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 Plasma from recovered COVID19 subjects inhibits spike protein binding to ACE2 in a microsphere-based inhibition assay

2020 ◽  
Author(s):  
Edward P. Gniffke ◽  
Whitney E. Harrington ◽  
Nicholas Dambrauskas ◽  
Yonghou Jiang ◽  
Olesya Trakhimets ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antibody Response ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Inhibition Assay ◽  
Serological Tests ◽  
Angiotensin Converting Enzyme 2 ◽  
A Cell ◽  
Inhibitory Antibodies

AbstractHigh throughput serological tests that can establish the presence and functional activity of anti-SARS-COV2 antibodies are urgently needed. Here we present microsphere-based Flow Cytometry assays that quantify both anti-spike IgGs in plasma, and the ability of plasma to inhibit the binding of spike protein to angiotensin converting enzyme 2 (ACE2). First, we detected anti-spike-trimer IgGs in 22/24 and anti-spike-receptor-binding-domain (RBD) IgGs in 21/24 COVID+ subjects at a median of 36 (range 14-73) days following documented SARS-CoV-2 RNA (+) secretions. Next, we find that plasma from all 22/24 subjects with anti-trimer IgGs inhibited ACE2-trimer binding to a greater degree than controls, and that the degree of inhibition correlated with anti-trimer IgG levels. Depletion of trimer-reactive Igs from plasma reduced ACE2-trimer inhibitory capacity to a greater degree than depletion of RBD-reactive Igs, suggesting that inhibitory antibodies act by binding both within and outside of the RBD. Amongst the 24 subjects, presence of fever was associated with higher levels of anti-trimer IgG and inhibition of binding to human ACE2. This inhibition assay may be broadly useful to quantify the functional antibody response of recovered COVID19 patients or vaccine recipients in a cell-free assay system.

scholarly journals A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding

2021 ◽  
Author(s):  
Nanami Kikuchi ◽  
Or Willinger ◽  
Naor Granik ◽  
Noa Navon ◽  
Shanny Ackerman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Detection Limit ◽  
Angiotensin Converting Enzyme ◽  
Rapid Screening ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Fluorescent Signal ◽  
A Cell ◽  
Particle Preparation

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

Thermodynamics of the interaction between the spike protein of severe acute respiratory syndrome- coronavirus-2 and the receptor of human angiotensin converting enzyme 2. Effects of possible ligands

2020 ◽  
Author(s):  
Cristina Garcia-Iriepa ◽  
Cecilia Hognon ◽  
Antonio Francés-Monerris ◽  
Isabel Iriepa ◽  
Tom Miclot ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Molecular Mechanisms ◽  
Molecular Design ◽  
Binding Free Energy ◽  
Transmembrane Protein ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Rational Molecular Design ◽  
Rational Evaluation

<div><p>Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 180,000 deaths all over the world, still lacking a medical treatment despite the concerns of the whole scientific community. Human Angiotensin-Converting Enzyme 2 (ACE2) was recently recognized as the transmembrane protein serving as SARS-CoV-2 entry point into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the complex and of the effects of possible ligands. Moreover, binding free energy between ACE2 and the active Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein is evaluated quantitatively, assessing the molecular mechanisms at the basis of the recognition and the ligand-induced decreased affinity. These results boost the knowledge on the molecular grounds of the SARS-CoV-2 infection and allow to suggest rationales useful for the subsequent rational molecular design to treat severe COVID-19 cases.</p></div>

scholarly journals The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

2021 ◽  
Vol 22 (15) ◽  
pp. 8226
Author(s):  
John Tsu-An Hsu ◽  
Chih-Feng Tien ◽  
Guann-Yi Yu ◽  
Santai Shen ◽  
Yi-Hsuan Lee ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Viral Entry ◽  
Negative Impact ◽  
Spike Protein ◽  
Infection Model ◽  
Converting Enzyme ◽  
Viral Receptor ◽  
Angiotensin Converting Enzyme 2 ◽  
People With Dementia ◽  
The Impact

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer’s disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ1-42, but not Aβ1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ1-42. Furthermore, Aβ1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ1-42 show that the clearance of Aβ1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ1-42 in the blood is beneficial to the fight against COVID-19 and AD.

scholarly journals SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity

2021 ◽  
Vol 8 ◽  
Author(s):  
Somasundaram Raghavan ◽  
Divya Borsandra Kenchappa ◽  
M. Dennis Leo
Keyword(s):  
Endothelial Cells ◽  
Angiotensin Converting Enzyme ◽  
Spike Protein ◽  
Endothelial Barrier ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Barrier Integrity ◽  
Junctional Proteins ◽  
Junction Protein ◽  
Junctional Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

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.

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