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1.
bioRxiv ; 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-39026801

RESUMO

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

3.
bioRxiv ; 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38559139

RESUMO

Neural circuits in many brain regions are refined by experience. Sensory circuits support higher plasticity at younger ages during critical periods - times of circuit refinement and maturation - and limit plasticity in adulthood for circuit stability. The mechanisms underlying these differing plasticity levels and how they serve to maintain and stabilize the properties of sensory circuits remain largely unclear. By combining a transcriptomic approach with ex vivo electrophysiology and in vivo imaging techniques, we identify that astrocytes release cellular communication network factor 1 (CCN1) to maintain synapse and circuit stability in the visual cortex. By overexpressing CCN1 in critical period astrocytes, we find that it promotes the maturation of inhibitory circuits and limits ocular dominance plasticity. Conversely, by knocking out astrocyte CCN1 in adults, binocular circuits are destabilized. These studies establish CCN1 as a novel astrocyte-secreted factor that stabilizes neuronal circuits. Moreover, they demonstrate that the composition and properties of sensory circuits require ongoing maintenance in adulthood, and that these maintenance cues are provided by astrocytes.

4.
Nat Commun ; 14(1): 6030, 2023 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-37758692

RESUMO

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.


Assuntos
COVID-19 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Proteômica , Replicação Viral/genética , SARS-CoV-2 , Antivirais/metabolismo , Interações Hospedeiro-Patógeno/genética
5.
Res Sq ; 2023 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-37720034

RESUMO

SARS-CoV-2 initiates infection in the conducting airways, which rely on mucocilliary clearance (MCC) to minimize pathogen penetration. However, it is unclear how MCC impacts SARS-CoV-2 spread after infection is established. To understand viral spread at this site, we performed live imaging of SARS-CoV-2 infected differentiated primary human bronchial epithelium cultures for up to 9 days. Fluorescent markers for cilia and mucus allowed longitudinal monitoring of MCC, ciliary motion, and infection. The number of infected cells peaked at 4 days post-infection in characteristic foci that followed mucus movement. Inhibition of MCC using physical and genetic perturbations limited foci. Later in infection, MCC was diminished despite relatively subtle ciliary function defects. Resumption of MCC and infection spread after mucus removal suggests that mucus secretion mediates this effect. We show that MCC facilitates SARS-CoV-2 spread early in infection while later decreases in MCC inhibit spread, suggesting a complex interplay between SARS-CoV-2 and MCC.

6.
Geriatr Nurs ; 53: 72-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37454421

RESUMO

The study aimed to study the influence of musculoskeletal pain on kinesiophobia in patients with heart failure. This cross-sectional study recruited 107 heart failure patients aged 73.18±12.68 years (57% men) from an outpatient setting. Participants self-reported pain using the Musculoskeletal System Assessment Inventory and the Cornell Musculoskeletal Discomfort Questionnaire. Kinesiophobia was assessed with the Tampa Scale for Kinesiophobia-11. About 62% reported musculoskeletal pain, with knees (16.8%) and lower back (12.%) being the most painful locations. About 31% reported moderate levels and 24% indicated high levels of kinesiophobia. There were positive and significant associations between the indicators of pain and kinesiophobia. Results showed an adequate structural equation model fit to the data with musculoskeletal pain factors explaining 22.09% of the variance in kinesiophobia. Assessment of kinesiophobia in patients with heart failure with musculoskeletal pain is essential to improve self-care and overall quality of life.


Assuntos
Insuficiência Cardíaca , Dor Musculoesquelética , Masculino , Humanos , Idoso , Feminino , Medo , Cinesiofobia , Qualidade de Vida , Estudos Transversais , Medição da Dor , Insuficiência Cardíaca/complicações
7.
Stem Cells Dev ; 32(9-10): 225-236, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36803114

RESUMO

SARS-CoV-2 infection during pregnancy has been associated with poor maternal and neonatal outcomes and placental defects. The placenta, which acts as a physical and immunological barrier at the maternal-fetal interface, is not established until the end of the first trimester. Therefore, localized viral infection of the trophoblast compartment early in gestation could trigger an inflammatory response resulting in altered placental function and consequent suboptimal conditions for fetal growth and development. In this study, we investigated the effect of SARS-CoV-2 infection in early gestation placentae using placenta-derived human trophoblast stem cells (TSCs), a novel in vitro model, and their extravillous trophoblast (EVT) and syncytiotrophoblast (STB) derivatives. SARS-CoV-2 was able to productively replicate in TSC-derived STB and EVT, but not undifferentiated TSCs, which is consistent with the expression of SARS-CoV-2 entry host factors, ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane cellular serine protease) in these cells. In addition, both TSC-derived EVT and STB infected with SARS-CoV-2 elicited an interferon-mediated innate immune response. Combined, these results suggest that placenta-derived TSCs are a robust in vitro model to investigate the effect of SARS-CoV-2 infection in the trophoblast compartment of the early placenta and that SARS-CoV-2 infection in early gestation activates the innate immune response and inflammation pathways. Therefore, placental development could be adversely affected by early SARS-CoV-2 infection by directly infecting the developing differentiated trophoblast compartment, posing a higher risk for poor pregnancy outcomes.


