scholarly journals Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Identification of active compounds

2021 ◽  
Vol 29 ◽  
pp. 204020662110268
Author(s):  
Jana Van Dycke ◽  
Jasper Rymenants ◽  
Johan Neyts ◽  
Joana Rocha-Pereira
Keyword(s):  
Cell Culture ◽  
Small Molecules ◽  
Mechanism Of Action ◽  
Antiviral Effect ◽  
Viral Gastroenteritis ◽  
Human Norovirus ◽  
In Vitro System ◽  
Insight Into ◽  
Detailed Protocol

Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. As potent and safe anti-norovirus small molecules are urgently needed, we here describe the detailed protocol for a set of assays that will allow the identification of novel norovirus inhibitors.

scholarly journals Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Early mechanistic studies

2021 ◽  
Vol 29 ◽  
pp. 204020662110251
Author(s):  
Jana Van Dycke ◽  
Jasper Rymenants ◽  
Johan Neyts ◽  
Joana Rocha-Pereira
Keyword(s):  
Small Molecules ◽  
Continuous Improvement ◽  
Antiviral Effect ◽  
Viral Gastroenteritis ◽  
Mechanistic Studies ◽  
Preclinical Development ◽  
In Vitro System ◽  
Insight Into

Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. After the identification of novel small molecules with anti-norovirus activity (part 1 of this protocol), we here describe the logical next step which entails the generation of early information of their mode of action. This information together with a continuous improvement of the potency of compounds will contribute to the optimization of a compound class towards in vivo efficacy and a successful preclinical development.

scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Epigenetic Regulator ◽  
Novel Target ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

AbstractAging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

scholarly journals Enisamium Inhibits SARS-CoV-2 RNA Synthesis

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1254
Author(s):  
Stefano Elli ◽  
Denisa Bojkova ◽  
Marco Bechtel ◽  
Thomas Vial ◽  
David Boltz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Polymerase ◽  
Influenza A ◽  
Rna Synthesis ◽  
Herd Immunity ◽  
Virus Rna ◽  
Independent States ◽  
Dynamics Simulations ◽  
Insight Into

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.

scholarly journals Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Laura Schaedel ◽  
Charlotta Lorenz ◽  
Anna V. Schepers ◽  
Stefan Klumpp ◽  
Sarah Köster
Keyword(s):  
Intermediate Filaments ◽  
Cell Mechanics ◽  
Physical Nature ◽  
Cellular Functions ◽  
Interaction Forces ◽  
In Vitro System ◽  
Direct Mechanism ◽  
Cytoskeletal Dynamics ◽  
Insight Into

AbstractThe cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components—actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

scholarly journals Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials

2008 ◽  
Vol 52 (4) ◽  
pp. 1291-1296 ◽  
Author(s):  
Darren J. Creek ◽  
William N. Charman ◽  
Francis C. K. Chiu ◽  
Richard J. Prankerd ◽  
Yuxiang Dong ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Antimalarial Activity ◽  
Biologically Active ◽  
Radical Intermediate ◽  
Reaction Conditions ◽  
Artemisinin Derivatives ◽  
Major Reaction Product ◽  
Major Reaction ◽  
Insight Into

ABSTRACT The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R 2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study.

scholarly journals The Enterovirus Protease Inhibitor Rupintrivir Exerts Cross-Genotypic Anti-Norovirus Activity and Clears Cells from the Norovirus Replicon

2014 ◽  
Vol 58 (8) ◽  
pp. 4675-4681 ◽  
Author(s):  
J. Rocha-Pereira ◽  
M. S. J. Nascimento ◽  
Q. Ma ◽  
R. Hilgenfeld ◽  
J. Neyts ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Murine Norovirus ◽  
Norwalk Virus ◽  
Rna Dependent Rna Polymerase ◽  
Human Norovirus ◽  
Polymerase Inhibitors ◽  
Murine Noroviruses

ABSTRACTPotent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that thein vitroanti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2′-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

scholarly journals Impact of IL-27 on hepatocyte antiviral gene expression and function

2016 ◽  
Vol 1 ◽  
pp. 17 ◽  
Author(s):  
Narayan Ramamurthy ◽  
Sara Boninsegna ◽  
Rebecca Adams ◽  
Natasha Sahgal ◽  
Helen Lockstone ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antigen Presentation ◽  
Microarray Analysis ◽  
Enrichment Analysis ◽  
Cell Types ◽  
Antiviral Effect ◽  
Gene Set Enrichment Analysis ◽  
And Function ◽  
The Impact

Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.

scholarly journals Vimentin Intermediate Filaments Stabilize Dynamic Microtubules by Direct Interactions

2020 ◽  
Author(s):  
Laura Schaedel ◽  
Charlotta Lorenz ◽  
Anna V. Schepers ◽  
Stefan Klumpp ◽  
Sarah Köster
Keyword(s):  
Intermediate Filaments ◽  
Cell Mechanics ◽  
Physical Nature ◽  
Cellular Functions ◽  
In Vitro System ◽  
Direct Mechanism ◽  
Cytoskeletal Dynamics ◽  
Novel Strategy ◽  
Insight Into

AbstractThe cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components, known as actin filaments, intermediate filaments and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a re-constituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism for cells to establish the fundamental cross-talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a novel strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

scholarly journals Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance

2021 ◽  
Vol 14 (10) ◽  
pp. 1006
Author(s):  
Jana Van Dycke ◽  
Michela Puxeddu ◽  
Giuseppe La Regina ◽  
Eloise Mastrangelo ◽  
Delia Tarantino ◽  
...  
Keyword(s):  
Small Molecules ◽  
Large Scale ◽  
Selective Pressure ◽  
Rna Replication ◽  
Societal Cost ◽  
Viral Gastroenteritis ◽  
Good Activity ◽  
Design And Synthesis ◽  
Time Point

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre (Sapienza University, Rome). Compound 3-((3,5-dimethylphenyl)sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound 1) was identified as an inhibitor of MNV replication with an EC50 of 0.5 ± 0.1 µM. A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 ± 0.1 µM) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 ± 0.6 µM). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.

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