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1.
Biomed Res Int ; 2022: 1558860, 2022.
Article in English | MEDLINE | ID: covidwho-1622112

ABSTRACT

Increasing outbreaks of new pathogenic viruses have promoted the exploration of novel alternatives to time-consuming vaccines. Thus, it is necessary to develop a universal approach to halt the spread of new and unknown viruses as they are discovered. One such promising approach is to target lipid membranes, which are common to all viruses and bacteria. The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has reaffirmed the importance of interactions between the virus envelope and the host cell plasma membrane as a critical mechanism of infection. Metadichol®, a nanolipid emulsion of long-chain alcohols, has been demonstrated as a strong candidate that inhibits the proliferation of SARS-CoV-2. Naturally derived substances, such as long-chain saturated lipid alcohols, reduce viral infectivity, including that of coronaviruses (such as SARS-CoV-2) by modifying their lipid-dependent attachment mechanism to human host cells. The receptor ACE2 mediates the entry of SARS-CoV-2 into the host cells, whereas the serine protease TMPRSS2 primes the viral S protein. In this study, Metadichol® was found to be 270 times more potent an inhibitor of TMPRSS2 (EC50 = 96 ng/mL) than camostat mesylate (EC50 = 26000 ng/mL). Additionally, it inhibits ACE with an EC50 of 71 ng/mL, but it is a very weak inhibitor of ACE2 at an EC50 of 31 µg/mL. Furthermore, the live viral assay performed in Caco-2 cells revealed that Metadichol® inhibits SARS-CoV-2 replication at an EC90 of 0.16 µg/mL. Moreover, Metadichol® had an EC90 of 0.00037 µM, making it 2081 and 3371 times more potent than remdesivir (EC50 = 0.77 µM) and chloroquine (EC50 = 1.14 µM), respectively.


Subject(s)
Fatty Alcohols/pharmacology , SARS-CoV-2/drug effects , Viruses/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cell Line , Chlorocebus aethiops , Esters/pharmacology , Guanidines/pharmacology , Humans , Lipid Metabolism/physiology , Lipids/chemistry , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/drug effects , Serine Endopeptidases/metabolism , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Attachment/drug effects , Virus Internalization/drug effects
2.
Front Immunol ; 12: 781352, 2021.
Article in English | MEDLINE | ID: covidwho-1613552

ABSTRACT

After the outburst of the SARS-CoV-2 pandemic, a worldwide research effort has led to the uncovering of many aspects of the COVID-19, among which we can count the outstanding role played by inflammatory cytokine milieu in the disease progression. Despite that, molecular mechanisms that regulate SARS-CoV-2 pathogenesis are still almost unidentified. In this study, we investigated whether the pro-inflammatory milieu of the host affects the susceptibility of SARS-CoV-2 infection by modulating ACE2 and TMPRSS2 expression. Our results indicated that the host inflammatory milieu favors SARS-CoV-2 infection by directly increasing TMPRSS2 expression. We unveiled the molecular mechanism that regulates this process and that can be therapeutically advantageously targeted.


Subject(s)
GATA2 Transcription Factor/metabolism , Interleukin-1beta/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , A549 Cells , COVID-19 , Humans , p38 Mitogen-Activated Protein Kinases/metabolism
3.
J Neuroinflammation ; 19(1): 8, 2022 Jan 06.
Article in English | MEDLINE | ID: covidwho-1613238

ABSTRACT

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.


Subject(s)
Benzamidines , Guanidines , Inflammation/drug therapy , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors , Toll-Like Receptor 7/immunology , Virus Diseases/drug therapy , Animals , Benzamidines/pharmacology , Benzamidines/therapeutic use , COVID-19/complications , COVID-19/drug therapy , Guanidines/pharmacology , Guanidines/therapeutic use , Illness Behavior/drug effects , Imidazoles/administration & dosage , Imidazoles/immunology , Inflammation/metabolism , Inflammation/virology , Male , Mice , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/therapeutic use , Toll-Like Receptor 7/agonists , Virus Diseases/metabolism , Virus Diseases/virology
5.
Int J Mol Med ; 49(2)2022 02.
Article in English | MEDLINE | ID: covidwho-1594678

