cells by modulating peroxisome proliferator-activated receptor alpha (PPARα), possibly contributing to lipotoxicity, inflammation and untoward respiratory effects. Disruption of this process may affect palmitoylation of SARS-CoV spike protein and thus infectivity and viral assembly. COVID-19 is also increasingly being recognized as a vascular disease, with several studies noting prominent systemic endothelial dysfunction. The pathogenesis of endothelial dysfunction may also be linked to COVID-19-mediated metabolic and inflammatory effects. Herein, exercise will be compared to fenofibrate as a possible therapeutic strategy to bolster resilience against (and help manage recovery from) COVID-19. This paper will explore the hypothesis that exercise may be a useful adjuvant in a setting of COVID-19 management/rehabilitation due to its effects on PPARα and vascular endothelial function.


Assuntos
Infecções por Coronavirus/terapia , Terapia por Exercício/métodos , PPAR alfa/metabolismo , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus , Comorbidade , Infecções por Coronavirus/tratamento farmacológico , Citoplasma/metabolismo , Diabetes Mellitus Tipo 2/complicações , Exercício Físico , Fenofibrato/química , Humanos , Inflamação , Metabolismo dos Lipídeos , Lipoilação , Pulmão/metabolismo , Obesidade/complicações , Pandemias
9.
Front Immunol ; 11: 576622, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33117378

RESUMO

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Front Immunol ; 11: 570927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33123144

RESUMO

The emergence and rapid spread of SARS-CoV-2 in December 2019 has brought the world to a standstill. While less pathogenic than the 2002-2003 SARS-CoV, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. The identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to SARS-CoV has helped to determine pathogenesis and direct treatment strategies. The exponential increase in cases has necessitated fast and reliable testing procedures. Although RT-PCR remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. Various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. Non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. Adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. While no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the SARS-CoV-2 pandemic.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/terapia , Reservatórios de Doenças/virologia , Reposicionamento de Medicamentos/métodos , Humanos , Técnicas Imunoenzimáticas , Imunoterapia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Pneumonia Viral/terapia , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
J Phys Chem B ; 124(44): 9785-9792, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33095007

RESUMO

Over 50 peptides, which were known to inhibit SARS-CoV-1, were computationally screened against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Based on the binding affinity and interaction, 15 peptides were selected, which showed higher affinity compared to the α-helix of the human ACE2 receptor. Molecular dynamics simulation demonstrated that two peptides, S2P25 and S2P26, were the most promising candidates, which could potentially block the entry of SARS-CoV-2. Tyr489 and Tyr505 residues present in the "finger-like" projections of the RBD were found to be critical for peptide interaction. Hydrogen bonding and hydrophobic interactions played important roles in prompting peptide-protein binding and interaction. Structure-activity relationship indicated that peptides containing aromatic (Tyr and Phe), nonpolar (Pro, Gly, Leu, and Ala), and polar (Asn, Gln, and Cys) residues were the most significant contributors. These findings can facilitate the rational design of selective peptide inhibitors targeting the spike protein of SARS-CoV-2.


Assuntos
Antivirais/metabolismo , Betacoronavirus/química , Peptídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Antivirais/química , Sítios de Ligação , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Relação Estrutura-Atividade
12.
PLoS One ; 15(10): e0241172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33091066

RESUMO

The novel coronavirus 2019 (COVID-19) global pandemic has drastically affected the world economy, raised public anxiety, and placed a substantial psychological burden on the governments and healthcare professionals by affecting over 4.7 million people worldwide. As a preventive measure to minimise the risk of community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in India, a nationwide lockdown was imposed initially for 21 days to limit the movement of 1.3 billion people. These restrictions continue in most areas, with a conditional relaxation occurring in a few Indian states. In an attempt to assess the emerging mutants of SARS-CoV-2 and determine their spread in India, we analysed 112 complete genomes of SARS-CoV-2 in a time-lapse manner. We found 72 distinct SARS-CoV-2 haplotypes, defined by 143 polymorphic sites and high haplotype diversity, suggesting that this virus possesses a high evolutionary potential. We also demonstrated that early introduction of SARS-CoV-2 into India was from China, Italy and Iran and observed signs of community spread of the virus following its rapid demographic expansion since its first outbreak in the country. Additionally, we identified 18 mutations in the SARS-CoV-2 spike glycoprotein and a few selected mutations showed to increase stability, binding affinity, and molecular flexibility in the overall tertiary structure of the protein that may facilitate interaction between the receptor binding domain (RBD) of spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor. The study provides a pragmatic view of haplotype-dependent spread of SARS-CoV-2 in India which could be important in tailoring the pharmacologic treatments to be more effective for those infected with the most common haplotypes. The findings based on the time-lapse sentinel surveillance of SARS-CoV-2 will aid in the development of a real-time practical framework to tackle the ongoing, fast-evolving epidemic challenges in the country.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Vigilância de Evento Sentinela , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Genoma Viral/genética , Haplótipos , Humanos , Índia/epidemiologia , Simulação de Acoplamento Molecular , Mutação , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Polimorfismo Genético , Estrutura Terciária de Proteína , Quarentena/métodos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Commun Biol ; 3(1): 641, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110195

