scholarly journals Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection

2021 ◽  
Author(s):  
Qi Zhang ◽  
Peter Radvak ◽  
Juhyung Lee ◽  
Yue Xu ◽  
Vivian Cao-Dao ◽  
...  
Keyword(s):  
Viral Entry ◽  
Alternative Therapy ◽  
Single Agent ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Dna Topoisomerase ◽  
Nucleosome Dynamics ◽  
Protein Receptors ◽  
Attractive Therapeutic Target ◽  
A Cell

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

On the Role of Continuum Models in Mechanobiology

2000 ◽  
Author(s):  
R. D. Kamm ◽  
A. K. McVittie ◽  
M. Bathe
Keyword(s):  
Basolateral Membrane ◽  
Biological Response ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Stress Distributions ◽  
Dynamic Simulations ◽  
Mechanics Model ◽  
A Cell ◽  
Underlying Mechanisms

Abstract Models of cellular and subcellular mechanics are essential in gaining an understanding of the link between forces applied to a cell and its biological response. Various approaches can be used to construct these models, but one that holds considerable promise is through the use of continuum solid and fluid mechanics, coupled when necessary with molecular dynamic simulations. Here we present a continuum mechanics model of the effects of normal stress applied to a layer of airway epithelial cells. The model predicts widely differing stress distributions associated with basolateral membrane deformation, intercellular flow, or mechanical forcing by other methods such as in the use of adherent beads. When coupled with experimental results obtained from cell culture, these simulations can help to identify the site and possibly the underlying mechanisms of mechanotransduction. We also present some hypotheses concerning the nature of mechanotransduction at the molecular scale.

The novel antiviral properties of brassicasterol against human adenovirus

2021 ◽  
Author(s):  
Peifeng Yu ◽  
Dan Lou ◽  
Lifeng Qi ◽  
Zewei Chen
Keyword(s):  
Dna Replication ◽  
Epithelial Cells ◽  
Viral Entry ◽  
Human Adenovirus ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
The Novel ◽  
Airway Epithelial ◽  
Infected Cells ◽  
Dose Dependent

Aim: To investigate whether brassicasterol has inhibitory effects against adenovirus (AdV). Materials and methods: The antiviral effects of brassicasterol against AdV 3 and 7 were tested in human airway epithelial cells. Brassicasterol cytotoxicity was assessed by WST-1 assay. AdV DNA was quantified by qPCR. Results: Brassicasterol inhibited AdV 3 and 7 infection of airway epithelial cells in a dose-dependent manner. Similarly, brassicasterol also inhibited AdV 3 and 7 production in infected cells. No apparent cytotoxicity of brassicasterol was detected. Further study showed that brassicasterol inhibited AdV DNA replication, but had no impact on viral entry of cells and viral genome import to nucleus. Conclusion: Brassicasterol exerts anti-AdV effects probably through the inhibition of viral DNA replication.

scholarly journals Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures

2021 ◽  
Vol 4 (1) ◽  
pp. 7
Author(s):  
Kaitlyn A. Barrow ◽  
Lucille M. Rich ◽  
Elizabeth R. Vanderwall ◽  
Stephen R. Reeves ◽  
Jennifer A. Rathe ◽  
...  
Keyword(s):  
Viral Entry ◽  
Uv Light ◽  
Sodium Dodecyl ◽  
Airway Epithelial Cells ◽  
Protein Isolate ◽  
Airway Epithelial ◽  
Innate Immune Responses ◽  
Triton X100 ◽  
Control And Prevention ◽  
Level 3

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

scholarly journals TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice

2021 ◽  
Author(s):  
Young Joo Sun ◽  
Gabriel Velez ◽  
Dylan Parsons ◽  
Kun Li ◽  
Miguel Ortiz ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cell Infection ◽  
Effective Inhibition ◽  
Transmembrane Serine Protease

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats

2005 ◽  
Vol 288 (3) ◽  
pp. L523-L529 ◽  
Author(s):  
Sarit Offer ◽  
Saul Yedgar ◽  
Ouri Schwob ◽  
Miron Krimsky ◽  
Haim Bibi ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Inflammatory Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Membrane Phospholipids ◽  
Cytosolic Phospholipase ◽  
Inflammatory Processes ◽  
A Cell ◽  
First Time

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.

