Disruption of disulfides within RBD of SARS‐CoV‐2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs

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.

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Inhibitors of Thiol-Mediated Uptake

10.26434/chemrxiv.12906533 ◽

2020 ◽

Author(s):

Yangyang Cheng ◽

Anh-Tuan Pham ◽

Takehiro Kato ◽

Bumhee Lim ◽

Dimitri Moreau ◽

...

Keyword(s):

Viral Entry ◽

Spike Protein ◽

Cell Surfaces ◽

Disulfide Exchange

Viral entry into cells can involve thiol-disulfide exchange with exofacial thiols on cell surfaces. The importance of thiol-mediated uptake for viral entry and beyond is poorly understood because efficient inhibitors do not exist. Here we use fluorescent cyclic oligochalcogenides that enter cells by thiol-mediated uptake to systematically screen for inhibitors, including epidithiodiketopiperazines, benzopolysulfanes, disulfide-bridged g-turned peptides, heteroaromatic sulfones and cyclic thiosulfonates, thiosulfinates and disulfides. Different activities found with different reporters reveal thiol-mediated uptake as a complex multitarget process. Initial tests with pseudo-lentivectors expressing SARS-CoV-2 spike protein do not exclude potential for the development of new antivirals

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Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

eLife ◽

10.7554/elife.61552 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Qi Yang ◽

Thomas A Hughes ◽

Anju Kelkar ◽

Xinheng Yu ◽

Kai Cheng ◽

...

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Multiple Roles ◽

Spike Protein ◽

Receptor Binding Domain ◽

Mechanistic Studies ◽

Post Translational Modification ◽

Minor Contribution ◽

Chemical Inhibitors ◽

The Impact

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

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Denaturation of the SARS-CoV-2 spike protein under non-thermal microwave radiation

Scientific Reports ◽

10.1038/s41598-021-02753-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pooya Afaghi ◽

Michael Anthony Lapolla ◽

Khashayar Ghandi

Keyword(s):

Electromagnetic Field ◽

Body Temperature ◽

Microwave Radiation ◽

Human Body ◽

Viral Entry ◽

Thermal Denaturation ◽

Spike Protein ◽

Heating Effect ◽

Bulk Heating ◽

Human Body Temperature

AbstractSARS-CoV-2, the virus that causes COVID-19, is still a widespread threat to society. The spike protein of this virus facilitates viral entry into the host cell. Here, the denaturation of the S1 subunit of this spike protein by 2.45 GHz electromagnetic radiation was studied quantitatively. The study only pertains to the pure electromagnetic effects by eliminating the bulk heating effect of the microwave radiation in an innovative setup that is capable of controlling the temperature of the sample at any desired intensity of the electromagnetic field. This study was performed at the internal human body temperature, 37 °C, for a relatively short amount of time under a high-power electromagnetic field. The results showed that irradiating the protein with a 700 W, 2.45 GHz electromagnetic field for 2 min can denature the protein to around 95%. In comparison, this is comparable to thermal denaturation at 75 °C for 40 min. Electromagnetic denaturation of the proteins of the virus may open doors to potential therapeutic or sanitation applications.

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Covid-19 Mutations and How the Vaccine Enhances Immune Intelligence

Journal of Endocrinology and Metabolism Research ◽

10.37191/mapsci-2582-7960-2(1)-014 ◽

2021 ◽

Author(s):

Xanya Sofra

Keyword(s):

