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1.
J Toxicol Sci ; 45(9): 581-587, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879257

RESUMO

Complement component 8 γ (C8γ) is a subunit of complement protein 8 (C8), which itself is a subunit of the complement cytolytic membrane attack complex. However, C8γ is also suggested to be a carrier protein for the general clearance of endogenous and exogenous compounds because it belongs to the lipocalin family of small secreted proteins that have the common ability to bind small hydrophobic ligands. Although retinoic acid, a metabolite of vitamin A, has been suggested as a potential ligand of C8γ, it remains unclear which other substances are able to bind to C8γ as ligands. Here, we evaluated the binding affinity of several organotin compounds that are ligands of a receptor of retinoic acid, retinoid X receptor, by using radioligand binding assays. The amount of [14C]triphenyltin (TPT), a tri-substituted organotin, that bound to purified recombinant C8γ was increased with increasing protein concentration, whereas that of [3H]all-trans retinoic acid and [3H]9-cis retinoic acid was unchanged. Scatchard analysis revealed that [14C]TPT bound to C8γ with an equilibrium dissociation constant (Kd) of 56.2 ± 16.2 nM. Non-radiolabeled tributyltin (TBT), another tri-substituted organotin, blocked the binding of [14C]TPT to C8γ in a competitive manner, but non-radiolabeled mono- or di-substituted organotin compounds did not. Together, our present observations indicate that TBT and TPT, but not retinoic acid or mono- or di-substituted organotin compounds, are potent ligands of C8γ, suggesting that C8γ may be involved in the toxicities of these organotin compounds.


Assuntos
Proteínas de Transporte , Complemento C8 , Ligantes , Compostos Orgânicos de Estanho/toxicidade , Compostos de Trialquitina/toxicidade , Ligação Competitiva , Complexo de Ataque à Membrana do Sistema Complemento/química , Ligação Proteica , Receptores X Retinoide/metabolismo , Tretinoína
2.
Nature ; 585(7824): 303-308, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879488

RESUMO

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.


Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Barbitúricos/química , Barbitúricos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Microscopia Crioeletrônica , Receptores de GABA-A/química , Regulação Alostérica/efeitos dos fármacos , Anestésicos Gerais/metabolismo , Barbitúricos/metabolismo , Benzodiazepinas/metabolismo , Bicuculina/química , Bicuculina/metabolismo , Bicuculina/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Diazepam/química , Diazepam/metabolismo , Diazepam/farmacologia , Eletrofisiologia , Etomidato/química , Etomidato/metabolismo , Etomidato/farmacologia , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Fenobarbital/química , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Picrotoxina/química , Picrotoxina/metabolismo , Picrotoxina/farmacologia , Propofol/química , Propofol/metabolismo , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestrutura , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
3.
Science ; 369(6508): 1261-1265, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753553

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Engenharia de Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese , Mutação , Peptidil Dipeptidase A/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química
4.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
5.
Environ Pollut ; 266(Pt 2): 115179, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32717636

RESUMO

Hydrophobic pollutants are still present in agricultural soil. The Cucurbitaceae family accumulates hydrophobic pollutants through roots, resulting in the contamination of aerial parts. Major latex-like proteins (MLPs), found in the Cucurbitaceae family, play an important role in the contamination by binding to these hydrophobic pollutants. Thus far, efficient cultivation methods for the production of safe crops with lower concentrations of hydrophobic pollutants have not been developed. Herein, we competitively inhibited the binding of MLPs to hydrophobic pollutants, pyrene and dieldrin, in roots by using MLP binding pesticides. By conducting a chemical array screening, we found that MLPs bound compounds with indole- and quinazoline-like structures. Commercially available pesticides amisulbrom and pyrifluquinazon, which possess such structures, successfully inhibited the binding of MLPs to pyrene and dieldrin in vitro. When zucchini plants were cultivated in the contaminated soil with 1.25 mmol/kg pyrene and 12.5 µmol/kg dieldrin, the concentration of pyrene and dieldrin in xylem sap was significantly decreased by 30% and 15%, respectively. Our results demonstrate that the pesticides binding to MLPs competitively inhibited the binding of MLPs to pyrene and dieldrin in roots, resulting in the reduction of overall contamination. This study proposes a novel approach to cultivate safer crops and advances the utilization of unknown functions of pesticides.


