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Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Humanos , Animais , SARS-CoV-2/efeitos dos fármacos , COVID-19/virologia , Camundongos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/uso terapêutico , Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Antivirais/química , Linhagem Celular , Pneumonia/tratamento farmacológico , Pneumonia/virologia , Pneumonia/prevenção & controle , Pulmão/virologia , Pulmão/patologia , FemininoRESUMO
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a fast-spreading viral pathogen and poses a serious threat to human health. New SARS-CoV-2 variants have been arising worldwide; therefore, is necessary to explore more therapeutic options. The interaction of the viral spike (S) protein with the angiotensin-converting enzyme 2 (ACE2) host receptor is an attractive drug target to prevent the infection via the inhibition of virus cell entry. In this study, Ligand- and Structure-Based Virtual Screening (LBVS and SBVS) was performed to propose potential inhibitors capable of blocking the S receptor-binding domain (RBD) and ACE2 interaction. The best five lead compounds were confirmed as inhibitors through ELISA-based enzyme assays. The docking studies and molecular dynamic (MD) simulations of the selected compounds maintained the molecular interaction and stability (RMSD fluctuations less than 5 Å) with key residues of the S protein. The compounds DRI-1, DRI-2, DRI-3, DRI-4, and DRI-5 efficiently block the interaction between the SARS-CoV-2 spike protein and receptor ACE2 (from 69.90 to 99.65% of inhibition) at 50 µM. The most potent inhibitors were DRI-2 (IC50 = 8.8 µM) and DRI-3 (IC50 = 2.1 µM) and have an acceptable profile of cytotoxicity (CC50 > 90 µM). Therefore, these compounds could be good candidates for further SARS-CoV-2 preclinical experiments.
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Background: Respiratory Syncytial Virus (RSV) presents a significant health threat, especially to young children. In-depth understanding of RSV entry mechanisms is essential for effective antiviral development. This study introduces an innovative RSV variant, featuring the fusion of the beta-lactamase (BlaM) enzyme with the RSV-P phosphoprotein, providing a versatile tool for dissecting viral entry dynamics. Methods: Using the AlphaFold2 algorithm, we modeled the tertiary structure of the P-BlaM chimera, revealing structural similarities with both RSV-P and BlaM. Functional assessments, utilizing flow cytometry, quantified beta-lactamase activity and GFP expression in infected bronchial epithelial cells. Western blot analysis confirmed the integrity of P-BlaM within virions. Results: The modeled P-BlaM chimera exhibited structural parallels with RSV-P and BlaM. Functional assays demonstrated robust beta-lactamase activity in recombinant virions, confirming successful P-BlaM incorporation as a structural protein. Quercetin, known for its antiviral properties, impeded viral entry by affecting virion fusion. Additionally, Ulixertinib, an ERK-1/2 inhibitor, significantly curtailed viral entry, implicating ERK-1/2 pathway signaling. Conclusions: Our engineered RSV-P-BlaM chimera emerges as a valuable tool, illuminating RSV entry mechanisms. Structural and functional analyses unveil potential therapeutic targets. Quercetin and Ulixertinib, identified as distinct stage inhibitors, show promise for targeted antiviral strategies. Time-of-addition assays pinpoint quercetin's specific interference stage, advancing our comprehension of RSV entry and guiding future antiviral developments.
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Sialic acids and heparan sulfates make up the outermost part of the cell membrane and the extracellular matrix. Both structures are characterized by being negatively charged, serving as receptors for various pathogens, and are highly expressed in the respiratory and digestive tracts. Numerous viruses use heparan sulfates as receptors to infect cells; in this group are HSV, HPV, and SARS-CoV-2. Other viruses require the cell to express sialic acids, as is the case in influenza A viruses and adenoviruses. This review aims to present, in a general way, the participation of glycoconjugates in viral entry, and therapeutic strategies focused on inhibiting the interaction between the virus and the glycoconjugates. Interestingly, there are few studies that suggest the participation of both glycoconjugates in the viruses addressed here. Considering the biological redundancy that exists between heparan sulfates and sialic acids, we propose that it is important to jointly evaluate and design strategies that contemplate inhibiting the interactions of both glycoconjugates. This approach will allow identifying new receptors and lead to a deeper understanding of interspecies transmission.
