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1.
Molecules ; 25(17)2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32882868

RESUMO

Over the years, coronaviruses (CoV) have posed a severe public health threat, causing an increase in mortality and morbidity rates throughout the world. The recent outbreak of a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the current Coronavirus Disease 2019 (COVID-19) pandemic that affected more than 215 countries with over 23 million cases and 800,000 deaths as of today. The situation is critical, especially with the absence of specific medicines or vaccines; hence, efforts toward the development of anti-COVID-19 medicines are being intensively undertaken. One of the potential therapeutic targets of anti-COVID-19 drugs is the angiotensin-converting enzyme 2 (ACE2). ACE2 was identified as a key functional receptor for CoV associated with COVID-19. ACE2, which is located on the surface of the host cells, binds effectively to the spike protein of CoV, thus enabling the virus to infect the epithelial cells of the host. Previous studies showed that certain flavonoids exhibit angiotensin-converting enzyme inhibition activity, which plays a crucial role in the regulation of arterial blood pressure. Thus, it is being postulated that these flavonoids might also interact with ACE2. This postulation might be of interest because these compounds also show antiviral activity in vitro. This article summarizes the natural flavonoids with potential efficacy against COVID-19 through ACE2 receptor inhibition.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Produtos Biológicos/farmacologia , Infecções por Coronavirus/virologia , Flavonoides/farmacologia , Pneumonia Viral/virologia , Inibidores da Enzima Conversora de Angiotensina/química , Antivirais/química , Produtos Biológicos/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Suscetibilidade a Doenças , Flavonoides/química , Humanos , Estágios do Ciclo de Vida , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , Vigilância da População , Relação Estrutura-Atividade
2.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867854

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
3.
mBio ; 11(4)2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820005

RESUMO

We assessed various newly generated compounds that target the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC50] = 2.8 µM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography-mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds.IMPORTANCE Targeting the main protease (Mpro) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Indóis/farmacologia , Pneumonia Viral/tratamento farmacológico , Sequência de Aminoácidos , Animais , Betacoronavirus/enzimologia , Chlorocebus aethiops , Cloroquina/farmacologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Indóis/química , Indóis/uso terapêutico , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
4.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
5.
Nat Commun ; 11(1): 3894, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753597

RESUMO

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17-29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717-29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Amiloide/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/metabolismo , Benzotiazóis , Catelicidinas/farmacologia , Cristalografia por Raios X , Gorilla gorilla , Humanos , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Staphylococcus hominis/efeitos dos fármacos , Difração de Raios X
6.
Biophys Chem ; 265: 106441, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745829

RESUMO

The possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. For both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. However, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. To predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. In this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the SARS-CoV-2 spike protein on surfaces commonly used in lateral-flow devices. These calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays.


Assuntos
Betacoronavirus/metabolismo , Epitopos/química , Modelos Moleculares , Glicoproteína da Espícula de Coronavírus/metabolismo , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Epitopos/imunologia , Simulação de Acoplamento Molecular , Domínios Proteicos , Dióxido de Silício/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Propriedades de Superfície
7.
Nat Commun ; 11(1): 3958, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769971

RESUMO

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.


Assuntos
Dimetilaliltranstransferase/metabolismo , Liases Intramoleculares/metabolismo , Alternaria/enzimologia , Domínio Catalítico , Dimetilaliltranstransferase/química , Ensaios Enzimáticos , Escherichia coli/metabolismo , Fusarium/enzimologia , Indóis/química , Indóis/metabolismo , Liases Intramoleculares/química , Cinética , Ligantes , Modelos Moleculares , Prenilação , Terpenos/metabolismo
8.
Nat Commun ; 11(1): 3969, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769976

RESUMO

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg2+-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.


Assuntos
Carboxiliases/metabolismo , Enterococcus faecalis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Carboxiliases/química , Sequência Conservada , Cristalografia por Raios X , Ligantes , Metais/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Especificidade por Substrato
9.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769995

RESUMO

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Tripsina/metabolismo
10.
PLoS Negl Trop Dis ; 14(8): e0008517, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810153

RESUMO

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.


Assuntos
Anti-Helmínticos/farmacologia , Oxamniquine/análogos & derivados , Oxamniquine/farmacologia , Schistosoma/efeitos dos fármacos , Esquistossomose/parasitologia , Animais , Anti-Helmínticos/química , Simulação por Computador , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Oxamniquine/química , Ligação Proteica
11.
Sci Transl Med ; 12(557)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32747425

RESUMO

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cristalografia por Raios X , Cisteína Endopeptidases/química , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Modelos Moleculares , Pandemias , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas , Especificidade da Espécie , Eletricidade Estática , Pesquisa Médica Translacional , Células Vero , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/química
12.
Infect Dis Poverty ; 9(1): 88, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32741372