Assuntos
Cucurbita , Poluentes Ambientais , Praguicidas/análise , Poluentes do Solo/análise , Ligação Competitiva , Látex , Raízes de Plantas/química
6.
Nature ; 584(7821): 443-449, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32668443

RESUMO

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Betacoronavirus/química , Ligação Competitiva , Linhagem Celular , Reações Cruzadas , Modelos Animais de Doenças , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Pessoa de Meia-Idade , Testes de Neutralização , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Profilaxia Pré-Exposição , Vírus da SARS/química , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
PLoS One ; 15(6): e0234683, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32544184

RESUMO

Rapid resuscitation of an opioid overdose with naloxone, an opioid antagonist, is critical. We developed an opioid receptor quantitative systems pharmacology (QSP) model for evaluation of naloxone dosing. In this model we examined three opioid exposure levels that have been reported in the literature (25 ng/ml, 50 ng/ml, and 75 ng/ml of fentanyl). The model predicted naloxone-fentanyl interaction at the mu opioid receptor over a range of three naloxone doses. For a 2 mg intramuscular (IM) dose of naloxone at lower fentanyl exposure levels (25 ng/ml and 50 ng/ml), the time to decreasing mu receptor occupancy by fentanyl to 50% was 3 and 10 minutes, respectively. However, at a higher fentanyl exposure level (75 ng/ml), a dose of 2 mg IM of the naloxone failed to reduce mu receptor occupancy by fentanyl to 50%. In contrast, naloxone doses of 5 mg and 10 mg IM reduced mu receptor occupancy by fentanyl to 50% in 5.5 and 4 minutes respectively. These results suggest that the current doses of naloxone (2 mg IM or 4 mg intranasal (IN)) may be inadequate for rapid reversal of toxicity due to fentanyl exposure and that increasing the dose of naloxone is likely to improve outcomes.


Assuntos
Ligação Competitiva , Fentanila/metabolismo , Modelos Teóricos , Naloxona/administração & dosagem , Receptores Opioides mu/metabolismo , Analgésicos Opioides/metabolismo , Simulação por Computador , Relação Dose-Resposta a Droga , Overdose de Drogas/tratamento farmacológico , Fentanila/toxicidade , Humanos , Naloxona/uso terapêutico , Antagonistas de Entorpecentes/administração & dosagem , Resultado do Tratamento
8.
Nature ; 584(7819): 120-124, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32454512

RESUMO

An outbreak of coronavirus disease 2019 (COVID-19)1-3, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addition, CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme 2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/farmacologia , Betacoronavirus/química , Ligação Competitiva , Linhagem Celular , Chlorocebus aethiops , Cristalização , Cristalografia por Raios X , Feminino , Humanos , Técnicas In Vitro , Macaca mulatta/imunologia , Macaca mulatta/virologia , Masculino , Modelos Moleculares , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Carga Viral/imunologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-32371328

RESUMO

INTRODUCTION: Ultrafiltration (UF) is used to separate unbound drugs; however, non-specific binding (NSB) may be a limiting factor of this technique. Pretreatment of UF devices has been suggested to reduce NSB. Therefore, the pretreatment methodologies for UF devices were evaluated in order to test their effectiveness in reducing NSB of protease inhibitors (PIs). METHODOLOGY: Two PIs (lopinavir-LPV and ritonavir-RTV) were tested. UF devices were pretreated with ultrapure water, Tween-20 or Tween-80. To evaluate the NSB, after UF devices being pretreated, ultrafiltrate solutions containing the analytes at two concentrations (low and high) were used. Samples were quantified by LC-MS/MS. RESULTS: UF devices pretreated with Tween-5% had the lowest NSB for both analytes. NSB values varied between 7 and 11% at low concentration 16-34% at high LPV concentration, respectively. For RTV, NSB was approximately 6% for low concentration and 18% for high concentration. Failure to completely remove Tween in UF devices could results in an overestimation of NSB. CONCLUSION: Pretreatment of UF device with Tween and subsequent removal proved to be effective in reducing NSB of PI.


Assuntos
Inibidores da Protease de HIV/química , Lopinavir/química , Ritonavir/química , Ultrafiltração/métodos , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Humanos , Plasma/química , Ligação Proteica , Padrões de Referência , Espectrometria de Massas em Tandem
10.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L148-L158, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432921

RESUMO

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.


Assuntos
Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Líquido Amniótico/metabolismo , Asparagina/metabolismo , Ligação Competitiva , Cromatografia de Afinidade , Feminino , Glicosilação , Humanos , Polissacarídeos/metabolismo , Gravidez , Ligação Proteica , Multimerização Proteica , Proteinose Alveolar Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/isolamento & purificação , Relação Estrutura-Atividade
11.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: covidwho-116533

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
12.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332765

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Nat Commun ; 11(1): 1805, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286318

RESUMO

Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.


Assuntos
RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , DNA/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/química , Deleção de Sequência , Eletricidade Estática
14.
Food Chem ; 318: 126511, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126462

RESUMO

Interactions between taste compounds and nanofibrillar cellulose were studied. For this, a new fluorescent indicator displacement method was developed. Two fluorescent indicators, namely, Calcofluor white and Congo red, were chosen because of their specific binding to cellulose and intrinsic fluorescence. Seven taste compounds with different structures were successfully measured together with nanofibrillar cellulose (NFC) and ranked according to their binding constants. The most pronounced interactions were found between quinine and NFC (1.4 × 104 M-1), whereas sucrose, aspartame and glutamic acid did not bind at all. Naringin showed moderate binding while stevioside and caffeine exhibited low binding. The comparison with microcrystalline cellulose indicates that the larger surface area of nanofibrillated cellulose enables stronger binding between the binder and macromolecules. The developed method can be further utilized to study interactions of different compound classes with nanocellulose materials in food, pharmaceutical and dye applications, using a conventional plate reader in a high-throughput manner.


