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1.
Sci Adv ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523846

RESUMO

Here, we report the topology-matched design of heteromultivalent nanostructures as potent and broad-spectrum virus entry inhibitors based on the host cell membrane. Initially, we investigate the virus binding dynamics to validate the better binding performance of the heteromultivalent moieties as compared to homomultivalent ones. The heteromultivalent binding moieties are transferred to nanostructures with a bowl-like shape matching the viral spherical surface. Unlike the conventional homomultivalent inhibitors, the heteromultivalent ones exhibit a half maximal inhibitory concentration of 32.4 ± 13.7 µg/ml due to the synergistic multivalent effects and the topology-matched shape. At a dose without causing cellular toxicity, >99.99% reduction of virus propagation has been achieved. Since multiple binding sites have also been identified on the S protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), we envision that the use of heteromultivalent nanostructures may also be applied to develop a potent inhibitor to prevent coronavirus infection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/virologia , Nanopartículas/química , Neuraminidase/química , Animais , Antivirais/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Cães , Membrana Eritrocítica/virologia , Humanos , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Ligação Proteica , Glicoproteína da Espícula de Coronavírus , Vírion , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
2.
J Vet Sci ; 22(1): e12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33522164

RESUMO

BACKGROUND: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). OBJECTIVES: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor. METHODS: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. RESULTS: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%-67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. CONCLUSIONS: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.


Assuntos
Quirópteros/virologia , Coronavirus/genética , Vírus da SARS/genética , /genética , Animais , Genes Virais/genética , Genoma Viral/genética , Genômica , Humanos , Funções Verossimilhança , Filogenia , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , República da Coreia , Análise de Sequência de DNA , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Ligação Viral
3.
Arch Virol ; 166(3): 697-714, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33483791

RESUMO

Coronaviruses are the paradigm of emerging 21st century zoonotic viruses, triggering numerous outbreaks and a severe global health crisis. The current COVID-19 pandemic caused by SARS-CoV-2 has affected more than 51 million people across the globe as of 12 November 2020. The crown-like spikes on the surface of the virion are the unique structural feature of viruses in the family Coronaviridae. The spike (S) protein adopts distinct conformations while mediating entry of the virus into the host. This multifunctional protein mediates the entry process by recognizing its receptor on the host cell, followed by the fusion of the viral membrane with the host cell membrane. This review article focuses on the structural and functional comparison of S proteins of the human betacoronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we review the current state of knowledge about receptor recognition, the membrane fusion mechanism, structural epitopes, and glycosylation sites of the S proteins of these viruses. We further discuss various vaccines and other therapeutics such as monoclonal antibodies, peptides, and small molecules based on the S protein of these three viruses.


Assuntos
/transmissão , Coronavírus da Síndrome Respiratória do Oriente Médio/ultraestrutura , Vírus da SARS/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , /metabolismo , Antivirais/uso terapêutico , /patologia , Cristalografia por Raios X , Glicosilação , Humanos , Conformação Proteica , Internalização do Vírus
4.
Future Microbiol ; 16: 107-118, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33459559

RESUMO

Viruses have caused the death of millions of people worldwide. Specifically, human viruses are grouped into 21 families, including the family of coronaviruses (CoVs). In December 2019, in Wuhan, China, a new human CoV was identified, SARS-CoV-2. The first step of the infection mechanism of the SARS-CoV-2 in the human host is adhesion, which occurs through the S glycoprotein that is found in diverse human organs. Another way through which SARS-CoV-2 could possibly attach to the host's cells is by means of the histo-blood group antigens. In this work, we have reviewed the mechanisms by which some viruses bind to the histo-blood group antigens, which could be related to the susceptibility of the individual and are dependent on the histo-blood group.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , Animais , Quirópteros/virologia , Suscetibilidade a Doenças/sangue , Genoma Viral/genética , Glicoproteínas/metabolismo , Humanos , /genética
5.
Nat Commun ; 12(1): 134, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420022

RESUMO

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.


