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1.
Immunol Lett ; 237: 33-41, 2021 09.
Article in English | MEDLINE | ID: covidwho-1293862

ABSTRACT

OBJECTIVE: In this study, we focused on the interaction between SARS-CoV-2 and host Type I Interferon (IFN) response, so as to identify whether IFN effects could be influenced by the products of SARS-CoV-2. METHODS: All the structural and non-structural proteins of SARS-CoV-2 were transfected and overexpressed in the bronchial epithelial cell line BEAS-2B respectively, and typical antiviral IFN-stimulated gene (ISG) ISG15 expression was detected by qRT-PCR. RNA-seq based transcriptome analysis was performed between control and Spike (S) protein-overexpressed BEAS-2B cells. The expression of ACE2 and IFN effector JAK-STAT signaling activation were detected in control and S protein-overexpressed BEAS-2B cells by qRT-PCR or/and Western blot respectively. The interaction between S protein with STAT1 and STAT2, and the association between JAK1 with downstream STAT1 and STAT2 were measured in BEAS-2B cells by co-immunoprecipitation (co-IP). RESULTS: S protein could activate IFN effects and downstream ISGs expression. By transcriptome analysis, overexpression of S protein induced a set of genes expression, including series of ISGs and the SARS-CoV-2 receptor ACE2. Mechanistically, S protein enhanced the association between the upstream JAK1 and downstream STAT1 and STAT2, so as to promote STAT1 and STAT2 phosphorylation and ACE2 expression. CONCLUSION: SARS-CoV-2 S protein enhances ACE2 expression via facilitating IFN effects, which may help its infection.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Bronchi/drug effects , COVID-19/virology , Epithelial Cells/drug effects , Interferon alpha-2/pharmacology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Bronchi/enzymology , Bronchi/virology , COVID-19/enzymology , Cytokines/genetics , Cytokines/metabolism , Epithelial Cells/enzymology , Epithelial Cells/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Janus Kinase 1/metabolism , Phosphorylation , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Ubiquitins/genetics , Ubiquitins/metabolism , Up-Regulation
2.
J Biol Chem ; 296: 100701, 2021.
Article in English | MEDLINE | ID: covidwho-1198856

ABSTRACT

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.


Subject(s)
Ambroxol/pharmacology , Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , Sphingomyelin Phosphodiesterase/genetics , Vesiculovirus/drug effects , Virus Internalization/drug effects , Administration, Inhalation , Animals , Biological Transport , Ceramides/metabolism , Chlorocebus aethiops , Drug Repositioning , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/virology , Expectorants , Gene Expression , Humans , Primary Cell Culture , Reassortant Viruses/drug effects , Reassortant Viruses/physiology , SARS-CoV-2/physiology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/metabolism , Sphingomyelins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Vesiculovirus/physiology
3.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1109085

ABSTRACT

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Subject(s)
Antiviral Agents/pharmacology , Epithelial Cells/drug effects , Hepatocytes/drug effects , Phenylalanine/pharmacology , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Cell Line , Cell Survival/drug effects , Epithelial Cells/cytology , Epithelial Cells/enzymology , Gene Expression Regulation/drug effects , Hepatocytes/cytology , Hepatocytes/enzymology , Humans , Hydrogen Peroxide/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Intestinal Mucosa/enzymology , Occludin/genetics , Occludin/metabolism , Oxidation-Reduction/drug effects , Phenylalanine/analogs & derivatives , Primary Cell Culture , Serine Endopeptidases/genetics
4.
J Pharm Pharm Sci ; 24: 84-93, 2021.
Article in English | MEDLINE | ID: covidwho-1100474

ABSTRACT

Angiotensin converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. ACE2 is one of the key enzymes in renin-angiotensin system and plays a vital role in the maintenance of cardiovascular function. ACE/ACE2 balance is critical in the regulation of blood pressure, electrolyte homeostasis, vascular and cardiac remodeling and inflammation. ACE2 was shown to be abundantly present in human epithelial cells of the lung and enterocytes of the small intestine as well as in endothelial cells of the arterial and venous vessels. ACE2 and TMPRSS2 are colocalized on the cell surface and produced a critical step host cell entry of SARS-CoV-2. TMPRSS2-cleaved ACE2 permits SARS-CoV-2 host cell entry however, ADAM17-cleaved ACE2 produces protective effects in several organs. Differently, basigin (CD147) was suggested as a putative alternate receptor for SARS-CoV-2 entry into endothelial cells. The intestinal ACE2 receptor is associated with the neutral amino acid transporter B0AT1 and ACE2 is necessary for the expression of this transporter on the luminal surface of intestinal epithelial cells. There is a good association between the localization of SARS-CoV-2 binding receptor ACE2 and the disease target organs in respiratory, cardiovascular and gastrointestinal systems. Decreased expression of ACE2, being a receptor for coronavirus, would prevent cellular entry of the virus thereby reducing progression of the infection. However, increased ACE2 expression produces beneficial health effects. Further studies are needed to clarify this conflicting situation. Currently, it is recommended to continue the therapy with ACE2-increasing drugs in patients with COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/enzymology , Endothelial Cells/enzymology , Epithelial Cells/enzymology , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Virus Internalization , Animals , COVID-19/virology , Endothelial Cells/virology , Epithelial Cells/virology , Host-Pathogen Interactions , Humans , Signal Transduction
5.
J Biol Chem ; 296: 100449, 2021.
Article in English | MEDLINE | ID: covidwho-1091794

ABSTRACT

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.


Subject(s)
Curcumin/pharmacology , Drug Design , Epithelial Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Animals , Cell Line, Tumor , Cloning, Molecular , Curcumin/analogs & derivatives , Curcumin/chemical synthesis , Drug Stability , Epithelial Cells/enzymology , Epithelial Cells/pathology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HT29 Cells , Humans , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/chemical synthesis , /metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolism , RAW 264.7 Cells , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , src-Family Kinases/genetics , src-Family Kinases/metabolism
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