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1.
PLoS Comput Biol ; 16(12): e1008461, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33290397

RESUMO

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through 'repurposing' drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.


Assuntos
Catepsina B/química , Catepsina L/química , Serina Endopeptidases/química , Internalização do Vírus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Reposicionamento de Medicamentos , Humanos , Modelos Teóricos , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírion
2.
Molecules ; 25(21)2020 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-33137894

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), which caused novel corona virus disease-2019 (COVID-19) pandemic, necessitated a global demand for studies related to genes and enzymes of SARS-CoV2. SARS-CoV2 infection depends on the host cell Angiotensin-Converting Enzyme-2 (ACE2) and Transmembrane Serine Protease-2 (TMPRSS2), where the virus uses ACE2 for entry and TMPRSS2 for S protein priming. The TMPRSS2 gene encodes a Transmembrane Protease Serine-2 protein (TMPS2) that belongs to the serine protease family. There is no crystal structure available for TMPS2, therefore, a homology model was required to establish a putative 3D structure for the enzyme. A homology model was constructed using SWISS-MODEL and evaluations were performed through Ramachandran plots, Verify 3D and Protein Statistical Analysis (ProSA). Molecular dynamics simulations were employed to investigate the stability of the constructed model. Docking of TMPS2 inhibitors, camostat, nafamostat, gabexate, and sivelestat, using Molecular Operating Environment (MOE) software, into the constructed model was performed and the protein-ligand complexes were subjected to MD simulations and computational binding affinity calculations. These in silico studies determined the tertiary structure of TMPS2 amino acid sequence and predicted how ligands bind to the model, which is important for drug development for the prevention and treatment of COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Serina Endopeptidases/química , Inibidores de Serino Proteinase/farmacologia , Antivirais/química , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Gabexato/análogos & derivados , Gabexato/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Guanidinas/farmacologia , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Sulfonamidas/farmacologia
3.
Sci Rep ; 10(1): 20584, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239694

RESUMO

Plants are endowed with a large pool of structurally diverse small molecules known as secondary metabolites. The present study aims to virtually screen these plant secondary metabolites (PSM) for their possible anti-SARS-CoV-2 properties targeting four proteins/ enzymes which govern viral pathogenesis. Results of molecular docking with 4,704 ligands against four target proteins, and data analysis revealed a unique pattern of structurally similar PSM interacting with the target proteins. Among the top-ranked PSM which recorded lower binding energy (BE), > 50% were triterpenoids which interacted strongly with viral spike protein-receptor binding domain, > 32% molecules which showed better interaction with the active site of human transmembrane serine protease were belongs to flavonoids and their glycosides, > 16% of flavonol glycosides and > 16% anthocyanidins recorded lower BE against active site of viral main protease and > 13% flavonol glycoside strongly interacted with active site of viral RNA-dependent RNA polymerase. The primary concern about these PSM is their bioavailability. However, several PSM recorded higher bioavailability score and found fulfilling most of the drug-likeness characters as per Lipinski's rule (Coagulin K, Kamalachalcone C, Ginkgetin, Isoginkgetin, 3,3'-Biplumbagin, Chrysophanein, Aromoline, etc.). Natural occurrence, bio-transformation, bioavailability of selected PSM and their interaction with the target site of selected proteins were discussed in detail. Present study provides a platform for researchers to explore the possible use of selected PSM to prevent/ cure the COVID-19 by subjecting them for thorough in vitro and in vivo evaluation for the capabilities to interfering with the process of viral host cell recognition, entry and replication.


Assuntos
Antivirais/química , Simulação por Computador , Extratos Vegetais/química , Plantas/metabolismo , Metabolismo Secundário , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos/métodos , Flavonoides/química , Humanos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Plantas/química , Ligação Proteica , /enzimologia , Serina Endopeptidases/química , Glicoproteína da Espícula de Coronavírus/química
4.
Nat Commun ; 11(1): 6063, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247098

RESUMO

Opportunistic pathogens such as Streptococcus pneumoniae secrete a giant metalloprotease virulence factor responsible for cleaving host IgA1, yet the molecular mechanism has remained unknown since their discovery nearly 30 years ago despite the potential for developing vaccines that target these enzymes to block infection. Here we show through a series of cryo-electron microscopy single particle reconstructions how the Streptococcus pneumoniae IgA1 protease facilitates IgA1 substrate recognition and how this can be inhibited. Specifically, the Streptococcus pneumoniae IgA1 protease subscribes to an active-site-gated mechanism where a domain undergoes a 10.0 Å movement to facilitate cleavage. Monoclonal antibody binding inhibits this conformational change, providing a direct means to block infection at the host interface. These structural studies explain decades of biological and biochemical studies and provides a general strategy to block Streptococcus pneumoniae IgA1 protease activity to potentially prevent infection.


