scholarly journals METHYLENE BLUE INHIBITS THE INTERACTION BETWEEN HEPARAN SULFATE AND SARS-COV-2 SPIKE PROTEIN; A REVIEW OF EVIDENCE FOR A HYPOTHESIS.

2021 ◽  
Vol 5 (4) ◽  
Author(s):  
Neluwa-Liyanage Ruwan Indika ◽  
Nirmalie Gayathrie Fonseka
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Electrostatic Interactions ◽  
Treatment Protocol ◽  
Spike Protein ◽  
Shielding Effect ◽  
Blue Dye ◽  
Viral Attachment

The addition of methylene blue to the standard treatment protocol has been shown to improve respiratory rate and oxygen saturation in COVID-19 patients, reducing morbidity and mortality. Evidence to date suggests that methylene blue inhibits protein-protein interactions between SARS-CoV-2 Spike protein and angiotensin-converting enzyme 2, which in turn inhibits the cell entry of SARS-CoV-2. However, the methylene blue dye-binding characteristics of sulfated glycosaminoglycans suggest additional inhibitory effects of the spike protein-heparan sulfate interaction. We hypothesize that the binding of cationic methylene blue neutralizes polyanionic heparan sulfate molecules on the host cell surface. As a consequence, electrostatic interactions between negatively charged heparan sulfate and the positively charged receptor binding domain of SARS-CoV-2 spike protein will be inhibited. Thus, methylene blue will exhibit a "shielding effect" on the heparan sulfate proteoglycans, inhibiting viral attachment to the cell surface. The proposed mechanism corroborates the possible broad-spectrum antiviral activity of methylene blue against multiple human coronaviruses that exploit the electrostatic interactions with sulfated glycosaminoglycans for virus entry. Methylene blue would exhibit the same anti-adhesive activity at the blood-brain-barrier and olfactory neuroepithelium, corroborating potential benefits in ameliorating post-COVID-19 neurological impairment. However, as cationic dyes can bind to both free glycosaminoglycans in circulation as well as proteoglycans attached to the cell surface, co-administration of intravenous heparin could possibly antagonize the proposed antiviral activity. This critical review focuses on empirical evidence to support the hypothesized heparan sulfate-dependent antiviral activity of MB.

scholarly journals Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

2021 ◽  
Vol 8 ◽  
Author(s):  
Jingwen Yue ◽  
Weihua Jin ◽  
Hua Yang ◽  
John Faulkner ◽  
Xuehong Song ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
Effective Prevention ◽  
Heparin Derivatives ◽  
Host Cell Surface

The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular heparan sulfate (HS) has been found to bind SARS-CoV-2 spike protein (SV2-S) and co-operate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In this study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. This binding is required for SARS-Cov-2 virus to infect host cells and can be blocked by heparin lyase, HS antagonist surfen, heparin, and heparin derivatives. The binding of heparin/HS to SV2-S is mainly determined by its overall sulfation with potential, minor contribution of specific SV2-S binding motifs. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS-CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. The higher host cell infection by SARS-CoV-2 G614 variant pseudovirus and the increased infection caused by upregulated HS expression both can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives too. Our findings support blocking HS-SV2-S interaction may provide one addition to achieve effective prevention and/treatment of COVID-19.

scholarly journals Membrane anchoring of heparan sulfate proteoglycans by phosphatidylinositol and kinetics of synthesis of peripheral and detergent-solubilized proteoglycans in Schwann cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1891-1897 ◽  
Author(s):  
D J Carey ◽  
D M Evans
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Schwann Cells ◽  
Electrostatic Interactions ◽  
Heparan Sulfate Proteoglycans ◽  
Membrane Bound ◽  
Covalently Linked ◽  
Mode Of Attachment ◽  
Membrane Anchoring ◽  
Kinetics Of