ABSTRACT

The pathophysiology of coronavirus disease 2019 (COVID­19) is mainly dependent on the underlying mechanisms that mediate the entry of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) into the host cells of the various human tissues/organs. Recent studies have indicated a higher order of complexity of the mechanisms of infectivity, given that there is a wide­repertoire of possible cell entry mediators that appear to co­localise in a cell­ and tissue­specific manner. The present study provides an overview of the 'canonical' SARS­CoV­2 mediators, namely angiotensin converting enzyme 2, transmembrane protease serine 2 and 4, and neuropilin­1, expanding on the involvement of novel candidates, including glucose­regulated protein 78, basigin, kidney injury molecule­1, metabotropic glutamate receptor subtype 2, ADAM metallopeptidase domain 17 (also termed tumour necrosis factor­α convertase) and Toll­like receptor 4. Furthermore, emerging data indicate that changes in microRNA (miRNA/miR) expression levels in patients with COVID­19 are suggestive of further complexity in the regulation of these viral mediators. An in silico analysis revealed 160 candidate miRNAs with potential strong binding capacity in the aforementioned genes. Future studies should concentrate on elucidating the association between the cellular tropism of the SARS­CoV­2 cell entry mediators and the mechanisms through which they might affect the clinical outcome. Finally, the clinical utility as a biomarker or therapeutic target of miRNAs in the context of COVID­19 warrants further investigation.


Subject(s)
COVID-19/metabolism , MicroRNAs/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , /metabolism , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , Neuropilin-1/genetics , Neuropilin-1/metabolism , Receptors, Virus/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Viral Tropism
6.
Pediatr Infect Dis J ; 41(2): e36-e45, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1599648

ABSTRACT

Although there are many hypotheses for the age-related difference in the severity of COVID-19, differences in innate, adaptive and heterologous immunity, together with differences in endothelial and clotting function, are the most likely mechanisms underlying the marked age gradient. Children have a faster and stronger innate immune response to SARS-CoV-2, especially in the nasal mucosa, which rapidly controls the virus. In contrast, adults can have an overactive, dysregulated and less effective innate response that leads to uncontrolled pro-inflammatory cytokine production and tissue injury. More recent exposure to other viruses and routine vaccines in children might be associated with protective cross-reactive antibodies and T cells against SARS-CoV-2. There is less evidence to support other mechanisms that have been proposed to explain the age-related difference in outcome following SARS-CoV-2 infection, including pre-existing immunity from exposure to common circulating coronaviruses, differences in the distribution and expression of the entry receptors ACE2 and TMPRSS2, and difference in viral load.


Subject(s)
Adaptive Immunity , Age Factors , COVID-19/immunology , Immunity, Heterologous , Immunity, Innate , SARS-CoV-2/immunology , Adult , Angiotensin-Converting Enzyme 2/metabolism , Blood Coagulation/immunology , Child , Cross Protection , Cross Reactions , Endothelium/immunology , Humans , Patient Acuity , Serine Endopeptidases/metabolism , Viral Load/immunology
7.
Sci Rep ; 11(1): 23993, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585801

ABSTRACT

Previous work indicates that SARS-CoV-2 virus entry proteins angiotensin-converting enzyme 2 (ACE-2) and the cell surface transmembrane protease serine 2 (TMPRSS-2) are regulated by sex hormones. However, clinical studies addressing this association have yielded conflicting results. We sought to analyze the impact of sex hormones, age, and cardiovascular disease on ACE-2 and TMPRSS-2 expression in different mouse models. ACE-2 and TMPRSS-2 expression was analyzed by immunostaining in a variety of tissues obtained from FVB/N mice undergoing either gonadectomy or sham-surgery and being subjected to ischemia-reperfusion injury or transverse aortic constriction surgery. In lung tissues sex did not have a significant impact on the expression of ACE-2 and TMPRSS-2. On the contrary, following myocardial injury, female sex was associated to a lower expression of ACE-2 at the level of the kidney tubules. In addition, after myocardial injury, a significant correlation between younger age and higher expression of both ACE-2 and TMPRSS-2 was observed for lung alveoli and bronchioli, kidney tubules, and liver sinusoids. Our experimental data indicate that gonadal hormones and biological sex do not alter ACE-2 and TMPRSS-2 expression in the respiratory tract in mice, independent of disease state. Thus, sex differences in ACE-2 and TMPRSS-2 protein expression observed in mice may not explain the higher disease burden of COVID-19 among men.