Assuntos
COVID-19 , Vírus , Glicoconjugados/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2 , Ácidos Siálicos/metabolismo , Sulfatos , Ligação Viral , Vírus/metabolismoRESUMO
Patents of lectins with antiviral, antibacterial and antifungal applications were searched and reviewed. Lectins are proteins that reversibly bind to specific carbohydrates and have the potential for therapy of infectious diseases as biopharmaceuticals, biomedical tools or in drug design. Given the rising concerns over drug resistance and epidemics, our patent review aims to add information, open horizons and indicate our view of the future perspectives about the antimicrobial applications of lectins. Patents with publications until December 2020 were retrieved from Espacenet using defined search terms and Boolean operators. The documents were used to identify the geographical and temporal distribution of the patents, characterize their lectins, and classify and summarize their antiviral, antibiotic and antifungal applications. Lectins are promising antiviral agents against viruses with epidemics and drug resistance concerns. Mannose-binding lectins were the most suggested antiviral agents since glycans with mannose residues are commonly involved in viral entry mechanisms. They were also immobilized onto surfaces to trap viral particles and inhibit their spread and replication. Many patents described the extraction, isolation, amino acid and nucleotide sequences, and expression vectors of lectins with antibiotic and/or antifungal activities in terms of MIC and IC50 for in vitro assays. The inventions also included lectins as biological tools in nanosensors for antibiotics susceptibility tests, drug-delivery systems for the treatment of resistant bacteria, diagnostics of viral diseases and as a vaccine adjuvant. Although research and development of new medicines is highly expensive, antimicrobial lectins may be worth investments given the emergence of epidemics and drug resistance. For this purpose, less invasive routes should be developed as alternatives to the parenteral administration of biologics. While anti-glycan neutralizing antibodies are difficult to develop due to the low immunogenicity of carbohydrates, lectins can be produced more easily and have a broad-spectrum activity. Protein engineering technologies may make the antimicrobial applications of lectins more successful.
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Anti-Infecciosos , Doenças Transmissíveis , Adjuvantes de Vacinas , Antibacterianos , Anti-Infecciosos/farmacologia , Doenças Transmissíveis/tratamento farmacológico , Humanos , LectinasRESUMO
COVID-19 is a pandemic disease caused by the SARS-CoV-2 virus, which is potentially fatal for vulnerable individuals. Disease management represents a challenge for many countries, given the shortage of medicines and hospital resources. The objective of this work was to review the medicinal plants, foods and natural products showing scientific evidence for host protection against various types of coronaviruses, with a focus on SARS-CoV-2. Natural products that mitigate the symptoms caused by various coronaviruses are also presented. Particular attention was placed on natural products that stabilize the Renin-Angiotensin-Aldosterone System (RAAS), which has been associated with the entry of the SARS-CoV-2 into human cells.
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Produtos Biológicos/farmacologia , Coronavirus/efeitos dos fármacos , Fitoterapia/métodos , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antivirais/metabolismo , Antivirais/farmacologia , Produtos Biológicos/metabolismo , COVID-19/virologia , Humanos , Pandemias , Extratos Vegetais/metabolismo , Plantas/química , Sistema Renina-Angiotensina/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
PIM kinases are a family of serine/threonine protein kinases that potentiate the progression of the cell cycle and inhibit apoptosis. Because of this, they are considered to be proto-oncogenes, and they represent an interesting target for the development of anticancer drugs. In mammals, three PIM kinases exist (PIM-1, PIM-2 and PIM-3), and different inhibitors have been developed to block their activity. In addition to their involvement in cancer, some publications have reported that the PIM kinases have pro-viral activity, and different mechanisms where PIM kinases favour viral infections have been proposed. Zebrafish possess more than 300 Pim kinase members in their genome, and by using RNA-Seq analysis, we found a high number of Pim kinase genes that were significantly induced after infection with spring viraemia of carp virus (SVCV). Moreover, analysis of the miRNAs modulated by this infection revealed that some of them could be involved in the post-transcriptional regulation of Pim kinase abundance. To elucidate the potential role of the 16 overexpressed Pim kinases in the infectivity of SVCV, we used three different pan-PIM kinase inhibitors (SGI-1776, INCB053914 and AZD1208), and different experiments were conducted both in vitro and in vivo. We observed that the PIM kinase inhibitors had a protective effect against SVCV, indicating that, similar to what is observed in mammals, PIM kinases are beneficial for the virus in zebrafish. Moreover, zebrafish Pim kinases seem to facilitate viral entry into the host cells because when ZF4 cells were pre-incubated with the virus and then were treated with the inhibitors, the protective effect of the inhibitors was abrogated. Although more investigation is necessary, these results show that pan-PIM kinase inhibitors could serve as a useful treatment for preventing the spread of viral diseases.