Assuntos
/metabolismo , /transmissão , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , Animais , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Furina/metabolismo , Células HEK293 , Heparitina Sulfato/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/virologia , Pulmão/patologia , Pulmão/virologia , Síndrome Respiratória Aguda Grave/patologia , Células Vero , Internalização do Vírus , Replicação Viral/fisiologia
6.
PLoS Biol ; 18(12): e3001016, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33347434

RESUMO

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.


Assuntos
/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Ligação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Gatos , Bovinos , Cães , Cobaias , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Coelhos , Ratos , /virologia
7.
Mar Drugs ; 18(12)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327522

RESUMO

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment; first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.


Assuntos
Organismos Aquáticos/metabolismo , Produtos Biológicos/farmacologia , Polifosfatos/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Células A549 , Bactérias/metabolismo , Produtos Biológicos/uso terapêutico , Humanos , Imunidade Inata/efeitos dos fármacos , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Polifosfatos/metabolismo , Polifosfatos/uso terapêutico , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , /patogenicidade , Metabolismo Secundário , Ligação Viral/efeitos dos fármacos
8.
mBio ; 11(6)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33310780

RESUMO

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T 1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.


Assuntos
/química , Peptídeos/farmacologia , /fisiologia , Ligação Viral/efeitos dos fármacos , Células A549 , Animais , Sítios de Ligação , Chlorocebus aethiops , Humanos , Concentração Inibidora 50 , Peptídeos/síntese química , Ligação Proteica , Células Vero
9.
Nat Commun ; 11(1): 5752, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188207

RESUMO

Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, with SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with FcγRIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity is eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Potent prophylactic and therapeutic effects against SARS-CoV-2 are observed in rhesus monkeys. A single dose of MW05/LALA blocks infection of SARS-CoV-2 in prophylactic treatment and clears SARS-CoV-2 in three days in a therapeutic treatment setting. These results pave the way for the development of MW05/LALA as an antiviral strategy for COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/prevenção & controle , Receptores de IgG/genética , Receptores de IgG/imunologia , Receptores Virais/metabolismo , Células Vero , Ligação Viral
11.
Elife ; 92020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33164751

RESUMO

Pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 19 disease (COVID-19) which presents a large spectrum of manifestations with fatal outcomes in vulnerable people over 70-years-old and with hypertension, diabetes, obesity, cardiovascular disease, COPD, and smoking status. Knowledge of the entry receptor is key to understand SARS-CoV-2 tropism, transmission and pathogenesis. Early evidence pointed to angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-2 entry receptor. Here, we provide a critical summary of the current knowledge highlighting the limitations and remaining gaps that need to be addressed to fully characterize ACE2 function in SARS-CoV-2 infection and associated pathogenesis. We also discuss ACE2 expression and potential role in the context of comorbidities associated with poor COVID-19 outcomes. Finally, we discuss the potential co-receptors/attachment factors such as neuropilins, heparan sulfate and sialic acids and the putative alternative receptors, such as CD147 and GRP78.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/virologia , Ligação Viral , Basigina/fisiologia , Comorbidade , Infecções por Coronavirus/epidemiologia , Regulação Enzimológica da Expressão Gênica , Heparitina Sulfato/fisiologia , Humanos , Hipertensão/epidemiologia , Hipertensão/fisiopatologia , Neuropilina-1/fisiologia , Oligopeptídeos/fisiologia , Especificidade de Órgãos , Pandemias , Pneumonia Viral/epidemiologia , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Virais , Sistema Renina-Angiotensina/fisiologia , Sistema Respiratório/enzimologia , Ácidos Siálicos/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Internalização do Vírus
12.
Emerg Microbes Infect ; 9(1): 2433-2445, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33073694

RESUMO

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2) [1-3]. Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in the nasal turbinates, lung and brain, with high lethality, and cytokine/chemokine production. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the nasal turbinates and lung, and no clinical signs of infection. The K18-hACE2 model provides a stringent model for testing vaccines and antivirals, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/patologia , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Vírus da SARS/crescimento & desenvolvimento , Replicação Viral/genética , Células A549 , Adenoviridae/genética , Animais , Betacoronavirus/metabolismo , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias , Vírus da SARS/metabolismo , Células Vero , Ligação Viral
13.
PLoS One ; 15(10): e0240647, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112891