Subject(s)
Aging/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Cardiomyopathies/metabolism , Castration/adverse effects , Serine Endopeptidases/metabolism , Animals , Bronchioles/metabolism , Disease Models, Animal , Female , Gene Expression Regulation , Kidney Tubules/metabolism , Liver/metabolism , Male , Mice , Pulmonary Alveoli/metabolism , Virus Internalization
8.
Emerg Microbes Infect ; 11(1): 277-283, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1585239

ABSTRACT

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization , Virus Replication , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Chlorocebus aethiops , Chloroquine/pharmacology , Endocytosis/drug effects , Esters/pharmacology , Guanidines/pharmacology , Humans , Immune Evasion , Lung Neoplasms/pathology , Macrolides/pharmacology , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , Vero Cells , Virus Cultivation , Virus Internalization/drug effects , Whole Genome Sequencing
9.
Front Immunol ; 12: 714027, 2021.
Article in English | MEDLINE | ID: covidwho-1581346

ABSTRACT

In the coronavirus disease 2019 (COVID-19) health crisis, one major challenge is to identify the susceptibility factors of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in order to adapt the recommendations for populations, as well as to reduce the risk of COVID-19 development in the most vulnerable people, especially patients with chronic respiratory diseases such as cystic fibrosis (CF). Airway epithelial cells (AECs) play a critical role in the modulation of both immune responses and COVID-19 severity. SARS-CoV-2 infects the airway through the receptor angiotensin-converting enzyme 2, and a host protease, transmembrane serine protease 2 (TMPRSS2), plays a major role in SARS-CoV-2 infectivity. Here, we show that Pseudomonas aeruginosa increases TMPRSS2 expression, notably in primary AECs with deficiency of the ion channel CF transmembrane conductance regulator (CFTR). Further, we show that the main component of P. aeruginosa flagella, the protein flagellin, increases TMPRSS2 expression in primary AECs and Calu-3 cells, through activation of Toll-like receptor-5 and p38 MAPK. This increase is particularly seen in Calu-3 cells deficient for CFTR and is associated with an intracellular increased level of SARS-CoV-2 infection, however, with no effect on the amount of virus particles released. Considering the urgency of the COVID-19 health crisis, this result may be of clinical significance for CF patients, who are frequently infected with and colonized by P. aeruginosa during the course of CF and might develop COVID-19.


Subject(s)
Cystic Fibrosis , Flagellin/metabolism , Pseudomonas Infections/complications , Respiratory Mucosa/virology , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Bacterial Proteins/metabolism , COVID-19/complications , Cells, Cultured , Humans , Pseudomonas aeruginosa , Respiratory Mucosa/metabolism
10.
Gastroenterology ; 160(3): 925-928.e4, 2021 02.
Article in English | MEDLINE | ID: covidwho-1575253
11.
Biomed Pharmacother ; 146: 112513, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1575252

ABSTRACT

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.


Subject(s)
Antiviral Agents/metabolism , Cytochrome P-450 Enzyme System/metabolism , Protease Inhibitors/metabolism , Serine Endopeptidases/metabolism , Serum Albumin, Human/metabolism , Antiviral Agents/analysis , Antiviral Agents/pharmacology , Dose-Response Relationship, Drug , Humans , Isoenzymes/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Protease Inhibitors/analysis , Protease Inhibitors/pharmacology , Protein Binding/physiology , Serine Endopeptidases/analysis , Spectrometry, Fluorescence/methods , Tandem Mass Spectrometry/methods
12.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
13.
J Med Virol ; 93(12): 6671-6685, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544318

