scholarly journals Akt Kinase Intervenes in Flavivirus Replication by Interacting with Viral Protein NS5

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 896
Author(s):  
Laura Albentosa-González ◽  
Nereida Jimenez de Oya ◽  
Armando Arias ◽  
Pilar Clemente-Casares ◽  
Miguel Ángel Martin-Acebes ◽  
...  
Keyword(s):  
Cell Culture ◽  
West Nile Virus ◽  
Cell Metabolism ◽  
Polymerase Activity ◽  
Akt Pathway ◽  
Cellular Functions ◽  
The World ◽  
Extension Activity ◽  
Human Illness

Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target.

scholarly journals Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

2012 ◽  
Vol 56 (8) ◽  
pp. 4277-4288 ◽  
Author(s):  
Dawei Cai ◽  
Courtney Mills ◽  
Wenquan Yu ◽  
Ran Yan ◽  
Carol E. Aldrich ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Polymerase Activity ◽  
Circular Dna ◽  
Drug Candidates ◽  
Covalently Closed Circular Dna ◽  
Hbv Cccdna ◽  
B Virus

ABSTRACTHepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in thein vitroendogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

scholarly journals Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection in vitro and in vivo

2021 ◽  
Author(s):  
Aleksandr Ianevski ◽  
Rouan Yo ◽  
Hilde Lysvand ◽  
Gunnveig Grodeland ◽  
Nicolas Legrand ◽  
...  
Keyword(s):  
Public Health ◽  
Patient Outcome ◽  
Cell Culture ◽  
Antiviral Activity ◽  
Critical Role ◽  
Low Doses ◽  
Important Mediator ◽  
The World

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

scholarly journals Potential Therapeutic Agents for Feline Calicivirus Infection

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 433 ◽  
Author(s):  
Tulio M. Fumian ◽  
Daniel Enosi Tuipulotu ◽  
Natalie E. Netzler ◽  
Jennifer H. Lun ◽  
Alice G. Russo ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Throughput Screening ◽  
Polymerase Activity ◽  
In Vitro Assays ◽  
Feline Calicivirus ◽  
Upper Respiratory Tract ◽  
Direct Acting Antivirals ◽  
Ic50 Values ◽  
Direct Acting

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 μM and 2.7 μM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 μM and 2.5 μM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.

scholarly journals Iota-carrageenan and Xylitol inhibit SARS-CoV-2 in cell culture

2020 ◽  
Author(s):  
Shruti Bansal ◽  
Colleen B. Jonsson ◽  
Shannon L. Taylor ◽  
Juan Manuel Figueroa ◽  
Andrea Vanesa Dugour ◽  
...  
Keyword(s):  
Cell Culture ◽  
Severe Acute Respiratory Syndrome ◽  
Nasal Spray ◽  
Cell Cultures ◽  
Dosage Form ◽  
Unmet Need ◽  
Antiviral Action ◽  
Nasal Sprays ◽  
The World

AbstractCOVID-19 (coronavirus disease 2019) is a pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) infection affecting millions of persons around the world. There is an urgent unmet need to provide an easy-to-produce, affordable medicine to prevent transmission and provide early treatment for this disease. The nasal cavity and the rhinopharynx are the sites of initial replication of SARS-CoV-2. Therefore, a nasal spray may be a suitable dosage form for this purpose. The main objective of our study was to test the antiviral action of three candidate nasal spray formulations against SARS-CoV-2. We have found that iota-carrageenan in concentrations as low as 6 µg/ mL inhibits SARS-CoV-2 infection in Vero cell cultures. The concentrations found to be active in vitro against SARS-CoV-2 may be easily achieved by the application of nasal sprays already marketed in several countries. Xylitol at a concentration of 5 % m/V has proved to be viricidal on its own and the association with iota-carrageenan may be beneficial, as well.

scholarly journals The Effect of Chinese Medicine on Bone Cell Activities

2002 ◽  
Vol 30 (02n03) ◽  
pp. 271-285 ◽  
Author(s):  
Chun-Yu Lin ◽  
Jui-Sheng Sun ◽  
Shiow-Yunn Sheu ◽  
Feng-Huei Lin ◽  
Yng-Jiin Wang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Beneficial Effect ◽  
Bone Cells ◽  
Colorimetric Assay ◽  
Cell Metabolism ◽  
Cell Activity ◽  
Bone Cell ◽  
Chinese Medicines ◽  
Bone Cell Culture