Amino Acid ◽

Viral Entry ◽

Open Reading Frames ◽

Spike Protein ◽

Single Amino Acid ◽

Immune Memory ◽

Type I ◽

Specific Amino Acid ◽

Adaptive Efficiency ◽

Acid Exchange

We traced the coronavirus classification and evolution, analyzed the Covid-19 composition and its distinguishing characteristics when compared to SARS-CoV and MERS-CoV. Despite their close kinship, SARS-CoV and Covid-19 display significant structural differences, including 380 amino acid substitutions, and variable homology between certain open reading frames that are bound to diversify the pathogenesis and virulence of the two viral compounds. A single amino acid substitution such as replacing Aspartate (D) with Glycine (G) composes the D614G mutation that is around 20% more infectious than its predecessor 614D. The B117 variant, that exhibits a 70% transmissibility rate, harbours 23 mutants, each reflecting one amino acid exchange. We examined several globally spreading mutations, 501.V2, B1351, P1, and others, with respect to the specific amino acid conversions involved. Unlike previous versions of coronavirus, where random mutations eventually precipitate extinction, the multiplicity of over 300,000 mutations appears to have rendered Covid-19 more contagious, facilitating its ability to evade detection, thus challenging the effectiveness of a large variety of emerging vaccines. Vaccination enhances immune memory and intelligence to combat or obstruct viral entry by generating antibodies that will prohibit the cellular binding and fusion with the Spike protein, ultimately debilitating the virus from releasing its contents into the cell. Developing antibodies during the innate response, appears to be the most compelling solution in light of the hypothesis that Covid-19 inhibits the production of Interferon type I, compromising adaptive efficiency to recognize the virus, possibly provoking a cytokine storm that injures vital organs. With respect to that perspective, the safety and effectiveness of different vaccines is evaluated and compared, including the Spike protein mRNA version, the Adenovirus DNA, Spike protein subunits, the deactivated virus genres, or, finally, the live attenuated coronavirus that appears to demonstrate the greatest effectiveness, yet, encompass a relatively higher risk.

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Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016093117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28046-28055 ◽

Cited By ~ 7

Author(s):

Anum Glasgow ◽

Jeff Glasgow ◽

Daniel Limonta ◽

Paige Solomon ◽

Irene Lui ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Surface Display ◽

Random Mutagenesis ◽

Yeast Surface Display ◽

Host Cells ◽

Spike Protein ◽

Receptor Protein ◽

Yeast Surface

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

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Preventing SARS-CoV-2 infection by blocking a tissue serine protease

Therapeutic Advances in Infectious Disease ◽

10.1177/2049936120933076 ◽

2020 ◽

Vol 7 ◽

pp. 204993612093307

Author(s):

Katherine C. Jankousky ◽

Jonathan Schultz ◽

Samuel Windham ◽

Andrés F. Henao-Martínez ◽

Carlos Franco-Paredes ◽

...

Keyword(s):

Epithelial Cells ◽

Severe Acute Respiratory Syndrome ◽

Serine Protease ◽

Viral Entry ◽

Clinical Impact ◽

Spike Protein ◽

Viral Pathogens ◽

Pharmacological Targets ◽

Alternative Approach ◽

Viral Surface

Currently, there are no proven pharmacologic interventions to reduce the clinical impact and prevent complications of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, the cause of the ongoing Coronavirus Disease of 2019 (COVID-19) pandemic. Selecting specific pharmacological targets for the treatment of viral pathogens has traditionally relied in blockage of specific steps in their replicative lifecycle in human cells. However, an alternative approach is reducing the molecular cleavage of the viral surface spike protein of SARS-CoV-2 to prevent viral entry into epithelial cells.

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Conformational States of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Ectodomain

Journal of Virology ◽

10.1128/jvi.02744-05 ◽

2006 ◽

Vol 80 (14) ◽

pp. 6794-6800 ◽

Cited By ~ 76

Author(s):

Fang Li ◽

Marcelo Berardi ◽

Wenhui Li ◽

Michael Farzan ◽

Philip R. Dormitzer ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Viral Entry ◽

Protein S ◽

Spike Protein ◽

Fusion Peptides ◽

Conformational States ◽

Protease Cleavage

ABSTRACT The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.

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Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

10.1101/2021.06.21.449352 ◽

2021 ◽

Author(s):

Hsu-Yu Chen ◽

Chun Huang ◽

Lu Tian ◽

Xiaoli Huang ◽

Chennan Zhang ◽

...

Keyword(s):

Viral Entry ◽

High Titer ◽

Cytoplasmic Tail ◽

Cell Types ◽

Spike Protein ◽

Tangential Flow Filtration ◽

Tangential Flow ◽

Protein Functions ◽

Flow Filtration ◽

The Impact

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein’s function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions.

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