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 Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1745
Author(s):  
Luisa Diomede ◽  
Sara Baroni ◽  
Ada De Luigi ◽  
Arianna Piotti ◽  
Jacopo Lucchetti ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Pseudotyped Virus ◽  
Hek 293 ◽  
Angiotensin Converting Enzyme 2 ◽  
Preclinical Phase ◽  
Vitro Binding ◽  
Rapid Spread ◽  
Shed Light ◽  
Unusual Situation

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

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 Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

2021 ◽  
Author(s):  
Luisa Diomede ◽  
Sara Baroni ◽  
Ada De Luigi ◽  
Arianna Piotti ◽  
Jacopo Lucchetti ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Experimental Approach ◽  
Pseudotyped Virus ◽  
Biological Processes ◽  
Hek 293 ◽  
Angiotensin Converting Enzyme 2 ◽  
Preclinical Phase ◽  
Vitro Binding ◽  
Human Receptor

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials.

scholarly journals Binding of SARS-CoV-2 spike protein to ACE2 is disabled by thiol-based drugs; evidence from in vitro SARS-CoV-2 infection studies

2020 ◽  
Author(s):  
Kritika Khanna ◽  
Wilfred Raymond ◽  
Annabelle R. Charbit ◽  
Jing Jin ◽  
Irina Gitlin ◽  
...  
Keyword(s):  
Host Cells ◽  
Surface Glycoprotein ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
Cell Infection ◽  
Live Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Novel Treatments ◽  
Binding Interface

AbstractCoronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the SARS-CoV-2 spike protein is an envelope glycoprotein that binds angiotensin converting enzyme 2 as an entry receptor. The capacity of enveloped viruses to infect host cells depends on a precise thiol/disulfide balance in their surface glycoprotein complexes. To determine if cystines in the SARS-CoV-2 spike protein maintain a native binding interface that can be disrupted by drugs that cleave cystines, we tested if thiol-based drugs have efficacy in receptor binding and cell infection assays. We found that thiol-based drugs, cysteamine and WR-1065 (the active metabolite of amifostine) in particular, decrease binding of SARS-CoV-2 spike protein to its receptor, decrease the entry efficiency of SARS-CoV-2 spike pseudotyped virus, and inhibit SARS-CoV-2 live virus infection. Our findings uncover a vulnerability of SARS-CoV-2 to thiol-based drugs and provide rationale to test thiol-based drugs, especially cysteamine and amifostine, as novel treatments for COVID-19.One Sentence SummaryThiol-based drugs decrease binding of SARS-CoV-2 spike protein to its receptor and inhibit SARS-CoV-2 cell entry.

Inhibition of binding of gonadal steroids to serum binding proteins by non-esterified fatty acids: the influence of chain length and degree of unsaturation

1989 ◽  
Vol 120 (2) ◽  
pp. 175-179 ◽  
Author(s):  
C. Street ◽  
R. J. S. Howell ◽  
L. Perry ◽  
S. Al-Othman ◽  
T. Chard
Keyword(s):  
Fatty Acids ◽  
Protein Binding ◽  
Unsaturated Fatty Acids ◽  
Late Pregnancy ◽  
Sex Hormone Binding Globulin ◽  
Partial Purification ◽  
Normal Female ◽  
Esterified Fatty Acids ◽  
Vitro Binding

Abstract. The effect of non-esterified fatty acids (NEFA) on the in vitro binding of testosterone, 5-alpha dihydrotestosterone and estradiol E2 to sex hormone binding globulin (SHBG) was examined using pooled normal female serum, and SHBG and albumin fractions obtained from the partial purification of late pregnancy serum. A range of saturated and unsaturated fatty acids were examined for their effect on steroid-protein binding. In normal female serum, NEFA added at physiological concentrations disrupted steroid-protein binding. The shorter chain (C8–C12) saturated acids and the poly-unsaturated acids proved to be more effective inhibitors than the longer chain saturated or mono-unsaturated acids. The greatest inhibition was obtained with E2 whereas the binding of dihydrotestosterone was least affected. With partially purified SHBG, the same concentrations of NEFA were less effective at inhibiting the binding of dihydrotestosterone and testosterone but elicited the same effect with E2. The binding of steroids to albumin appeared to be unaffected by these concentrations of NEFA.

scholarly journals Virtual screening as therapeutic strategy of COVID-19 targeting angiotensin-converting enzyme 2

2021 ◽  
Vol 2 (1) ◽  
pp. 16-27
Author(s):  
Zahra Sharifinia ◽  
◽  
Samira Asadi ◽  
Mahyar Irani ◽  
Abdollah Allahverdi ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Angiotensin Converting Enzyme ◽  
Host Cells ◽  
Spike Protein ◽  
Potential Drug ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Approved Drugs ◽  
Fda Approved Drugs