In this experiment, we investigate the biochemical effects of traditional Chinese medicines via an in vitro bone cell culture. Ten different Chinese medicines were used in this study. The rat osteoblast-osteoclast co-culture system was used as the experimental model. After the cells grew to 80% confluence, various tested materials were added. The mitochondria activity of the bone cells after exposure to various preparations of Chinese medicines was determined by colorimetric assay. Biochemical markers such as protein content, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and acid phosphatase (ACP) titer were analyzed to evaluate the bone cell activity. When cultured with various Chinese medicines for 24 hours, only four of these ten Chinese medicines had potential beneficial effects on the bone cell culture; and only Drynaria fortunei (Kunze) J. Sm. had a universal beneficial effect on bone cell metabolism. The major beneficial effect of Drynaria fortunei (Kunze) J. Sm. on bone cells is probably mediated by the induction of apoptosis of the osteoclast cell population. Continued study of alterations in gene expression of bone cells caused by Chinese medicines will improve our understanding of bone cell responses to various pharmacological interventions.

scholarly journals NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells

2018 ◽  
Author(s):  
Julia Becker-Kettern ◽  
Nicole Paczia ◽  
Jean-François Conrotte ◽  
Chenchen Zhu ◽  
Oliver Fiehn ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Early Death ◽  
Human Cells ◽  
Repair System ◽  
Cellular Functions ◽  
Repair Deficiency ◽  
Domains Of Life ◽  
Metabolite Repair

ABSTRACTNADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenasesin vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both theS- andR-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivativesin vivoand their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD+and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair deficient children.

scholarly journals Nafamostat–Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1768
Author(s):  
Aleksandr Ianevski ◽  
Rouan Yao ◽  
Hilde Lysvand ◽  
Gunnveig Grødeland ◽  
Nicolas Legrand ◽  
...  
Keyword(s):  
Public Health ◽  
Patient Outcome ◽  
Cell Culture ◽  
Critical Role ◽  
Interferon Α ◽  
Low Doses ◽  
Important Mediator ◽  
The World

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

scholarly journals Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

2010 ◽  
Vol 6 (11) ◽  
pp. e1001176 ◽  
Author(s):  
Aartjan J. W. te Velthuis ◽  
Sjoerd H. E. van den Worm ◽  
Amy C. Sims ◽  
Ralph S. Baric ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Rna Polymerase Activity

Hormonal dependency of cerebral meningiomas

1990 ◽  
Vol 73 (5) ◽  
pp. 750-755 ◽  
Author(s):  
Eric F. Adams ◽  
Uwe M. H. Schrell ◽  
Rudolf Fahlbusch ◽  
Paul Thierauf
Keyword(s):  
Cell Culture ◽  
Dna Polymerase ◽  
Cell Cultures ◽  
Polymerase Activity ◽  
Proliferative Potential ◽  
Content Type ◽  
Androgen Antagonists ◽  
Epidermal Growth ◽  
Meningioma Cell

✓ Cell culture and biochemical techniques have been employed to examine the effects of steroids, bromocriptine, and epidermal growth factor (EGF) on the growth and proliferative potential of meningiomas. In cell culture, the growth of meningiomas was not altered by progestogens, antiprogestogens, or 17β-estradiol. The progestogen, norethisterone, had no effect on the uptake by meningioma cell cultures of 3H-thymidine. Furthermore, cytosolic deoxyribonucleic acid (DNA) polymerase activity of meningiomas did not correlate with the progesterone receptor status of the same tumors. In contrast, the androgen antagonists, cyproterone acetate and 11-α-hydroxyprogesterone, and the dopamine agonist, bromocriptine, all inhibited the in vitro growth of meningioma cells. The growth of meningioma cell cultures was stimulated by EGF, and there was a positive correlation between the EGF content and DNA polymerase activity in meningioma cytosols. These results demonstrate that female sex steroids do not influence growth of meningiomas in vitro, whereas antiandrogens and bromocriptine have an antiproliferative effect. Consequently, bromocriptine and antiandrogens may have a role in the medical treatment of meningiomas. In addition, these results suggest that EGF may be involved in the genesis and/or progression of meningiomas.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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