Molecular characterization of COVID-19 therapeutics: Luteolin as an allosteric modulator of the spike protein of SARS-CoV-2

2021 ◽  
Author(s):  
Juan J de Pablo ◽  
Walter Alvarado ◽  
Fabian Bylehn ◽  
Cintia Menendez ◽  
Gustavo Perez
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Molecular Characterization ◽  
Receptor Binding ◽  
Viral Entry ◽  
Spike Protein ◽  
Allosteric Modulator ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2

The interactions between the receptor binding domain (RBD) of SARS-CoV-2 and the angiotensin- converting enzyme 2 (ACE2) are crucial for viral entry and subsequent replication. Given the large and featureless...

scholarly journals Competitive SARS-CoV-2 Serology Reveals Most Antibodies Targeting the Spike Receptor-Binding Domain Compete for ACE2 Binding

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
James R. Byrnes ◽  
Xin X. Zhou ◽  
Irene Lui ◽  
Susanna K. Elledge ◽  
Jeff E. Glasgow ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Viral Entry ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Block Interaction ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Receptor

ABSTRACT As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual’s seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies. IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.

scholarly journals Nanoluciferase complementation-based biosensor reveals the importance of N- linked glycosylation of SARS-CoV-2 Spike for viral entry

2020 ◽  
Author(s):  
Taha Azad ◽  
Ragunath Singaravelu ◽  
Zaid Taha ◽  
Stephen Boulton ◽  
Mathieu J.F. Crupi ◽  
...  
Keyword(s):  
Virus Infection ◽  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Viral Entry ◽  
Critical Role ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Entry Receptor

Abstract The ongoing COVID-19 pandemic has highlighted the` immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 lifecycle. We developed a bioluminescence-based biosensor to interrogate the interaction between the SARS-CoV-2 viral spike protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The biosensor assay is based on a Nanoluciferase complementation reporter, composed of two subunits, Large BiT and Small BiT, fused to the spike receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this biosensor, we uncovered a critical role for glycosylation of asparagine residues within the RBD in mediating successful binding to the cellular ACE2 receptor and subsequent virus infection. Our findings support RBD glycosylation as a therapeutic and vaccine target to blunt SARSCoV- 2 infections.

scholarly journals Multidisciplinary Approaches Identify Compounds that Bind to Human ACE2 or SARS-CoV-2 Spike Protein as Candidates to Block SARS-CoV-2–ACE2 Receptor Interactions

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Christopher J. Day ◽  
Benjamin Bailly ◽  
Patrice Guillon ◽  
Larissa Dirr ◽  
Freda E.-C. Jen ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Evans Blue ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Entry Inhibitors ◽  
Protein Receptor

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 μM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency—Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.

Effective ACE2 Peptide-Nanoparticle Conjugation and Its Binding with SARS-Cov-2 RBD Quantified by Dynamic Light Scattering

2021 ◽  
Author(s):  
Vince St. Dollente Mesias ◽  
Hongni Zhu ◽  
Xiao Tang ◽  
Xin Dai ◽  
Yusong Guo ◽  
...  
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Spike Protein ◽  
Cellular Receptor ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Receptor

The infection of coronavirus initiates with the binding between its spike protein receptor binding domain (RBD) and a human cellular receptor called angiotensin-converting enzyme 2 (ACE2). Here, we construct truncated...

scholarly journals A Drug Repurposing Approach for Antimalarials Interfering with SARS-CoV-2 Spike Protein Receptor Binding Domain (RBD) and Human Angiotensin-Converting Enzyme 2 (ACE2)

2021 ◽  
Vol 14 (10) ◽  
pp. 954
Author(s):  
Paolo Coghi ◽  
Li Jun Yang ◽  
Jerome P. L. Ng ◽  
Richard K. Haynes ◽  
Maurizio Memo ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Cell Invasion ◽  
Binding Domain ◽  
Spike Protein ◽  
Interaction Patterns ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2

Host cell invasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by the interaction of the viral spike protein (S) with human angiotensin-converting enzyme 2 (ACE2) through the receptor-binding domain (RBD). In this work, computational and experimental techniques were combined to screen antimalarial compounds from different chemical classes, with the aim of identifying small molecules interfering with the RBD-ACE2 interaction and, consequently, with cell invasion. Docking studies showed that the compounds interfere with the same region of the RBD, but different interaction patterns were noted for ACE2. Virtual screening indicated pyronaridine as the most promising RBD and ACE2 ligand, and molecular dynamics simulations confirmed the stability of the predicted complex with the RBD. Bio-layer interferometry showed that artemisone and methylene blue have a strong binding affinity for RBD (KD = 0.363 and 0.226 μM). Pyronaridine also binds RBD and ACE2 in vitro (KD = 56.8 and 51.3 μM). Overall, these three compounds inhibit the binding of RBD to ACE2 in the μM range, supporting the in silico data.