RESUMO

The World Health Organization declared the COVID-19 epidemic a public health emergency of international concern on March 11th, 2020, and the pandemic is rapidly spreading worldwide. COVID-19 is caused by a novel coronavirus SARS-CoV-2, which enters human target cells via angiotensin converting enzyme 2 (ACE2). We used a number of bioinformatics tools to computationally characterize ACE2 by determining its cell-specific expression in trachea, lung, and small intestine, derive its putative functions, and predict transcriptional regulation. The small intestine expressed higher levels of ACE2 mRNA than any other organ. By immunohistochemistry, duodenum, kidney and testis showed strong signals, whereas the signal was weak in the respiratory tract. Single cell RNA-Seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ACE2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. Gene ontology analysis suggested that, besides its classical role in the renin-angiotensin system, ACE2 may be functionally associated with angiogenesis/blood vessel morphogenesis. Using a novel tool for the prediction of transcription factor binding sites we identified several putative binding sites within two tissue-specific promoters of the ACE2 gene as well as a new putative short form of ACE2. These include several interferon-stimulated response elements sites for STAT1, IRF8, and IRF9. Our results also confirmed that age and gender play no significant role in the regulation of ACE2 mRNA expression in the lung.


Assuntos
Betacoronavirus/fisiologia , Biologia Computacional , Infecções por Coronavirus/virologia , Pandemias , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/virologia , Receptores Virais/fisiologia , Envelhecimento/metabolismo , Sítios de Ligação , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Ontologia Genética , Humanos , Interferons/fisiologia , Pulmão/metabolismo , Masculino , Metaloproteases/biossíntese , Metaloproteases/genética , Neovascularização Fisiológica/fisiologia , Especificidade de Órgãos , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Receptores Virais/biossíntese , Receptores Virais/genética , Sistema Renina-Angiotensina/fisiologia , Caracteres Sexuais , Análise de Célula Única , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Ligação Viral
14.
Int J Antimicrob Agents ; 56(2): 106020, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32862840

RESUMO

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.


Assuntos
Azitromicina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Gangliosídeo G(M1)/metabolismo , Hidroxicloroquina/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Alinhamento de Sequência
15.
Virus Res ; 289: 198146, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866534

RESUMO

The rapid emergence of novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2), originated from Wuhan, China, imposed a global health emergency. Angiotensin-converting enzyme 2 (ACE2) receptor serves as an entry point for this deadly virus while the proteases like furin, transmembrane protease serine 2 (TMPRSS2) and 3 chymotrypsin-like protease (3CLpro) are involved in the further processing and replication of SARS-CoV-2. The interaction of SP with ACE2 and these proteases results in the SARS-CoV-2 invasion and fast epidemic spread. The small molecular inhibitors are reported to limit the interaction of SP with ACE2 and other proteases. Arbidol, a membrane fusion inhibitor approved for influenza virus is currently undergoing clinical trials against COVID-19. In this context, we report some analogues of arbidol designed by scaffold morphing and structure-based designing approaches with a superior therapeutic profile. The representative compounds A_BR4, A_BR9, A_BR18, A_BR22 and A_BR28 restricted the interaction of SARS-CoV-2 SP with ACE2 and host proteases furin and TMPRSS2. For 3CLPro, Compounds A_BR5, A_BR6, A_BR9 and A_BR18 exhibited high binding affinity, docking score and key residue interactions. Overall, A_BR18 and A_BR28 demonstrated multi-targeting potential against all the targets. Among these top-scoring molecules A_BR9, A_BR18, A_BR22 and A_BR28 were predicted to confer favorable ADME properties.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Indóis/química , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Receptores Virais/efeitos dos fármacos , Ligação Viral/efeitos dos fármacos , Algoritmos , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/fisiologia , Disponibilidade Biológica , Desenho de Fármacos , Humanos , Indóis/metabolismo , Indóis/farmacologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/fisiologia , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade , Internalização do Vírus , Replicação Viral
16.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32994342

RESUMO

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de Vírus
17.
Molecules ; 25(19)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977525

