Structural and functional modelling of SARS-CoV-2 entry in animal models

Abstract SARS-CoV-2 is the novel coronavirus responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 26th July 2020, has infected over 16 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor, and TMPRSS2 and Furin proteases usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret, mouse, hamster and macaque. Here we showed that ACE2, but not TMPRSS2 or Furin, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Using molecular docking simulations we compared the SARS-CoV and SARS-CoV-2 Spike proteins in complex with the ACE2 receptor and showed that the SARS-CoV-2 Spike glycoprotein is compatible to bind the human ACE2 with high specificity. In contrast, TMPRSS2 and Furin are sufficiently similar in the considered hosts not to drive susceptibility differences. Computational analysis of binding modes and protein contacts indicates that macaque, ferrets and hamster are the most suitable models for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 and Furin are similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the hACE2 transgenic mouse, are also likely to be useful models for studies investigating viral entry.

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Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex

10.1101/2020.06.30.179663 ◽

2020 ◽

Cited By ~ 4

Author(s):

Ravikanth Vishnubhotla ◽

Naveen Vankadari ◽

Vijayasarathy Ketavarapu ◽

Ramars Amanchy ◽

Steffie Avanthi ◽

...

Keyword(s):

Exome Sequencing ◽

Protease Inhibitors ◽

Viral Entry ◽

Genetic Variants ◽

Spike Protein ◽

Cleavage Sites ◽

Spike Glycoprotein ◽

Whole Exome ◽

Potential Therapeutics

AbstractSARS-CoV-2, a highly transmittable pathogen has infected over 3.8 million people around the globe. The spike glycoprotein of SARS-CoV-2 engages host ACE2 for adhesion, TMPRSS2 for activation and entry. With the aid of whole-exome sequencing, we report a variant rs12329760 in TMPRSS2 gene and its mutant V160M, which might impede viral entry. Furthermore, we identified TMPRSS2 cleavage sites in S2 domain of spike glycoprotein and report the structure of TMPRSS2 in complex with spike glycoprotein. We also report the structures of protease inhibitors in complex with TMPRSS2, which could hamper the interaction with spike protein. These findings advance our understanding on the role of TMPRSS2 and in the development of potential therapeutics.

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Druggability of cavity pockets within SARS-CoV-2 spike glycoprotein and pharmacophore-based drug discovery

Future Virology ◽

10.2217/fvl-2020-0394 ◽

2021 ◽

Author(s):

Alireza Mohebbi ◽

Fatemeh Sana Askari ◽

Ali Salehnia Sammak ◽

Mohsen Ebrahimi ◽

Zahra Najafimemar

Keyword(s):

Drug Discovery ◽

Viral Entry ◽

Spike Protein ◽

Lead Compound ◽

Autodock Vina ◽

Spike Glycoprotein ◽

Viral Glycoprotein ◽

Chemical Derivatives ◽

Good Target ◽

Hit Identification

Aim: Virus spike glycoprotein of SARS-CoV-2 is a good target for drug discovery. Objective: To examine the potential for druggability of spike protein for pharmacophore-based drug discovery and to investigate the binding affinity of natural products with SARS-CoV-2 spike protein. Methods: Druggable cavities were searched though CavityPlus. A pharmacophore was built and used for hit identification. Autodock Vina was used to evaluate the hits' affinities. 10 chemical derivatives were also made from the chemical backbone to optimize the lead compound. Results: 10 druggable cavities were found within the glycoprotein spike. Only one cavity with the highest score at the binding site was selected for pharmacophore extraction. Hit identification resulted in the identification of 410 hits. Discussion: This study provides a druggable region within viral glycoprotein and a candidate compound to block viral entry.

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907.v2 ◽

2017 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907 ◽

2018 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18

Biomolecules ◽

10.3390/biom10050686 ◽

2020 ◽

Vol 10 (5) ◽

pp. 686 ◽

Cited By ~ 3

Author(s):

Alexander Neumann ◽

Viktor Engel ◽

Andhika B. Mahardhika ◽

Clara T. Schoeder ◽

Vigneshwaran Namasivayam ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

G Protein ◽

Phenyl Ring ◽

Binding Mode ◽

Dynamics Simulation ◽

Simulation Studies ◽

Binding Modes ◽

G Protein Coupled Receptor ◽

G Protein Coupled

GPR18 is an orphan G protein-coupled receptor (GPCR) expressed in cells of the immune system. It is activated by the cannabinoid receptor (CB) agonist ∆9-tetrahydrocannabinol (THC). Several further lipids have been proposed to act as GPR18 agonists, but these results still require unambiguous confirmation. In the present study, we constructed a homology model of the human GPR18 based on an ensemble of three GPCR crystal structures to investigate the binding modes of the agonist THC and the recently reported antagonists which feature an imidazothiazinone core to which a (substituted) phenyl ring is connected via a lipophilic linker. Docking and molecular dynamics simulation studies were performed. As a result, a hydrophobic binding pocket is predicted to accommodate the imidazothiazinone core, while the terminal phenyl ring projects towards an aromatic pocket. Hydrophobic interaction of Cys251 with substituents on the phenyl ring could explain the high potency of the most potent derivatives. Molecular dynamics simulation studies suggest that the binding of imidazothiazinone antagonists stabilizes transmembrane regions TM1, TM6 and TM7 of the receptor through a salt bridge between Asp118 and Lys133. The agonist THC is presumed to bind differently to GPR18 than to the distantly related CB receptors. This study provides insights into the binding mode of GPR18 agonists and antagonists which will facilitate future drug design for this promising potential drug target.

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The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

International Journal of Molecular Sciences ◽

10.3390/ijms22126490 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6490

Author(s):

Olga A. Postnikova ◽

Sheetal Uppal ◽

Weiliang Huang ◽

Maureen A. Kane ◽

Rafael Villasmil ◽

...

Keyword(s):

Amino Acid ◽

Insertion Sequence ◽

Experimental Studies ◽

Spike Protein ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

New Host ◽

S Protein ◽

Furin Cleavage Site ◽

Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

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Spanning the gap: unraveling RSC dynamics in vivo

Current Genetics ◽

10.1007/s00294-020-01144-1 ◽

2021 ◽

Author(s):

Heinz Neumann ◽

Bryan J. Wilkins

Keyword(s):

Cell Cycle ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Binding Mode ◽

Binding Modes ◽

Binding Domains ◽

Binding Interface ◽

The Many ◽

And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

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Transgenic animal models of tauopathies

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease ◽

10.1016/j.bbadis.2004.06.014 ◽

2005 ◽

Vol 1739 (2-3) ◽

pp. 251-259 ◽

Cited By ~ 81

Author(s):

Virginia M.-Y. Lee ◽

Theresa K. Kenyon ◽

John Q. Trojanowski

Keyword(s):

Animal Models ◽

Transgenic Animal ◽

Transgenic Animal Models

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

International Journal of Molecular Sciences ◽

10.3390/ijms22158226 ◽

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.

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