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Rim/enzimologia , Proteínas Proto-Oncogênicas c-pim-1/genética , Infecções por Rhabdoviridae/veterinária , Internalização do Vírus/efeitos dos fármacos , Peixe-Zebra/virologia , Animais , Apoptose , Compostos de Bifenilo/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Rim/virologia , Poli I-C/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridazinas/farmacologia , RNA-Seq , Rhabdoviridae , Tiazolidinas/farmacologia , Peixe-Zebra/anatomia & histologiaRESUMO
Arboviruses have been a huge threat for human health since the discovery of yellow fever virus in 1901. Arboviruses are arthropod born viruses, mainly transmitted by mosquitoes and ticks, responsible for more than thousands of deaths annually. The Flavivirideae family is probably the most clinically relevant, as it is composed of very important agents, such as dengue, yellow fever, West Nile, Japanese encephalitis, and, recently, Zika virus. Intriguingly, despite their structural and genomic similarities, flaviviruses may cause conditions ranging from mild infections with fever, cutaneous rash, and headache, to very severe cases, such as hemorrhagic fever, encephalitis, Guillain-Barré syndrome, and microcephaly. These differences may greatly rely on viral burden, tissue tropism, and mechanisms of immune evasion that may depend on both viral and host genetic factors. Unfortunately, very little is known about the biology of these factors, and how they orchestrate these differences. In this context, viral structural proteins and host cellular receptors may have a great relevance, as their interaction dictates not only viral tissue tropism, but also a plethora on intracellular mechanisms that may greatly account for either failure or success of infection. A great number of viral receptors have been described so far, although there is still a huge gap in understanding their overall role during infection. Here we discuss some important aspects triggered after the interaction of flaviviruses and host membrane receptors, and how they change the overall outcome of the infection.
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Flavivirus/metabolismo , Receptores Virais/metabolismo , Animais , Humanos , Integrinas/metabolismo , Fosfatidilserinas/metabolismoRESUMO
We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.
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Vírus da Necrose Pancreática Infecciosa/fisiologia , Macrófagos/virologia , Pinocitose , Salmão/virologia , Internalização do Vírus , Actinas/metabolismo , Animais , Infecções por Birnaviridae/virologia , Linhagem Celular , Doenças dos Peixes/virologia , Rim Cefálico/virologia , Macrófagos/citologiaRESUMO
Dengue is the most relevant mosquito-borne viral disease in the world. It has been estimated that 390 million infections of dengue occur each year. Dengue virus (DENV) infection can be asymptomatic or can produce a self-limited febrile illness called dengue fever (DF) or a severe form of the infection called severe dengue. In some viruses, the entry and egress from cells, occur in a specific domain of polarized endothelial and epithelial cells. In this study, we investigated whether the entry and release of DENV was polarized in epithelial cells, and evaluated the effect of DENV infection on cellular junctions of epithelial cells. We used MDCK epithelial cells, which serve as an excellent model to study a functional barrier due to the presence of an apical junctional complex (AJC), and showed that entry and release of DENV from the cells, is bipolar. Additionally, we performed paracellular flux, diffusion of membrane lipid, immunofluorescence and immunoblotting assays to evaluate the integrity of the AJC during DENV infection. We observed that at later stages of infection, DENV altered the barrier function causing a decrease in the transepithelial electrical resistance and the degradation and delocalization of TJ and AJ proteins. The present study contributes to understand how DENV traverse epithelia in order to cause a productive infection, and provides insights into the mechanism of DENV pathogenesis.
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Vírus da Dengue/fisiologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Internalização do Vírus , Animais , Dengue/virologia , Cães , Células Madin Darby de Rim CaninoRESUMO
The respiratory syncytial virus (RSV) is the most prevalent etiological agent of lower respiratory tract infections and the first cause of hospitalization in infants due to respiratory disease worldwide. However, efforts to develop safe and effective vaccines and antivirals have been challenged by an incomplete understanding of the RSV pathogenesis and the host immune response to RSV infection in the airways. Here, we discuss recent advances in understanding the interaction between RSV and the epithelium to induce pathogenesis in the airways, such as the role of the RSV NS2 protein in the airway epithelium, as well as the events involved in the RSV entry process. In addition, we summarize the cellular factors produced by airway epithelial cells (AECs) in response to RSV infection that lead to the activation of innate and adaptive immune responses, inducing lung inflammation and disease. Further, we discuss the possible contribution of a recently identified cytokine, thymic stromal lymphopoitein (TSLP), in the lung immunopathology caused by RSV.