ABSTRACT

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Gastrointestinal Tract/virology , Genitalia/virology , Hepatitis A Virus Cellular Receptor 1/metabolism , Immune System/virology , Neuropilin-1/metabolism , Respiratory System/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization , Blotting, Western , COVID-19/metabolism , COVID-19/virology , Cell Line , Cells, Cultured , Fluorescent Antibody Technique , Gastrointestinal Tract/cytology , Genitalia/cytology , Humans , Immune System/cytology , Respiratory System/cytology
14.
Sovrem Tekhnologii Med ; 12(6): 98-108, 2021.
Article in English | MEDLINE | ID: covidwho-1527052

ABSTRACT

The rapidly accumulating information about the new coronavirus infection and the ambiguous results obtained by various authors necessitate further research aiming at prevention and treatment of this disease. At the moment, there is convincing evidence that the pathogen affects not only the respiratory but also the central nervous system (CNS). The aim of the study is to provide an insight into the molecular mechanisms underlying the damage to the CNS caused by the new coronavirus SARS-CoV-2. Results: By analyzing the literature, we provide evidence that the brain is targeted by this virus. SARS-CoV-2 enters the body with the help of the target proteins: angiotensin-converting enzyme 2 (ACE2) and associated serine protease TMPRSS2 of the nasal epithelium. Brain damage develops before the onset of pulmonary symptoms. The virus spreads through the brain tissue into the piriform cortex, basal ganglia, midbrain, and hypothalamus. Later, the substantia nigra of the midbrain, amygdala, hippocampus, and cerebellum become affected. Massive death of neurons, astrogliosis and activation of microglia develop at the next stage of the disease. By day 4, an excessive production of proinflammatory cytokines in the brain, local neuroinflammation, breakdown of the blood-brain barrier, and impaired neuroplasticity are detected. These changes imply the involvement of a vascular component driven by excessive activity of matrix metalloproteinases, mediated by CD147. The main players in the pathogenesis of COVID-19 in the brain are products of angiotensin II (AT II) metabolism, largely angiotensin 1-7 (AT 1-7) and angiotensin IV (AT IV). There are conflicting data regarding their role in damage to the CNS in various diseases, including the coronavirus infection.The second participant in the pathogenesis of brain damage in COVID-19 is CD147 - the inducer of extracellular matrix metalloproteinases. This molecule is expressed on the endothelial cells of cerebral microvessels, as well as on leukocytes present in the brain during neuroinflammation. The CD147 molecule plays a significant role in maintaining the structural and functional integrity of the blood-brain barrier by controlling the basal membrane permeability and by mediating the astrocyte-endothelial interactions. Via the above mechanisms, an exposure to SARS-CoV-2 leads to direct damage to the neurovascular unit of the brain.


Subject(s)
Astrocytes/metabolism , Brain/metabolism , COVID-19/metabolism , SARS-CoV-2/metabolism , Angiotensin I/metabolism , Angiotensin II/analogs & derivatives , Angiotensin II/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Basigin , Humans , Peptide Fragments/metabolism , Serine Endopeptidases/metabolism
15.
Biosci Rep ; 41(10)2021 10 29.
Article in English | MEDLINE | ID: covidwho-1510636

ABSTRACT

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30-60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Computational Biology/methods , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/immunology , COVID-19/immunology , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , HSP70 Heat-Shock Proteins/antagonists & inhibitors , Humans , Mitochondrial Proteins/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , SARS-CoV-2/immunology , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
16.
Front Biosci (Landmark Ed) ; 26(10): 740-751, 2021 10 30.
Article in English | MEDLINE | ID: covidwho-1498507