Objective: The receptor-binding domain (RBD) of the S1 domain of the SARS-CoV- 2 Spike protein performs a key role in the interaction with Angiotensin-converting enzyme 2 (ACE2), leading to both subsequent S2 domain-mediated membrane fusion and incorporation of viral RNA in host cells. Methods: In this study, we investigated the inhibitor’s targeted compounds through existing human ACE2 drugs to use as a future viral invasion. 54 FDA approved drugs were selected to assess their binding affinity to the ACE2 receptor. The structurebased methods via computational ones have been used for virtual screening of the best drugs from the drug database. Key Findings: The ligands “Cinacalcet” and “Levomefolic acid” highaffinity scores can be a potential drug preventing Spike protein of SARS-CoV-2 and human ACE2 interaction. Levomefolic acid from vitamin B family was proved to be a potential drug as a spike protein inhibitor in previous clinical and computational studies. Besides that, in this study, the capability of Levomefolic acid to avoid ACE2 and Spike protein of SARS-CoV-2 interaction is indicated. Therefore, it is worth to consider this drug for more in vitro investigations as ACE2 and Spike protein inhibition candidate. Conclusion: The two Cinacalcet and Levomefolic acid are the two ligands that have highest energy binding for human ACE2 blocking among 54 FDA approved drugs.

scholarly journals Plasma From Recovered COVID-19 Patients Inhibits Spike Protein Binding to ACE2 in a Microsphere-Based Inhibition Assay

2020 ◽  
Vol 222 (12) ◽  
pp. 1965-1973 ◽  
Author(s):  
Edward P Gniffke ◽  
Whitney E Harrington ◽  
Nicholas Dambrauskas ◽  
Yonghou Jiang ◽  
Olesya Trakhimets ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Angiotensin Converting Enzyme ◽  
Protein Binding ◽  
Immunoglobulin G ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Inhibition Assay ◽  
Flow Cytometry Assay ◽  
Angiotensin Converting Enzyme 2 ◽  
A Cell

Abstract We present a microsphere-based flow cytometry assay that quantifies the ability of plasma to inhibit the binding of spike protein to angiotensin-converting enzyme 2. Plasma from 22 patients who had recovered from mild coronavirus disease 2019 (COVID-19) and expressed anti–spike protein trimer immunoglobulin G inhibited angiotensin-converting enzyme 2–spike protein binding to a greater degree than controls. The degree of inhibition was correlated with anti–spike protein immunoglobulin G levels, neutralizing titers in a pseudotyped lentiviral assay, and the presence of fever during illness. This inhibition assay may be broadly useful to quantify the functional antibody response of patients recovered from COVID-19 or vaccine recipients in a cell-free assay system.

scholarly journals De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

Science ◽  
2020 ◽  
Vol 370 (6521) ◽  
pp. 1208-1214 ◽  
Author(s):  
Thomas W. Linsky ◽  
Renan Vergara ◽  
Nuria Codina ◽  
Jorgen W. Nelson ◽  
Matthew J. Walker ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
High Specificity ◽  
Spike Protein ◽  
Host Proteins ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
De Novo Protein Design ◽  
Single Spike

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo–electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

scholarly journals Protein-DNA Binding and CpG Methylation at Nucleotide Resolution of Latency-Associated Promoters Qp, Cp, and LMP1p of Epstein-Barr Virus

2001 ◽  
Vol 75 (6) ◽  
pp. 2584-2596 ◽  
Author(s):  
Daniel Salamon ◽  
Maria Takacs ◽  
Dorina Ujvari ◽  
Jörg Uhlig ◽  
Hans Wolf ◽  
...  
Keyword(s):  
Protein Binding ◽  
Promoter Activity ◽  
Cpg Methylation ◽  
Barr Virus ◽  
In Vitro Binding ◽  
Binding Data ◽  
Vitro Binding ◽  
Epstein Barr

ABSTRACT Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity.

scholarly journals Murine Coronavirus with an Extended Host Range Uses Heparan Sulfate as an Entry Receptor

2005 ◽  
Vol 79 (22) ◽  
pp. 14451-14456 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Zhen Li ◽  
Eddie te Lintelo ◽  
Berend Jan Bosch ◽  
Bert Jan Haijema ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Host Range ◽  
Binding Sites ◽  
Spike Protein ◽  
Heparin Binding ◽  
Furin Cleavage ◽  
Cleavage Motif ◽  
Entry Receptor ◽  
Murine Coronavirus

ABSTRACT Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif. The adaptation of the virus through the use of heparan sulfate as an attachment/entry receptor was demonstrated by increased heparin binding as well as by inhibition of infection through treatment of cells and the virus with heparinase and heparin, respectively.

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