Assuntos
COVID-19 , SARS-CoV-2 , Recém-Nascido , Gravidez , Feminino , Humanos , COVID-19/metabolismo , Trofoblastos/metabolismo , Interferons , Placenta
8.
Ann Vasc Surg ; 92: 195-200, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36566912

RESUMO

BACKGROUND: A prolonged operative time (OT) is a well-recognized risk factor of postoperative complications after many open surgical procedures, although little is known about its impact in less-invasive endovascular procedures. We aimed to define the characteristics related to a prolonged OT in the endovascular treatment of aorto-iliac aneurysms (EVAR) and to evaluate the influence of OT on postoperative outcomes. METHODS: Retrospective analysis of 284 consecutive patients (mean age 75 years, 95% male) who underwent an elective EVAR between 2000 and 2019. Operative characteristics related to OT and the impact of OT in postoperative results was studied using multiple lineal and logistic regression analyses, respectively. RESULTS: The mean surgical time was 200 min. OT was associated (regression model) with the implantation of straight endografts (-38 min, P = 0.007), femoral artery surgery (+80 min, P < 0.001), hypogastric preservation procedures (+70 min, P < 0.001), associated peripheral arterial disease (+22 min, P = 0.013), general anesthesia (+34 min, P < 0.001), and aneurysm diameter (+9 min/cm, P = 0.002). During the postoperative period (<30 days or at discharge), 21% presented a complication and 2.8% died. OT was independently associated with a higher incidence of postoperative complications (odds ratio [OR] for each additional 30' of surgery = 1.34, P < 0.001), such as immediate (OR = 1.48, P = 0.003) and 6-month mortality (OR = 1.28, P = 0.025). CONCLUSIONS: A prolonged OT is an independent risk factor for complications and mortality after EVAR. Surgeons must take this factor into consideration when defining the best therapeutic strategy for abdominal aortic aneurysms.


Assuntos
Aneurisma da Aorta Abdominal , Implante de Prótese Vascular , Procedimentos Endovasculares , Humanos , Masculino , Idoso , Feminino , Duração da Cirurgia , Estudos Retrospectivos , Resultado do Tratamento , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/cirurgia , Aneurisma da Aorta Abdominal/complicações , Fatores de Risco , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia
9.
PLoS Biol ; 20(11): e3001845, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36327326

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was rapidly declared a pandemic by the World Health Organization (WHO). Early clinical symptomatology focused mainly on respiratory illnesses. However, a variety of neurological manifestations in both adults and newborns are now well-documented. To experimentally determine whether SARS-CoV-2 could replicate in and affect human brain cells, we infected iPSC-derived human brain organoids. Here, we show that SARS-CoV-2 can productively replicate and promote death of neural cells, including cortical neurons. This phenotype was accompanied by loss of excitatory synapses in neurons. Notably, we found that the U.S. Food and Drug Administration (FDA)-approved antiviral Sofosbuvir was able to inhibit SARS-CoV-2 replication and rescued these neuronal alterations in infected brain organoids. Given the urgent need for readily available antivirals, these results provide a cellular basis supporting repurposed antivirals as a strategic treatment to alleviate neurocytological defects that may underlie COVID-19- related neurological symptoms.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Recém-Nascido , Humanos , Sofosbuvir/farmacologia , Sofosbuvir/uso terapêutico , Organoides , Antivirais/farmacologia , Antivirais/uso terapêutico , Encéfalo , Morte Celular , Sinapses
10.
Sci Adv ; 8(40): eabm5859, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36197970

RESUMO

Molecular responses to influenza A virus (IAV) infections vary between mammalian species. To identify conserved and species-specific molecular responses, we perform a comparative study of transcriptomic data derived from blood cells, primary epithelial cells, and lung tissues collected from IAV-infected humans, ferrets, and mice. The molecular responses in the human host have unique functions such as antigen processing that are not observed in mice or ferrets. Highly conserved gene coexpression modules across the three species are enriched for IAV infection-induced pathways including cell cycle and interferon (IFN) signaling. TDRD7 is predicted as an IFN-inducible host factor that is up-regulated upon IAV infection in the three species. TDRD7 is required for antiviral IFN response, potentially modulating IFN signaling via the JAK/STAT/IRF9 pathway. Identification of the common and species-specific molecular signatures, networks, and regulators of IAV infection provides insights into host-defense mechanisms and will facilitate the development of novel therapeutic interventions against IAV infection.