Assuntos
Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Streptococcus pneumoniae/enzimologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/ultraestrutura , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Modelos Moleculares , Ligação Proteica , Serina Endopeptidases/química , Serina Endopeptidases/ultraestrutura
5.
Molecules ; 25(20)2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33050360

RESUMO

The current pandemic, caused by SARS-CoV-2 virus, is a severe challenge for human health and the world economy. There is an urgent need for development of drugs that can manage this pandemic, as it has already infected 19 million people and led to the death of around 711,277 people worldwide. At this time, in-silico studies are providing lots of preliminary data about potential drugs, which can be a great help in further in-vitro and in-vivo studies. Here, we have selected three polyphenolic compounds, mangiferin, glucogallin, and phlorizin. These compounds are isolated from different natural sources but share structural similarities and have been reported for their antiviral activity. The objective of this study is to analyze and predict the anti-protease activity of these compounds on SARS-CoV-2main protease (Mpro) and TMPRSS2 protein. Both the viral protein and the host protein play an important role in the viral life cycle, such as post-translational modification and viral spike protein priming. This study has been performed by molecular docking of the compounds using PyRx with AutoDock Vina on the two aforementioned targets chosen for this study, i.e., SARS-CoV-2 Mpro and TMPRSS2. The compounds showed good binding affinity and are further analyzed by (Molecular dynamic) MD and Molecular Mechanics Poisson-Boltzmann Surface Area MM-PBSA study. The MD-simulation study has predicted that these natural compounds will have a great impact on the stabilization of the binding cavity of the Mpro of SARS-CoV-2. The predicted pharmacokinetic parameters also show that these compounds are expected to have good solubility and absorption properties. Further predictions for these compounds also showed no involvement in drug-drug interaction and no toxicity.


Assuntos
Betacoronavirus/isolamento & purificação , Produtos Biológicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Pneumonia Viral/tratamento farmacológico , Polifenóis/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Antivirais/farmacologia , Simulação por Computador , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo
6.
Arch Biochem Biophys ; 695: 108631, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33080173

RESUMO

Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.


Assuntos
Nucleosídeo-Trifosfatase/química , RNA Helicases/química , Serina Endopeptidases/química , Proteínas Virais/química , Zika virus/enzimologia , Coenzimas , Nucleosídeo-Trifosfatase/genética , Domínios Proteicos , RNA Helicases/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genética
7.
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
8.
J Clin Pathol ; 73(12): 773-776, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32873700

RESUMO

Transmembrane serine protease 2 is encoded by the TMPRSS2 gene. The gene is widely conserved and has two isoforms, both being autocatalytically activated from the inactive zymogen form. A fusion gene between the TMPRSS2 gene and ERG (erythroblast-specific-related gene), an oncogenic transcription factor, is the most common chromosomal aberration detected in prostate cancer, responsible for driving carcinogenesis. The other key role of TMPRSS2 is in priming the viral spike protein which facilitates viral entry essential for viral infectivity. The protease activates a diverse range of viruses. Both SARS-CoV and SARS-CoV-2 (COVID-19) use angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 to facilitate entry to cells, but with SARS-CoV-2 human-to-human transmission is much higher than SARS-CoV. As TMPRSS2 is expressed outside of the lung, and can therefore contribute to extrapulmonary spread of viruses, it warrants further exploration as a potential target for limiting viral spread and infectivity.


Assuntos
/virologia , Serina Endopeptidases/genética , Internalização do Vírus , Marcadores Genéticos , Humanos , Masculino , Neoplasias da Próstata/genética , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo
9.
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
10.
Hum Cell ; 33(4): 1068-1080, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32779152

RESUMO

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.