Assuntos
Celulose/metabolismo , Corantes Fluorescentes/química , Nanoestruturas/química , Aspartame/química , Aspartame/metabolismo , Benzenossulfonatos/química , Ligação Competitiva , Cafeína/metabolismo , Celulose/química , Vermelho Congo/química , Diterpenos de Caurano/metabolismo , Flavanonas/metabolismo , Glucosídeos/metabolismo , Espectrofotometria Ultravioleta , Paladar
15.
Immunity ; 52(3): 499-512.e5, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32187518

RESUMO

Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.


Assuntos
Interleucina-17/imunologia , Multimerização Proteica/imunologia , Receptores de Interleucina-17/imunologia , Transdução de Sinais/imunologia , Ligação Competitiva , Cristalografia por Raios X , Células HEK293 , Humanos , Interleucina-17/química , Interleucina-17/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores de Interleucina-17/química , Receptores de Interleucina-17/metabolismo
16.
J Med Chem ; 63(8): 3935-3955, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32212732

RESUMO

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.


Assuntos
5'-Nucleotidase/antagonistas & inibidores , Descoberta de Drogas/métodos , Triazóis/química , Triazóis/farmacologia , 5'-Nucleotidase/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cristalografia por Raios X/métodos , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos
17.
Chemosphere ; 249: 126034, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32062553

RESUMO

Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin αvß3. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin αvß3. Based on different probes, two assays were modified: a fluorescence competitive binding assay and a radioligand competitive binding assay. The chemicals tested included the known TH, 3,3',5,5'-tetraiodo-l-thyronine (T4); a deaminated analog of T4, tetraiodothyroacetic acid (tetrac); and phthalate esters (PAEs). The relative binding potency of T4 was studied, and the concentration required to displace 50% of the ligands from their receptors (RIC50) of T4 was 4.9 × 105 and 9.7 × 104 nM for the fluorescence and radioligand competitive binding assays, respectively, suggesting that the radioligand competitive binding assay might be more sensitive for the evaluation of the binding affinity for integrin αvß3. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin αvß3 in the following order of potency: DnBP > DEHP > BBP tested by the radioligand competitive binding assay. A docking simulation of each of the three PAEs with integrin αvß3 confirmed the calculated binding energies, which had a strong positive relationship with the log RIC20 values of the 3 PAEs (R = 0.99, p < 0.001). The present study shows that the established radioligand competitive binding assay could be used as a valuable tool for quantifying the affinity of TDCs for integrin αvß3.


Assuntos
Bioensaio , Integrina alfaVbeta3/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Glândula Tireoide/efeitos dos fármacos , Ligação Competitiva , Dibutilftalato , Ésteres , Ácidos Ftálicos/toxicidade , Glândula Tireoide/metabolismo , Tiroxina/análogos & derivados
18.
PLoS Genet ; 16(2): e1008589, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059010

RESUMO

Nervous systems have the ability to select appropriate actions and action sequences in response to sensory cues. The circuit mechanisms by which nervous systems achieve choice, stability and transitions between behaviors are still incompletely understood. To identify neurons and brain areas involved in controlling these processes, we combined a large-scale neuronal inactivation screen with automated action detection in response to a mechanosensory cue in Drosophila larva. We analyzed behaviors from 2.9x105 larvae and identified 66 candidate lines for mechanosensory responses out of which 25 for competitive interactions between actions. We further characterize in detail the neurons in these lines and analyzed their connectivity using electron microscopy. We found the neurons in the mechanosensory network are located in different regions of the nervous system consistent with a distributed model of sensorimotor decision-making. These findings provide the basis for understanding how selection and transition between behaviors are controlled by the nervous system.


Assuntos
Potenciais de Ação/fisiologia , Ligação Competitiva , Mecanotransdução Celular/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Células Receptoras Sensoriais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Geneticamente Modificados , Ligação Competitiva/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Mapeamento Encefálico , Sinais (Psicologia) , Drosophila melanogaster/genética , Vias Neurais/metabolismo , Neurônios/metabolismo , Fenótipo
19.
Nucleic Acids Res ; 48(5): e28, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980824

RESUMO

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.


Assuntos
Bioensaio , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Repressores Lac/deficiência , Repressores Lac/genética , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
20.
PLoS Biol ; 18(1): e3000593, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31995552

RESUMO

During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.


Assuntos
Guanosina Tetrafosfato/farmacologia , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Ligação Competitiva , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Interações Hospedeiro-Patógeno/fisiologia , Cinética , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética
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