Effects of dexamethasone on Muc5ac mucin production by primary airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. L52-L60 ◽  
Author(s):  
Wenju Lu ◽  
Erik P. Lillehoj ◽  
K. Chul Kim
Keyword(s):  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucus Production ◽  
Mucin Production ◽  
Proinflammatory Mediators ◽  
A Cell ◽  
The Stability

Mucus hypersecretion associated with airway inflammation is reduced by glucocorticoids. Two mechanisms of glucocorticoid-mediated inhibition of mucus production have been proposed, direct inhibition of mucus production by airway epithelial cells and indirectly through inhibition of proinflammatory mediators that stimulate mucus production. In this study, we examined the effect of dexamethasone (DEX) on mRNA expression and synthesis of MUC5AC by A549 human lung adenocarcinoma cells as well as Muc5ac and total high-molecular-weight (HMW) mucins by primary rat tracheal surface epithelial (RTSE) cells. Our results showed that in primary RTSE cells, DEX 1) dose dependently suppressed Muc5ac mRNA levels, but the levels of cellular Muc5ac protein and HMW mucins were unaffected; 2) did not affect constitutive or UTP-stimulated mucin secretion; 3) enhanced the translation of Muc5ac; and 4) increased the stability of intracellular Muc5ac protein by a mechanism other than the inhibition of the proteasomal degradation. In A549 cells, however, DEX suppressed both MUC5AC mRNA levels and MUC5AC protein secretion in a dose-dependent manner. We conclude that whereas DEX inhibits the levels of Muc5ac mRNA in primary RTSE cells, the levels of Muc5ac protein remain unchanged as a consequence of increases in both translation and protein stability. Interestingly, some of the effects of DEX were opposite in a cell line.

scholarly journals SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krzysztof Pyrć ◽  
Aleksandra Milewska ◽  
Emilia Barreto Duran ◽  
Paweł Botwina ◽  
Agnieszka Dabrowska ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
Statistical Significance ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
In Vivo Studies ◽  
Repeat Dose ◽  
Airway Epithelial ◽  
Viral Inhibition

AbstractThere are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of − 3 to − 4 at a concentration of 10–100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

scholarly journals Targeting a central feature of asthma using a cell type-selective IL-13-responsive enhancer

2021 ◽  
Author(s):  
Kyung Duk Koh ◽  
Luke R Bonser ◽  
Walter L Eckalbar ◽  
Jiangshan Shen ◽  
Ofer Yizhar-Barnea ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Airway Epithelial Cells ◽  
Secretory Cells ◽  
Central Feature ◽  
Airway Epithelial ◽  
Cell Type ◽  
Mucin Production ◽  
Genome Wide ◽  
A Cell

IL-13 is a central mediator of asthma. Here, we used genome-wide approaches to characterize genes and regulatory elements modulated by IL-13 and other asthma-associated cytokines in airway epithelial cells and showed how they can be used for therapeutic purposes. Using bulk and single cell RNA-seq, we found distinctive responses to IL-13, IL-17, and interferons in human bronchial epithelial basal, ciliated, and secretory cells. H3K27ac ChIP-seq revealed that IL-13 had widespread effects on regulatory elements. Detailed characterization of an enhancer of SPDEF, a transcription factor required for pathologic mucin production, revealed that STAT6 and KLF5 binding sites cooperate to drive IL-13-dependent transcription selectively in secretory cells. Using this enhancer to drive CRISPRi and knockdown either SPDEF or the mucin MUC5AC showed the potential use of this approach for asthma therapeutics. This work identifies numerous genes and regulatory elements involved in cell type-selective cytokine responses and showcases their use for therapeutic purposes.

scholarly journals SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

2020 ◽  
Author(s):  
Krzysztof Pyrć ◽  
Aleksandra Milewska ◽  
Emilia Barreto Duran ◽  
Paweł Botwina ◽  
Rui Lopes ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
Repeat Dose ◽  
Airway Epithelial ◽  
Viral Inhibition ◽  
Human Airway ◽  
Adverse Effect Level ◽  
Human Airway Epithelial Cells

AbstractThere are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of −3 to −4 at a concentration of 10 – 100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

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