scholarly journals SARS-CoV-2 variant with higher affinity to ACE2 shows reduced sera neutralization susceptibility

2021 ◽  
Author(s):  
Monique Vogel ◽  
Xinyue Chang ◽  
Gilles Sousa Augusto ◽  
Mona O. Mohsen ◽  
Daniel E. Speiser ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Polyclonal Antibodies ◽  
Mutant Virus ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Serum Antibodies ◽  
Angiotensin Converting Enzyme 2 ◽  
Receptor Affinity

AbstractBackgroundSeveral new variants of SARS-CoV-2 have emerged since fall 2020 which have multiple mutations in the receptor binding domain (RBD) of the spike protein.ObjectiveWe aimed to assess how mutations in the SARS-CoV-2 RBD affect receptor affinity to angiotensin-converting enzyme 2 (ACE2) and neutralization by anti-RBD serum antibodies.MethodsWe produced a SARS-CoV-2 RBD mutant (RBDmut) with key mutations (E484K, K417N, N501Y) from the newly emerged Brazilian variant. Using Biolayer Interferometry, we analyzed the binding of this mutant to ACE2, and the susceptibility to neutralization by sera from vaccinated mice and COVID-19 convalescent patients.ResultsKinetic profiles showed increased RBDmut - ACE2 affinity compared to RBDwt, and binding of vaccine-elicited or convalescent sera was significantly reduced. Likewise, both sera types showed significantly reduced ability to block RBDmut - ACE2 binding indicating that antibodies induced by RBDwt have reduced capability to neutralize mutant virus.ConclusionOur physiochemical data show enhanced infectivity and reduced neutralization by polyclonal antibodies of the Brazilian variant of SARS-CoV-2.Capsule summarySARS-CoV-2 variant with Brazilian RBD mutations shows increased ACE2 affinity and reduced susceptibility to blockage by vaccine-elicited and convalescent sera.

Design of Inhibitory Peptides of the Interaction between the RBD Domain of the S1 Protein of SARS-CoV-2 and the Angiotensin Converting Enzyme 2 (ACE2) Receptor

2020 ◽  
Vol 17 ◽  
Author(s):  
Carlos Andrés Rodríguez-Salazar ◽  
Delia Piedad Recalde-Reyes ◽  
Jhon Carlos Castaño-Osorio
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Alveolar Cells ◽  
Protein Database ◽  
Angiotensin Converting Enzyme 2 ◽  
Inhibitory Peptides

Background: The recent outbreak caused by SARS-CoV-2, known as COVID-19, has been cataloged as a global catastrophe due to the growing number of infected cases and deaths since November 2019; this infectious contagious disease, to date, does not have a vaccine available or specific treatment, which is why the number of cases continues increasing. SARS-CoV-2 infects humans as a result of the interaction between the receptor-binding domain of the viral spike protein and the receptor of the angiotensin-converting enzyme-2 (rACE2), located predominantly in the alveolar cells. Objective: To identify through computational tools, inhibitory peptides of the protein-protein interaction between the Receptor-binding-domain of the Spike protein of SARS-CoV-2 and the angiotensin-converting enzyme-2 receptor. Methods: through the Research Collaboratory for Structural Bioinformatics protein database, crystals were selected and interaction models were carried out between the viral protein and the ACE2; thereafter, the study sought and designed inhibitory peptides of the interaction through the Rosetta web server, validated their interaction through ClusPro and, finally, determined the theoretical physicochemical and cytotoxic properties. Results: A protein complex was generated modeled through ClusPro; the balanced model was selected with the lowest binding energy. From the protein interactions of each of the crystals and from the model eight peptides of 20 residues were obtained. The theoretical evaluation showed non-toxic peptides, six soluble in water, and two insoluble. Conclusion: We found eight peptides interacted with the receptor-binding-domain of the Spike Protein of SARS-CoV-2, which could avoid contact with the cell receptor and generate interference in the infection process.

scholarly journals Interaction of human ACE2 to membrane-bound SARS-CoV-1 and SARS-CoV-2 S glycoproteins

2020 ◽  
Author(s):  
Sai Priya Anand ◽  
Yaozong Chen ◽  
Jérémie Prévost ◽  
Romain Gasser ◽  
Guillaume Beaudoin-Bussières ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Viral Entry ◽  
Conformational Transitions ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Spike Glycoprotein ◽  
Angiotensin Converting Enzyme 2 ◽  
Membrane Bound

AbstractA novel severe acute respiratory (SARS)-like coronavirus (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor binding domain of its trimeric Spike glycoprotein and the human angiotensin converting enzyme 2 (ACE2) receptor. A better understanding of the Spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance in the development of therapeutics that block the Spike/ACE2 interaction.

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