RESUMO

The emergence of SARS-CoV-2 has caused over a million human deaths and massive global disruption. The viral infection may also represent a threat to our closest living relatives, nonhuman primates. The contact surface of the host cell receptor, ACE2, displays amino acid residues that are critical for virus recognition, and variations at these critical residues modulate infection susceptibility. Infection studies have shown that some primate species develop COVID-19-like symptoms; however, the susceptibility of most primates is unknown. Here, we show that all apes and African and Asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ACE2. Monkeys in the Americas, and some tarsiers, lemurs and lorisoids, differ at critical contact residues, and protein modeling predicts that these differences should greatly reduce SARS-CoV-2 binding affinity. Other lemurs are predicted to be closer to catarrhines in their susceptibility. Our study suggests that apes and African and Asian monkeys, and some lemurs, are likely to be highly susceptible to SARS-CoV-2. Urgent actions have been undertaken to limit the exposure of great apes to humans, and similar efforts may be necessary for many other primate species.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/veterinária , Especificidade de Hospedeiro/genética , Pandemias/veterinária , Peptidil Dipeptidase A/genética , Pneumonia Viral/veterinária , Doenças dos Primatas/enzimologia , Primatas/genética , Receptores Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Betacoronavirus/fisiologia , Evolução Biológica , Quirópteros/genética , Sequência Conservada , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Predisposição Genética para Doença , Mamíferos/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/química , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Mutação Puntual , Doenças dos Primatas/virologia , Ligação Proteica , Conformação Proteica , Risco , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
J Phys Chem Lett ; 11(21): 9272-9281, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33085491

RESUMO

Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.


Assuntos
Betacoronavirus/metabolismo , Ligantes , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Diosmina/química , Diosmina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Plicamicina/química , Plicamicina/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Termodinâmica
15.
Cells ; 9(11)2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105869

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease (COVID-19) pandemic, has so far resulted in more than 1.1 M deaths and 40 M cases worldwide with no confirmed remedy yet available. Since the first outbreak in Wuhan, China in December 2019, researchers across the globe have been in a race to develop therapies and vaccines against the disease. SARS-CoV-2, similar to other previously identified Coronaviridae family members, encodes several structural proteins, such as spike, envelope, membrane, and nucleocapsid, that are responsible for host penetration, binding, recycling, and pathogenesis. Structural biology has been a key player in understanding the viral infection mechanism and in developing intervention strategies against the new coronavirus. The spike glycoprotein has drawn considerable attention as a means to block viral entry owing to its interactions with the human angiotensin-converting enzyme 2 (ACE2), which acts as a receptor. Here, we review the current knowledge of SARS-CoV-2 and its interactions with ACE2 and antibodies. Structural information of SARS-CoV-2 spike glycoprotein and its complexes with ACE2 and antibodies can provide key input for the development of therapies and vaccines against the new coronavirus.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Humanos , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
Sci Rep ; 10(1): 16615, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024203

RESUMO

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Assuntos
Anticorpos Antivirais/análise , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/virologia , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fatores de Transcrição NFATC/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Elementos de Resposta , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção
17.
Sci Rep ; 10(1): 16862, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033344