Assuntos
Doenças Transmissíveis , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Antivirais , Furões/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/genética , Interferons/metabolismo , Camundongos , Infecções por Orthomyxoviridae/genética , Ribonucleoproteínas
11.
Nat Neurosci ; 25(9): 1163-1178, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36042312

RESUMO

Astrocytes negatively impact neuronal development in many models of neurodevelopmental disorders (NDs); however, how they do this, and if mechanisms are shared across disorders, is not known. In this study, we developed a cell culture system to ask how astrocyte protein secretion and gene expression change in three mouse models of genetic NDs (Rett, Fragile X and Down syndromes). ND astrocytes increase release of Igfbp2, a secreted inhibitor of insulin-like growth factor (IGF). IGF rescues neuronal deficits in many NDs, and we found that blocking Igfbp2 partially rescues inhibitory effects of Rett syndrome astrocytes, suggesting that increased astrocyte Igfbp2 contributes to decreased IGF signaling in NDs. We identified that increased BMP signaling is upstream of protein secretion changes, including Igfbp2, and blocking BMP signaling in Fragile X and Rett syndrome astrocytes reverses inhibitory effects on neurite outgrowth. This work provides a resource of astrocyte-secreted proteins in health and ND models and identifies novel targets for intervention in diverse NDs.


Assuntos
Transtornos do Neurodesenvolvimento , Síndrome de Rett , Animais , Astrócitos/metabolismo , Camundongos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Neurogênese , Neurônios/metabolismo , Síndrome de Rett/metabolismo
12.
ACS Infect Dis ; 8(7): 1265-1279, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35766385

RESUMO

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.


Assuntos
Tratamento Farmacológico da COVID-19 , Vírus da Influenza A , Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Antivirais/uso terapêutico , Depsipeptídeos , Humanos , Pandemias , SARS-CoV-2 , Infecção por Zika virus/tratamento farmacológico
13.
iScience ; 25(5): 104311, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35502318

RESUMO

Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal and synthetic dosage lethal (SL/SDL) partners of such altered host genes. Pursuing this disparate antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL/SDL with altered host genes. The predicted SL/SDL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. We further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming noninfected healthy cells.

14.
Viruses ; 14(3)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35337019

RESUMO

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus-host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.


Assuntos
COVID-19 , SARS-CoV-2 , Biotinilação , Humanos , Pandemias , Proteômica
15.
Mol Syst Biol ; 17(11): e10260, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34709707

RESUMO

Tremendous progress has been made to control the COVID-19 pandemic caused by the SARS-CoV-2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS-CoV-2 infection using genome-scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS-CoV-2 infection. We next applied the GEM-based metabolic transformation algorithm to predict anti-SARS-CoV-2 targets that counteract the virus-induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco-2 cells. Further generating and analyzing RNA-sequencing data of remdesivir-treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti-SARS-CoV-2 drug. Our study provides clinical data-supported candidate anti-SARS-CoV-2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/uso terapêutico , COVID-19/metabolismo , Redes e Vias Metabólicas/genética , Pandemias , SARS-CoV-2/fisiologia , Monofosfato de Adenosina/uso terapêutico , Alanina/uso terapêutico , Animais , COVID-19/virologia , Células CACO-2 , Chlorocebus aethiops , Conjuntos de Dados como Assunto , Desenvolvimento de Medicamentos , Reposicionamento de Medicamentos , Interações Hospedeiro-Patógeno , Humanos , RNA Interferente Pequeno , Análise de Sequência de RNA , Células Vero , Tratamento Farmacológico da COVID-19
16.
bioRxiv ; 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34545363

RESUMO

Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal (SL) partners of such altered host genes. Pursuing this antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL with altered host genes. The predicted SL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. Integrating our predictions with the results of these screens, we further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells. Our results are made publicly available, to facilitate their in vivo testing and further validation.

17.
bioRxiv ; 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34580671

RESUMO

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.

18.
Nat Microbiol ; 6(10): 1319-1333, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34556855

RESUMO

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.


Assuntos
Autofagia , Evasão da Resposta Imune , Vírus da Influenza A/fisiologia , Antivirais/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Lisossomos/metabolismo , Ligação Proteica , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
19.
J Virol ; 95(22): e0099621, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34468177

RESUMO

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Replicação Viral/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Zika virus/efeitos dos fármacos , Animais , Astrócitos , Chlorocebus aethiops , Células Dendríticas , Células HEK293 , Humanos , Cultura Primária de Células , Células Vero , Compartimentos de Replicação Viral/efeitos dos fármacos
20.
Mol Cell ; 81(11): 2261-2265, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34087174

RESUMO

COVID-19 altered our lives and pushed scientific research to operate at breakneck speed, leading to significant breakthroughs in record time. We asked experts in the field about the challenges they faced in transitioning, rapidly but safely, to working on the virus while navigating the shutdown. Their voices converge on the importance of teamwork, forging new collaborations, and working toward a shared goal.


Assuntos
Pesquisa Biomédica , COVID-19/epidemiologia , COVID-19/prevenção & controle , Pandemias , Quarentena , SARS-CoV-2 , Humanos , Poesia como Assunto
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