RESUMO

The problem of treating viral infections is extremely relevant due to both the emergence of new viral diseases and to the low effectiveness of existing approaches to the treatment of known viral infections. This review focuses on the application of porphyrin, chlorin, and phthalocyanine series for combating viral infections by chemical and photochemical inactivation methods. The purpose of this review paper is to summarize the main approaches developed to date in the chemical and photodynamic inactivation of human and animal viruses using porphyrins and their analogues and to analyze and discuss the information on viral targets and antiviral activity of porphyrins, chlorins, of their conjugates with organic/inorganic compounds obtained in the last 10-15 years in order to identify the most promising areas.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Fotoquimioterapia/métodos , Pneumonia Viral/tratamento farmacológico , Porfirinas/farmacologia , Antivirais/química , Humanos , Indóis/química , Indóis/farmacologia , Pandemias , Processos Fotoquímicos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Ligação Viral/efeitos dos fármacos
18.
Nat Commun ; 11(1): 4541, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917884

RESUMO

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , Internalização do Vírus , Células A549 , Betacoronavirus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/metabolismo , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/química
19.
Virus Res ; 289: 198154, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918944

RESUMO

Recent reports have shown that small and big felines could be infected by SARS-CoV-2, while other animals, like swines and mice, are apparently not susceptible to this infection. These findings raise the question of the role of cell factors associated with early stages of the viral infection in host selectivity. The cellular receptor for SARS-CoV-2 is the Angiotensin Converting Enzyme (ACE2). Transmembrane protease serine 2 (TMPRSS2) has been shown to prime the viral spike for its interaction with its receptor. GRP78 has also been proposed as a possible co-receptor. In this study, we used several bioinformatics approaches to bring clues in the interaction of ACE2, TMPRSS2, and GRP78 with SARS-CoV-2. We selected several mammalian hosts that could play a key role in viral spread by acting as secondary hosts (cats, dogs, pigs, mice, and ferrets) and evaluated their predicted permissiveness by in silico analysis. Results showed that ionic pairs (salt bridges, N-O pair, and long-range interactions) produced between ACE2 and the viral spike has an essential function in the host interaction. On the other hand, TMPRSS2 and GRP78 are proteins with high homology in all the evaluated hosts. Thus, these proteins do not seem to play a role in host selectivity, suggesting that other factors may play a role in the non-permissivity in some of these hosts. These proteins represent however interesting cell targets that could be explored in order to control the virus replication in humans and in the intermediary hosts.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Proteínas de Choque Térmico/química , Mamíferos/metabolismo , Peptidil Dipeptidase A/química , Pneumonia Viral/virologia , Receptores Virais/química , Serina Endopeptidases/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Gatos , Cães , Furões , Guanidinas/farmacologia , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Receptores Virais/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Especificidade da Espécie , Suínos , Ligação Viral , Internalização do Vírus
20.
PLoS Negl Trop Dis ; 14(9): e0008602, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886656

RESUMO

Besides the common Fc receptor (FcR)-mediated mechanism of antibody-dependent enhancement (ADE), Ebola virus (EBOV) is known to utilize the complement component C1q for ADE of infection. This mechanism is FcR-independent and mediated by cross-linking of virus-antibody-C1q complexes to cell surface C1q receptors, leading to enhanced viral entry into cells. Using confocal microscopy, we found that virus-like particles (VLPs) consisting of EBOV glycoprotein, nucleoprotein, and matrix protein attached to the surface of human kidney 293 cells more efficiently in the presence of an ADE monoclonal antibody and C1q than with the antibody or C1q alone, and that there was no significant difference in the efficiency of VLP uptake into endosomes between the C1q-mediated ADE and non-ADE entry. Accordingly, both ADE and non-ADE infection were similarly decreased by inhibitors of the signaling pathways known to be required for endocytosis. These results suggest that C1q-mediated ADE of EBOV infection is simply caused by increased attachment of virus particles to the cell surface, which is distinct from the mechanism of FcR-mediated ADE requiring intracellular signaling to promote phagocytosis/macropinocytosis.


Assuntos
Anticorpos Facilitadores/imunologia , Complemento C1q/metabolismo , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Ebolavirus/patogenicidade , Endocitose/imunologia , Células HEK293 , Doença pelo Vírus Ebola/patologia , Humanos , Células Vero , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Ligação Viral
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