RESUMO

The prevalence of a novel ß-coronavirus (SARS-CoV-2) was declared as a public health emergency of international concern on 30 January 2020 and a global pandemic on 11 March 2020 by WHO. The spike glycoprotein of SARS-CoV-2 is regarded as a key target for the development of vaccines and therapeutic antibodies. In order to develop anti-viral therapeutics for SARS-CoV-2, it is crucial to find amino acid pairs that strongly attract each other at the interface of the spike glycoprotein and the human angiotensin-converting enzyme 2 (hACE2) complex. In order to find hot spot residues, the strongly attracting amino acid pairs at the protein-protein interaction (PPI) interface, we introduce a reliable inter-residue interaction energy calculation method, FMO-DFTB3/D/PCM/3D-SPIEs. In addition to the SARS-CoV-2 spike glycoprotein/hACE2 complex, the hot spot residues of SARS-CoV-1 spike glycoprotein/hACE2 complex, SARS-CoV-1 spike glycoprotein/antibody complex, and HCoV-NL63 spike glycoprotein/hACE2 complex were obtained using the same FMO method. Following this, a 3D-SPIEs-based interaction map was constructed with hot spot residues for the hACE2/SARS-CoV-1 spike glycoprotein, hACE2/HCoV-NL63 spike glycoprotein, and hACE2/SARS-CoV-2 spike glycoprotein complexes. Finally, the three 3D-SPIEs-based interaction maps were combined and analyzed to find the consensus hot spots among the three complexes. As a result of the analysis, two hot spots were identified between hACE2 and the three spike proteins. In particular, E37, K353, G354, and D355 of the hACE2 receptor strongly interact with the spike proteins of coronaviruses. The 3D-SPIEs-based map would provide valuable information to develop anti-viral therapeutics that inhibit PPIs between the spike protein of SARS-CoV-2 and hACE2.


Assuntos
Betacoronavirus/metabolismo , Biologia Computacional/métodos , Infecções por Coronavirus/epidemiologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/epidemiologia , Mapas de Interação de Proteínas , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/metabolismo , Humanos , Pandemias , Pneumonia Viral/virologia , Prevalência , Domínios Proteicos , Receptores Virais/metabolismo , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/virologia
18.
Sci Rep ; 10(1): 17538, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067518

RESUMO

The binding affinity and adhesive strength between the spike (S) glycoproteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the human angiotensin-converting enzyme 2 (ACE2) receptor is computed using molecular dynamics (MD) simulations. The calculations indicate that the binding affinity is [Formula: see text] [Formula: see text] with a maximum adhesive force of [Formula: see text] pN. Our analysis suggests that only 27 (13 in S-protein, 14 in ACE2) residues are active during the initial fusion process between the S-protein and ACE2 receptor. With these insights, we investigated the effect of possible therapeutics in the size and wrapping time of virus particles by reducing the binding energy. Our analysis indicates that this energy has to be reduced significantly, around 50% or more, to block SARS-CoV-2 particles with radius in the order of [Formula: see text] nm. Our study provides concise target residues and target binding energy reduction between S-proteins and receptors for the development of new therapeutics treatments for COVID-19 guided by computational design.


Assuntos
Infecções por Coronavirus/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/virologia , Endocitose , Humanos , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/virologia , Ligação Proteica , Eletricidade Estática , Termodinâmica
19.
Sci Rep ; 10(1): 17699, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077836

RESUMO

Angiotensin converting enzyme 2 (ACE2) (EC:3.4.17.23) is a transmembrane protein which is considered as a receptor for spike protein binding of novel coronavirus (SARS-CoV2). Since no specific medication is available to treat COVID-19, designing of new drug is important and essential. In this regard, in silico method plays an important role, as it is rapid and cost effective compared to the trial and error methods using experimental studies. Natural products are safe and easily available to treat coronavirus affected patients, in the present alarming situation. In this paper five phytochemicals, which belong to flavonoid and anthraquinone subclass, have been selected as small molecules in molecular docking study of spike protein of SARS-CoV2 with its human receptor ACE2 molecule. Their molecular binding sites on spike protein bound structure with its receptor have been analyzed. From this analysis, hesperidin, emodin and chrysin are selected as competent natural products from both Indian and Chinese medicinal plants, to treat COVID-19. Among them, the phytochemical hesperidin can bind with ACE2 protein and bound structure of ACE2 protein and spike protein of SARS-CoV2 noncompetitively. The binding sites of ACE2 protein for spike protein and hesperidin, are located in different parts of ACE2 protein. Ligand spike protein causes conformational change in three-dimensional structure of protein ACE2, which is confirmed by molecular docking and molecular dynamics studies. This compound modulates the binding energy of bound structure of ACE2 and spike protein. This result indicates that due to presence of hesperidin, the bound structure of ACE2 and spike protein fragment becomes unstable. As a result, this natural product can impart antiviral activity in SARS CoV2 infection. The antiviral activity of these five natural compounds are further experimentally validated with QSAR study.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Antraquinonas/química , Antraquinonas/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Emodina/química , Emodina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química
20.
Sci Rep ; 10(1): 17698, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077899

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 µg/g and 130 µg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Tabaco/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Folhas de Planta/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
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