ABSTRACT

Objectives: To quantify the integrated levels of ACE2 and TMPRSS2, the two well-recognized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry-related genes, and to further identify key factors contributing to SARS-CoV-2 susceptibility in head and neck squamous cell carcinoma (HNSC). Methods: We developed a metric of the potential for tissue infected with SARS-CoV-2 ("TPSI") based on ACE2 and TMPRSS2 transcript levels and compared TPSI levels between tumor and matched normal tissues across 11 tumor types. For further analysis of HNSC, weighted gene co-expression network analysis (WGCNA), functional analysis, and single sample gene set enrichment analysis (ssGSEA) were conducted to investigate TPSI-relevant biological processes and their relationship with the immune landscape. TPSI-related factors were identified from clinical and mutational domains, followed by lasso regression to determine their relative effects on TPSI levels. Results: TPSI levels in tumors were generally lower than in the normal tissues. In HNSC, the genes highly associated with TPSI were enriched in viral entry-related processes, and TPSI levels were positively correlated with both eosinophils and T helper 17 (Th17) cell infiltration. Furthermore, the site of onset, human papillomaviruses (HPV) status, and nuclear receptor binding SET domain protein 1 (NSD1) mutations were identified as the most important factors shaping TPSI levels. Conclusions: This study identified the infection risk of SARS-CoV-2 between tumor and normal tissues, and provided evidence for the risk stratification of HNSC.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Serine Endopeptidases/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/virology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/virology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/virology , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization
18.
J Virol ; 95(19): e0086121, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1486519

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.


Subject(s)
Antiviral Agents/pharmacology , Esters/pharmacology , Guanidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/chemistry , Serine Endopeptidases/pharmacology , Serine Proteases/pharmacology , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/prevention & control , Carcinoma, Squamous Cell , Esters/chemistry , Esters/metabolism , Guanidines/chemistry , Guanidines/metabolism , Humans , Molecular Dynamics Simulation , Mouth Neoplasms , Protein Domains , Sequence Alignment , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteases/chemistry , Serine Proteases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Virus Internalization/drug effects
19.
Viruses ; 13(9)2021 09 04.
Article in English | MEDLINE | ID: covidwho-1478110

ABSTRACT

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.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzamidines/pharmacology , COVID-19/metabolism , COVID-19/virology , Guanidines/pharmacology , Interferon-alpha/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Benzamidines/therapeutic use , COVID-19/drug therapy , Cricetinae , Disease Models, Animal , Drug Therapy, Combination , Female , Guanidines/therapeutic use , Host-Pathogen Interactions/drug effects , Humans , Interferon-alpha/therapeutic use , Virus Replication/drug effects
20.
Biomed Res Int ; 2021: 9982729, 2021.
Article in English | MEDLINE | ID: covidwho-1476892

ABSTRACT

The human transmembrane protease serine 2 (TMPRSS2) protein plays an important role in prostate cancer progression. It also facilitates viral entry into target cells by proteolytically cleaving and activating the S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the current study, we used different available tools like SIFT, PolyPhen2.0, PROVEAN, SNAP2, PMut, MutPred2, I-Mutant Suite, MUpro, iStable, ConSurf, ModPred, SwissModel, PROCHECK, Verify3D, and TM-align to identify the most deleterious variants and to explore possible effects on the TMPRSS2 stability, structure, and function. The six missense variants tested were evaluated to have deleterious effects on the protein by SIFT, PolyPhen2.0, PROVEAN, SNAP2, and PMut. Additionally, V160M, G181R, R240C, P335L, G432A, and D435Y variants showed a decrease in stability by at least 2 servers; G181R, G432A, and D435Y are highly conserved and identified posttranslational modifications sites (PTMs) for proteolytic cleavage and ADP-ribosylation using ConSurf and ModPred servers. The 3D structure of TMPRSS2 native and mutants was generated using 7 meq as a template from the SwissModeller group, refined by ModRefiner, and validated using the Ramachandran plot. Hence, this paper can be advantageous to understand the association between these missense variants rs12329760, rs781089181, rs762108701, rs1185182900, rs570454392, and rs867186402 and susceptibility to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Mutation, Missense , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Binding Sites , Computational Biology/methods , Evolution, Molecular , Genetic Predisposition to Disease , Humans , Models, Molecular , Phylogeny , Polymorphism, Single Nucleotide , Protein Conformation , Protein Stability , Serine Endopeptidases/metabolism
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