Assuntos
Ácidos/farmacologia , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Serina Endopeptidases/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Humanos , Mutação , Domínios Proteicos/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Células Tumorais Cultivadas
11.
Molecules ; 25(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842606

RESUMO

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Catepsina L/química , Compostos Fitoquímicos/química , Inibidores de Proteases/química , Receptores Virais/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Sítios de Ligação , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Catepsina L/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cumarínicos/química , Cumarínicos/isolamento & purificação , Cumarínicos/farmacologia , Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Índia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monossacarídeos/química , Monossacarídeos/isolamento & purificação , Monossacarídeos/farmacologia , Pandemias , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Plantas Medicinais/química , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Termodinâmica , Internalização do Vírus/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 117(36): 22146-22156, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848056

RESUMO

Packing interaction is a critical driving force in the folding of helical membrane proteins. Despite the importance, packing defects (i.e., cavities including voids, pockets, and pores) are prevalent in membrane-integral enzymes, channels, transporters, and receptors, playing essential roles in function. Then, a question arises regarding how the two competing requirements, packing for stability vs. cavities for function, are reconciled in membrane protein structures. Here, using the intramembrane protease GlpG of Escherichia coli as a model and cavity-filling mutation as a probe, we tested the impacts of native cavities on the thermodynamic stability and function of a membrane protein. We find several stabilizing mutations which induce substantial activity reduction without distorting the active site. Notably, these mutations are all mapped onto the regions of conformational flexibility and functional importance, indicating that the cavities facilitate functional movement of GlpG while compromising the stability. Experiment and molecular dynamics simulation suggest that the stabilization is induced by the coupling between enhanced protein packing and weakly unfavorable lipid desolvation, or solely by favorable lipid solvation on the cavities. Our result suggests that, stabilized by the relatively weak interactions with lipids, cavities are accommodated in membrane proteins without severe energetic cost, which, in turn, serve as a platform to fine-tune the balance between stability and flexibility for optimal activity.


Assuntos
Proteínas de Ligação a DNA/química , Endopeptidases/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Domínio Catalítico , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Serina Endopeptidases/química
13.
Prostate Cancer Prostatic Dis ; 23(4): 561-563, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32709978

RESUMO

A new coronavirus, named SARS-CoV-2, emerged in Wuhan city, China, in December 2019 causing atypical pneumonia and affecting multiple body organs. The rapidly increasing numbers of infected patients and deaths due to COVID-19 disease necessitated declaring it as a global pandemic. Efforts were combined since then to rapidly develop a treatment and/or a vaccine to combat the deadly virus. Drug repurposing approach has been pursued as a temporary management tactic to treat COVID-19 patients. However, reports about the efficacy of many of the used drugs had been controversial with a dire need to keep the ongoing efforts for rapid development of new treatments. Promising data came out pointing to a possible hidden liaison between prostate cancer (PCa) and COVID-19, where androgen-deprivation therapies (ADT) used in PCa had been shown to instigate a protective role against COVID-19. Delving into the possible mechanisms underlying the crosstalk between COVID-19 and PCa alludes a potential association between SARS-CoV-2 targets on host epithelial cells and PCa genetic aberrations and molecular signatures, including AR and TMPRSS2. The question remains: Can PCa treatments serve as potential therapeutic options for COVID-19 patients?


Assuntos
Antagonistas de Androgênios/uso terapêutico , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Neoplasias da Próstata/epidemiologia , Betacoronavirus/efeitos dos fármacos , China/epidemiologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Humanos , Masculino , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/virologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo
14.
Food Chem ; 333: 127452, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32673951

RESUMO

Aimed to study the characteristics of prolyl endopeptidase (PEP, EC 3.4.21.26) and its possible role in the degradation of collagen, we cloned the full-length cDNA sequence of PEP from abalone (Haliotis discus hannai) (Hdh-PEP). Recombinant Hdh-PEP (rHdh-PEP) was expressed in vitro, its enzymatic properties were detected, and its secondary structure was analyzed by Circular Dichroism (CD). We for the first time determined the 1.5 Å crystal structure of rHdh-PEP. The decomposition effect of rHdh-PEP on collagen peptides was analyzed. Our data revealed that the molecular weight of rHdh-PEP is 85 kDa, consisting of a catalytic domain and a ß-propeller domain. The optimal pH and temperature of rHdh-PEP were pH 6.0 and 20 °C, respectively. Using small collagen peptides as substrates, HPLC-ESI-MS analysis confirmed that rHdh-PEP specifically cleaved at the carboxyl side of proline residues, suggesting its role in the degradation of collagen peptides during autolysis.