RESUMO

BACKGROUND: An outbreak of infection caused by SARS-CoV-2 recently has brought a great challenge to public health. Rapid identification of immune epitopes would be an efficient way to screen the candidates for vaccine development at the time of pandemic. This study aimed to predict the protective epitopes with bioinformatics methods and resources for vaccine development. METHODS: The genome sequence and protein sequences of SARS-CoV-2 were retrieved from the National Center for Biotechnology Information (NCBI) database. ABCpred and BepiPred servers were utilized for sequential B-cell epitope analysis. Discontinuous B-cell epitopes were predicted via DiscoTope 2.0 program. IEDB server was utilized for HLA-1 and HLA-2 binding peptides computation. Surface accessibility, antigenicity, and other important features of forecasted epitopes were characterized for immunogen potential evaluation. RESULTS: A total of 63 sequential B-cell epitopes on spike protein were predicted and 4 peptides (Spike315-324, Spike333-338, Spike648-663, Spike1064-1079) exhibited high antigenicity score and good surface accessibility. Ten residues within spike protein (Gly496, Glu498, Pro499, Thr500, Leu1141, Gln1142, Pro1143, Glu1144, Leu1145, Asp1146) are forecasted as components of discontinuous B-cell epitopes. The bioinformatics analysis of HLA binding peptides within nucleocapsid protein produced 81 and 64 peptides being able to bind MHC class I and MHC class II molecules respectively. The peptides (Nucleocapsid66-75, Nucleocapsid104-112) were predicted to bind a wide spectrum of both HLA-1 and HLA-2 molecules. CONCLUSIONS: B-cell epitopes on spike protein and T-cell epitopes within nucleocapsid protein were identified and recommended for developing a protective vaccine against SARS-CoV-2.


Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Biologia Computacional/métodos , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Sequência de Aminoácidos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Humanos , Imunogenicidade da Vacina/imunologia , Modelos Moleculares , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Alinhamento de Sequência , Análise de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas do Envelope Viral/imunologia
13.
Structure ; 28(8): 874-878, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32755569

RESUMO

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.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Proteínas Virais/química , Cisteína Endopeptidases/química , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Biologia Molecular , Conformação Proteica , RNA Replicase/química , Glicoproteína da Espícula de Coronavírus/química , Proteínas não Estruturais Virais/química
14.
Int J Mol Sci ; 21(15)2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731361

RESUMO

Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.


Assuntos
Betacoronavirus/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Betacoronavirus/química , Domínio Catalítico , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica
15.
J Comput Chem ; 41(24): 2158-2161, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32779780

RESUMO

D614G spike glycoprotein (sgp) mutation in rapidly spreading severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) is associated with enhanced fitness and higher transmissibility in new cases of COVID-19 but the underlying mechanism is unknown. Here, using atomistic simulation, a plausible mechanism has been delineated. In G614 sgp but not wild type, increased D(G)614-T859 Cα-distance within 65 ns is interpreted as S1/S2 protomer dissociation. Overall, ACE2-binding, post-fusion core, open-state and sub-optimal antibody-binding conformations were preferentially sampled by the G614 mutant, but not wild type. Furthermore, in the wild type, only one of the three sgp chains has optimal communication route between residue 614 and the receptor-binding domain (RBD); whereas, two of the three chains communicated directly in G614 mutant. These data provide evidence that D614G sgp mutant is more available for receptor binding, cellular invasion and reduced antibody interaction; thus, providing framework for enhanced fitness and higher transmissibility in D614G SARS-COV-2 mutant.


Assuntos
Betacoronavirus/metabolismo , Simulação por Computador , Infecções por Coronavirus/virologia , Modelos Químicos , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares , Mutação , Pandemias , Ligação Proteica , Conformação Proteica , Domínios Proteicos
16.
PLoS One ; 15(8): e0237300, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785274

RESUMO

The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H+ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct "core" structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Betacoronavirus/isolamento & purificação , Simulação por Computador , Infecções por Coronavirus/virologia , Humanos , Hidrogênio , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pneumonia Viral/virologia , Mutação Puntual , Conformação Proteica , Homologia Estrutural de Proteína , Vacinas Atenuadas , Vacinas de Produtos Inativados , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Água/química
17.
Viruses ; 12(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32825063

RESUMO

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.


Assuntos
Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Amino Açúcares/metabolismo , Betacoronavirus/fisiologia , Sítios de Ligação , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/química , Antígeno Sialil Lewis X/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Internalização do Vírus
18.
Phys Rev Lett ; 125(3): 033604, 2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32745420

RESUMO

We demonstrate Bragg diffraction of the antibiotic ciprofloxacin and the dye molecule phthalocyanine at a thick optical grating. The observed patterns show a single dominant diffraction order with the expected dependence on the incidence angle as well as oscillating population transfer between the undiffracted and diffracted beams. We achieve an equal-amplitude splitting of 14ℏk (photon momenta) and maximum momentum transfer of 18ℏk. This paves the way for efficient, large-momentum beam splitters and mirrors for hot and complex molecules.


Assuntos
Ciprofloxacino/química , Indóis/química , Modelos Químicos , Antibacterianos/química , Interferometria/métodos , Modelos Moleculares , Pigmentos Biológicos/química , Espalhamento de Radiação
19.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
20.
Nat Commun ; 11(1): 4169, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820172

RESUMO

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.


Assuntos
Temperatura Alta , Ativação do Canal Iônico/fisiologia , Canais de Cátion TRPV/metabolismo , Sensação Térmica/fisiologia , Sítios de Ligação/genética , Capsaicina/química , Capsaicina/metabolismo , Dicroísmo Circular , Humanos , Ativação do Canal Iônico/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética , Sensação Térmica/genética
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