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1.
PLoS One ; 15(10): e0240004, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002032

RESUMO

The SARS-CoV-2 virus has caused a pandemic and is public health emergency of international concern. As of now, no registered therapies are available for treatment of coronavirus infection. The viral infection depends on the attachment of spike (S) glycoprotein to human cell receptor angiotensin-converting enzyme 2 (ACE2). We have designed a protein inhibitor (ΔABP-D25Y) targeting S protein using computational approach. The inhibitor consists of two α helical peptides homologues to protease domain (PD) of ACE2. Docking studies and molecular dynamic simulation revealed that the inhibitor binds exclusively at the ACE2 binding site of S protein. The computed binding affinity of the inhibitor is higher than the ACE2 and thus will likely out compete ACE2 for binding to S protein. Hence, the proposed inhibitor ΔABP-D25Y could be a potential blocker of S protein and receptor binding domain (RBD) attachment.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Desenho de Fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Homologia Estrutural de Proteína , Sítios de Ligação , Simulação por Computador , Infecções por Coronavirus , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral , Domínios Proteicos
2.
Nat Commun ; 11(1): 4578, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929090

RESUMO

Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of ß-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.


Assuntos
Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Estereoisomerismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Talidomida/química , Talidomida/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
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
4.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776918

RESUMO

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Uridina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Uridina/metabolismo
5.
J Mol Graph Model ; 100: 107710, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32829149

RESUMO

The emergence of SARS-CoV-2 has prompted a worldwide health emergency. There is an urgent need for therapeutics, both through the repurposing of approved drugs and the development of new treatments. In addition to the viral drug targets, a number of human drug targets have been suggested. In theory, targeting human proteins should provide an advantage over targeting viral proteins in terms of drug resistance, which is commonly a problem in treating RNA viruses. This paper focuses on the human protein TMPRSS2, which supports coronavirus life cycles by cleaving viral spike proteins. The three-dimensional structure of TMPRSS2 is not known and so we have generated models of the TMPRSS2 in the apo state as well as in complex with a peptide substrate and putative inhibitors to aid future work. Importantly, many related human proteases have 80% or higher identity with TMPRSS2 in the S1-S1' subsites, with plasminogen and urokinase-type plasminogen activator (uPA) having 95% identity. We highlight 376 approved, investigational or experimental drugs targeting S1A serine proteases that may also inhibit TMPRSS2. Whilst the presence of a relatively uncommon lysine residue in the S2/S3 subsites means that some serine protease inhibitors will not inhibit TMPRSS2, this residue is likely to provide a handle for selective targeting in a focused drug discovery project. We discuss how experimental drugs targeting related serine proteases might be repurposed as TMPRSS2 inhibitors to treat coronaviruses.


Assuntos
Antivirais/química , Betacoronavirus/química , Inibidores de Proteases/química , Serina Endopeptidases/química , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Simulação de Dinâmica Molecular , Pandemias , Plasminogênio/antagonistas & inibidores , Plasminogênio/química , Plasminogênio/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
6.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32647346

RESUMO

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Peptidil Dipeptidase A/química , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Quirópteros/virologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Evolução Molecular , Furina/química , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
7.
Biophys Chem ; 264: 106420, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32622243

RESUMO

One notable feature of the SARS-CoV-2 genome, the spike (S) protein of SARS-CoV-2 has a polybasic furin cleavage site (FCS) at its S1-S2 boundary through the insertion of 12 nucleotides encoding four amino acid residues PRRA. Quite intriguingly, this polybasic FCS is absent in coronaviruses of the same clade as SARS-CoV-2. Thus, with currently available experimental structural data for S protein, this short article presents a set of comprehensive structural characterization of the insertion of FCS into S protein, and argues against a hypothesis of the origin of SARS-CoV-2 from purposeful manipulation: (1), the inserted FCS is spatially located at a random coil loop region, mostly distantly solvent-exposed (instead of deeply buried), with no structural proximity to the other part of the S protein; (2), the insertion of FCS itself does not alter, neither stabilize nor de-stabilize, the three-dimensional structure of S; (3), the net result here is the insertion of a furin cleavage site into S protein, whose S1 and S2 subunits will still be strongly electrostatically bonded together from a structural and biophysical point of view, even if the polybasic FCS is actually cleaved by furin protease before or after viral cell entry.


Assuntos
Betacoronavirus/química , Furina/química , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Betacoronavirus/genética , Expressão Gênica , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
8.
Nat Commun ; 11(1): 3422, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647223

RESUMO

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Autoantígeno Ku/química , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Homologia Estrutural de Proteína
9.
Mol Cell ; 79(3): 406-415.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692975

RESUMO

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.


Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
10.
PLoS Biol ; 18(7): e3000755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644996

RESUMO

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
11.
Mol Cell ; 79(3): 390-405.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32619402

RESUMO

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.


Assuntos
Moléculas de Adesão Celular/química , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores da Família Eph/antagonistas & inibidores , Receptores da Família Eph/química , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato
12.
Molecules ; 25(11)2020 May 29.
Artigo em Inglês | MEDLINE | ID: covidwho-436971

RESUMO

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.


Assuntos
Anticoagulantes/química , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Hepacivirus/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/química , Trombina/genética , Trombina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
13.
J Allergy Clin Immunol ; 146(2): 330-331, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593491
14.
Virus Res ; 286: 198058, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32531235

RESUMO

The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.