Assuntos
Colágeno/metabolismo , Gastrópodes/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Animais , Dicroísmo Circular , Cristalografia por Raios X , DNA Complementar/genética , Concentração de Íons de Hidrogênio , Prolina/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Espectrometria de Massas por Ionização por Electrospray , Temperatura
15.
J Biol Chem ; 295(33): 11742-11753, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32587094

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has challenged the speed at which laboratories can discover the viral composition and study health outcomes. The small ∼30-kb ssRNA genome of coronaviruses makes them adept at cross-species spread while enabling a robust understanding of all of the proteins the viral genome encodes. We have employed protein modeling, molecular dynamics simulations, evolutionary mapping, and 3D printing to gain a full proteome- and dynamicome-level understanding of SARS-CoV-2. We established the Viral Integrated Structural Evolution Dynamic Database (VIStEDD at RRID:SCR_018793) to facilitate future discoveries and educational use. Here, we highlight the use of VIStEDD for nsp6, nucleocapsid (N), and spike (S) surface glycoprotein. For both nsp6 and N, we found highly conserved surface amino acids that likely drive protein-protein interactions. In characterizing viral S protein, we developed a quantitative dynamics cross-correlation matrix to gain insights into its interactions with the angiotensin I-converting enzyme 2 (ACE2)-solute carrier family 6 member 19 (SLC6A19) dimer. Using this quantitative matrix, we elucidated 47 potential functional missense variants from genomic databases within ACE2/SLC6A19/transmembrane serine protease 2 (TMPRSS2), warranting genomic enrichment analyses in SARS-CoV-2 patients. These variants had ultralow frequency but existed in males hemizygous for ACE2. Two ACE2 noncoding variants (rs4646118 and rs143185769) present in ∼9% of individuals of African descent may regulate ACE2 expression and may be associated with increased susceptibility of African Americans to SARS-CoV-2. We propose that this SARS-CoV-2 database may aid research into the ongoing pandemic.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/metabolismo , Bases de Dados de Proteínas , Simulação de Dinâmica Molecular , Pneumonia Viral/metabolismo , Proteoma , Grupo com Ancestrais do Continente Africano/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Infecções por Coronavirus/virologia , Predisposição Genética para Doença , Variação Genética , Interações Hospedeiro-Patógeno , Humanos , Masculino , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfoproteínas , Pneumonia Viral/virologia , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
Molecules ; 25(10)2020 May 12.
Artigo em Inglês | MEDLINE | ID: covidwho-244987

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of -14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.


Assuntos
Betacoronavirus/enzimologia , Desenho de Fármacos , Serina Endopeptidases/química , Inibidores de Serino Proteinase/química , Bibliotecas de Moléculas Pequenas , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Gabexato/análogos & derivados , Gabexato/química , Gabexato/metabolismo , Gabexato/farmacologia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/metabolismo , Inibidores de Serino Proteinase/farmacologia
17.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140441, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32371149

RESUMO

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.


Assuntos
Trifosfato de Adenosina/química , Vírus da Dengue/química , Fosfatos/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Vírus da Dengue/enzimologia , Hidrólise , Simulação de Dinâmica Molecular , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/metabolismo
18.
Molecules ; 25(10)2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32408547

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of -14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.


Assuntos
Betacoronavirus/enzimologia , Desenho de Fármacos , Serina Endopeptidases/química , Inibidores de Serino Proteinase/química , Bibliotecas de Moléculas Pequenas , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Gabexato/análogos & derivados , Gabexato/química , Gabexato/metabolismo , Gabexato/farmacologia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/metabolismo , Inibidores de Serino Proteinase/farmacologia
19.
Proc Natl Acad Sci U S A ; 117(11): 5895-5906, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123115

RESUMO

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key ß-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Mycobacterium tuberculosis/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Cristalografia por Raios X , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Escherichia coli , Homeostase , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise
20.
Microbiol Res ; 236: 126468, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32208189

RESUMO

Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.


Assuntos
Halococcus/genética , Serina Endopeptidases , Serina Proteases/biossíntese , Sequência de Aminoácidos/genética , Proteínas de Bactérias/análise , Biotecnologia , Domínio Catalítico/genética , Fermentação , Produtos Pesqueiros , Serina Endopeptidases/biossíntese , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Proteases/química , Serina Proteases/genética
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