Previous studies have shown that Schwann cells synthesize both peripheral and integral hydrophobic cell surface heparan sulfate proteoglycans (HSPGs). The experiments reported here were undertaken to investigate the mode of attachment of these proteins to the cell surface and their potential interrelationship. The binding of the hydrophobic HSPGs to membranes appears to be via covalently linked phosphatidylinositol based on the observation that incubation of the detergent-solubilized protein with purified phosphatidylinositol-specific phospholipase C significantly reduces the ability of the HSPGs to associate with phospholipid vesicles in a reconstitution assay. The peripherally associated HSPGs were released from the cells by incubation in the presence of heparin (10 mg/ml), 10 mM phytic acid (inositol hexaphosphate), or 2 M NaCl. These treatments also solubilized basement membrane HSPGs synthesized by the Schwann cells. These data suggest that the peripheral HSPGs are bound to the surface by electrostatic interactions. The peripheral and hydrophobic HSPGs were identical in overall size, net charge, length of glycosaminoglycan chains, and patterns of N-sulfation. To determine whether the peripheral HSPGs were derived from the membrane-bound form by cleavage of the membrane anchor, we examined the kinetics of synthesis and degradation of the two forms of HSPGs. The results obtained indicated the existence of two pools of detergent-solubilized HSPG with fast (t1/2 = 6 h) and slow (t1/2 = 55 h) turnover kinetics. The data were consistent with a model in which the peripheral HSPGs were derived from the slowly turning over pool of detergent-solubilized HSPGs.

scholarly journals Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

2012 ◽  
Vol 56 (10) ◽  
pp. 5278-5288 ◽  
Author(s):  
Manuela Donalisio ◽  
Marco Rusnati ◽  
Valeria Cagno ◽  
Andrea Civra ◽  
Antonella Bugatti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
A549 Cells ◽  
Epithelial Tissue ◽  
Virus Infectivity ◽  
Human Respiratory Tract ◽  
Rsv Infection ◽  
Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC50s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery.

scholarly journals Heparan Sulfate Proteoglycans and Viral Attachment: True Receptors or Adaptation Bias?

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 596 ◽  
Author(s):  
Cagno ◽  
Tseligka ◽  
Jones ◽  
Tapparel
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Blood Coagulation ◽  
Biological Activities ◽  
Extracellular Matrix Proteins ◽  
Heparan Sulfate Proteoglycans ◽  
Matrix Proteins ◽  
Cell Homeostasis ◽  
Viral Attachment

Heparan sulfate proteoglycans (HSPG) are composed of unbranched, negatively charged heparan sulfate (HS) polysaccharides attached to a variety of cell surface or extracellular matrix proteins. Widely expressed, they mediate many biological activities, including angiogenesis, blood coagulation, developmental processes, and cell homeostasis. HSPG are highly sulfated and broadly used by a range of pathogens, especially viruses, to attach to the cell surface.

scholarly journals Heparan Sulfate Proteoglycans Are Required for Cellular Binding of the Hepatitis E Virus ORF2 Capsid Protein and for Viral Infection

2009 ◽  
Vol 83 (24) ◽  
pp. 12714-12724 ◽  
Author(s):  
Manjula Kalia ◽  
Vivek Chandra ◽  
Sheikh Abdul Rahman ◽  
Deepak Sehgal ◽  
Shahid Jameel
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Capsid Protein ◽  
Marked Reduction ◽  
Electrostatic Interactions ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Heparan Sulfate Proteoglycans ◽  
Productive Infection ◽  
Target Cells

ABSTRACT The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an ∼56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.

scholarly journals SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

2020 ◽  
Author(s):  
Thomas Mandel Clausen ◽  
Daniel R. Sandoval ◽  
Charlotte B. Spliid ◽  
Jessica Pihl ◽  
Chelsea D. Painter ◽  
...  
Keyword(s):  
Protein Binding ◽  
Heparan Sulfate ◽  
Docking Studies ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Vitro Binding ◽  
Domain Docking

AbstractWe show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

scholarly journals Methylene Blue Inhibits the SARS-CoV-2 Spike–ACE2 Protein-Protein Interaction–a Mechanism that can Contribute to its Antiviral Activity Against COVID-19

2021 ◽  
Vol 11 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Peter Buchwald
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viral Attachment ◽  
Protein Protein Interaction

Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction. Methylene blue inhibited the entry of a SARS-CoV-2 spike bearing pseudovirus into ACE2-expressing cells with similar IC50 (3.5 μM). Hence, this PPI inhibitory activity could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.

Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells

2007 ◽  
Vol 35 (4) ◽  
pp. 788-793 ◽  
Author(s):  
G.M.K. Poon ◽  
J. Gariépy
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Electrostatic Interactions ◽  
Cell Penetrating Peptides ◽  
Cationic Polymers ◽  
Protein Transduction Domains ◽  
Core Proteins ◽  
Wide Range ◽  
Entry Mechanism ◽  
Endocytic Pathways

Polycationic macromolecules and cationic peptides acting as PTDs (protein transduction domains) and CPPs (cell-penetrating peptides) represent important classes of agents used for the import and delivery of a wide range of molecular cargoes into cells. Their entry into cells is typically initiated through interaction with cell-surface HS (heparan sulfate) molecules via electrostatic interactions, followed by endocytosis of the resulting complexes. However, the endocytic mechanism employed (clathrin-mediated endocytosis, caveolar uptake or macropinocytosis), defining the migration of these peptides into cells, depends on parameters such as the nature of the cationic agent itself and complex formation with cargo, as well as the nature and distribution of proteoglycans expressed on the cell surface. Moreover, a survey of the literature suggests that endocytic pathways should not be considered as mutually exclusive, as more than one entry mechanism may be operational for a given cationic complex in a particular cell type. Specifically, the observed import may best be explained by the distribution and uptake of cell-surface HSPGs (heparan sulfate proteoglycans), such as syndecans and glypicans, which have been shown to mediate the uptake of many ligands besides cationic polymers. A brief overview of the roles of HSPGs in ligand internalization is presented, as well as mechanistic hypotheses based on the known properties of these cell-surface markers. The identification and investigation of interactions made by glycosaminoglycans and core proteins of HSPGs with PTDs and cationic polymers will be crucial in defining their uptake by cells.

scholarly journals Bacterial modification of the host glycosaminoglycan heparan sulfate modulates SARS-CoV-2 infectivity

2020 ◽  
Author(s):  
Cameron Martino ◽  
Benjamin P. Kellman ◽  
Daniel R. Sandoval ◽  
Thomas Mandel Clausen ◽  
Clarisse A. Marotz ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Culture Media ◽  
Microbial Community Composition ◽  
Lavage Fluid ◽  
Spike Protein ◽  
Viral Attachment ◽  
Protein Receptor ◽  
Close Relationship ◽  
The Impact

AbstractThe human microbiota has a close relationship with human disease and it remodels components of the glycocalyx including heparan sulfate (HS). Studies of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike protein receptor binding domain suggest that infection requires binding to HS and angiotensin converting enzyme 2 (ACE2) in a codependent manner. Here, we show that commensal host bacterial communities can modify HS and thereby modulate SARS-CoV-2 spike protein binding and that these communities change with host age and sex. Common human-associated commensal bacteria whose genomes encode HS-modifying enzymes were identified. The prevalence of these bacteria and the expression of key microbial glycosidases in bronchoalveolar lavage fluid (BALF) was lower in adult COVID-19 patients than in healthy controls. The presence of HS-modifying bacteria decreased with age in two large survey datasets, FINRISK 2002 and American Gut, revealing one possible mechanism for the observed increase in COVID-19 susceptibility with age. In vitro, bacterial glycosidases from unpurified culture media supernatants fully blocked SARS-CoV-2 spike binding to human H1299 protein lung adenocarcinoma cells. HS-modifying bacteria in human microbial communities may regulate viral adhesion, and loss of these commensals could predispose individuals to infection. Understanding the impact of shifts in microbial community composition and bacterial lyases on SARS-CoV-2 infection may lead to new therapeutics and diagnosis of susceptibility. Graphical Abstract. Diagram of hypothesis for bacterial mediation of SARS-CoV-2 infection through heparan sulfate (HS).It is well known that host microbes groom the mucosa where they reside. Recent investigations have shown that HS, a major component of mucosal layers, is necessary for SARS-CoV-2 infection. In this study we examine the impact of microbial modification of HS on viral attachment.

scholarly journals Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

2020 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Peter Buchwald
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viral Attachment

AbstractDue to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction with an activity similar, possibly slightly higher, than those found for it for other PPIs. This PPI inhibitory activity of methylene blue could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.

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