Assuntos
Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Peptidil Dipeptidase A/química , Receptores Virais/química , Vírus da SARS/química , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Quirópteros , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Evolução Molecular , Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Vírus da SARS/genética , Vírus da SARS/metabolismo , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína , Termodinâmica , Ligação Viral
15.
Interdiscip Sci ; 12(3): 368-376, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32488835

RESUMO

A novel coronavirus, called 2019-nCoV, was recently found in Wuhan, Hubei Province of China, and now is spreading across China and other parts of the world. Although there are some drugs to treat 2019-nCoV, there is no proper scientific evidence about its activity on the virus. It is of high significance to develop a drug that can combat the virus effectively to save valuable human lives. It usually takes a much longer time to develop a drug using traditional methods. For 2019-nCoV, it is now better to rely on some alternative methods such as deep learning to develop drugs that can combat such a disease effectively since 2019-nCoV is highly homologous to SARS-CoV. In the present work, we first collected virus RNA sequences of 18 patients reported to have 2019-nCoV from the public domain database, translated the RNA into protein sequences, and performed multiple sequence alignment. After a careful literature survey and sequence analysis, 3C-like protease is considered to be a major therapeutic target and we built a protein 3D model of 3C-like protease using homology modeling. Relying on the structural model, we used a pipeline to perform large scale virtual screening by using a deep learning based method to accurately rank/identify protein-ligand interacting pairs developed recently in our group. Our model identified potential drugs for 2019-nCoV 3C-like protease by performing drug screening against four chemical compound databases (Chimdiv, Targetmol-Approved_Drug_Library, Targetmol-Natural_Compound_Library, and Targetmol-Bioactive_Compound_Library) and a database of tripeptides. Through this paper, we provided the list of possible chemical ligands (Meglumine, Vidarabine, Adenosine, D-Sorbitol, D-Mannitol, Sodium_gluconate, Ganciclovir and Chlorobutanol) and peptide drugs (combination of isoleucine, lysine and proline) from the databases to guide the experimental scientists and validate the molecules which can combat the virus in a shorter time.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Aprendizado Profundo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Domínio Catalítico , Infecções por Coronavirus/epidemiologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Produtos Farmacêuticos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Pneumonia Viral/epidemiologia , Alinhamento de Sequência , Homologia Estrutural de Proteína , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
16.
Molecules ; 25(11)2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32485894

RESUMO

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.


Assuntos
Anticoagulantes/química , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Hepacivirus/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/química , Trombina/genética , Trombina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
17.
Biomed Res Int ; 2020: 4389089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32596311

RESUMO

The Coronavirus Disease 2019 (COVID-19) is a new viral infection caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2). Genomic analyses have revealed that SARS-CoV-2 is related to Pangolin and Bat coronaviruses. In this report, a structural comparison between the Sars-CoV-2 Envelope and Membrane proteins from different human isolates with homologous proteins from closely related viruses is described. The analyses here reported show the high structural similarity of Envelope and Membrane proteins to the counterparts from Pangolin and Bat coronavirus isolates. However, the comparisons have also highlighted structural differences specific of Sars-CoV-2 proteins which may be correlated to the cross-species transmission and/or to the properties of the virus. Structural modelling has been applied to map the variant sites onto the predicted three-dimensional structure of the Envelope and Membrane proteins.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Alphacoronavirus/química , Alphacoronavirus/classificação , Alphacoronavirus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Quirópteros/virologia , Coronaviridae/química , Coronaviridae/classificação , Coronaviridae/genética , Eutérios/virologia , Humanos , Modelos Moleculares , Pandemias , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Homologia Estrutural de Proteína , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética
18.
Infect Genet Evol ; 84: 104389, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32502733

RESUMO

The newly identified SARS-CoV-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. Spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment. In this study, 483 unique variations have been identified among the genomes of SARS-CoV-2 including 25 nonsynonymous mutations and one deletion in the spike (S) protein. Among the 26 variations detected in S, 12 variations were located at the N-terminal domain (NTD) and 6 variations at the receptor-binding domain (RBD) which might alter the interaction of S protein with the host receptor angiotensin-converting enzyme 2 (ACE2). Besides, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on results reported herein, potential inhibitors against S protein can be designed by considering these variations and their impact on protein structure.


Assuntos
Alphacoronavirus/genética , Betacoronavirus/genética , Genoma Viral , Peptidil Dipeptidase A/química , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/química , Alphacoronavirus/classificação , Alphacoronavirus/metabolismo , Animais , Sequência de Bases , Betacoronavirus/classificação , Betacoronavirus/metabolismo , Sítios de Ligação , Quirópteros/virologia , Expressão Gênica , Humanos , Modelos Moleculares , Mutação , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/classificação , Vírus da SARS/metabolismo , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína , Ligação Viral
19.
Nat Commun ; 11(1): 2450, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415073

RESUMO

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Polissacarídeos/metabolismo , Acetilação , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Domínio Catalítico , Sequência Conservada , Regulação Fúngica da Expressão Gênica , Glicosaminoglicanos/biossíntese , Metais/metabolismo , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Tempo
20.
PLoS One ; 15(5): e0233866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470085

RESUMO

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.


Assuntos
Vacinas contra a AIDS/imunologia , Edição de Genes , Proteólise , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , Cricetulus , Fator VIII/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Trombina/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
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