scholarly journals The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor

2020 ◽  
Author(s):  
Yang Yang ◽  
Yi Du ◽  
Igor A. Kaltashov
Keyword(s):  
Host Cell ◽  
Gas Phase ◽  
Viral Entry ◽  
Protein A ◽  
Cell Receptor ◽  
Critical Element ◽  
S Protein ◽  
Host Cell Receptor ◽  
Native Ms ◽  
Gas Phase Ion

ABSTRACTThe emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over six million cases and nearly four hundred thousand deaths reported world-wide by the end of May 2020. A rush to find the cures prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the non-covalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease of its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.Abstract Figure

scholarly journals ACE2 glycans preferentially interact with the RBD of SARS-CoV-2 over SARS-CoV

2021 ◽  
Author(s):  
Atanu Acharya ◽  
Diane Lynch ◽  
Anna Pavlova ◽  
Yui Tik Pang ◽  
James Gumbart
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Cell Receptor ◽  
Glycosylation Site ◽  
Distinct Difference ◽  
S Protein ◽  
Binding Domains ◽  
Host Cell Receptor ◽  
Protein Receptor

We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2 glycan at N90 may offer protection against infections of both coronaviruses, while the ACE2 glycan at N322 enhances interactions with the SARS-CoV-2 RBD. The interactions of the ACE2 glycan at N322 with SARS-CoV RBD are blocked by the presence of the RBD glycan at N357 of the SARS-CoV RBD. The absence of this glycosylation site on SARS-CoV-2 RBD may enhance its binding with ACE2.

ACE2 glycans preferentially interact with SARS-CoV-2 over SARS-CoV

2021 ◽  
Author(s):  
Atanu Acharya ◽  
Diane L. Lynch ◽  
Anna Pavlova ◽  
Yui Tik Pang ◽  
James Gumbart
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Cell Receptor ◽  
Distinct Difference ◽  
S Protein ◽  
Binding Domains ◽  
Host Cell Receptor ◽  
Protein Receptor

We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2...

scholarly journals Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2’ for viral fusion/entry: indirect role of TMPRSS2

2020 ◽  
Author(s):  
Rachid Essalmani ◽  
Jaspreet Jain ◽  
Delia Susan-Resiga ◽  
Ursula Andréo ◽  
Alexandra Evagelidis ◽  
...  
Keyword(s):  
Viral Entry ◽  
Virus Entry ◽  
Critical Role ◽  
Cell Receptor ◽  
Viral Titer ◽  
Viral Fusion ◽  
S Protein ◽  
Cytopathic Effects ◽  
Host Cell Receptor

AbstractThe Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic “priming” (S1/S2) and “fusion-activation” (S2’) for viral entry. The S-protein furin-like motifs PRRAR685↓ and KPSKR815↓ indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2’ processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (μS1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2’, but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1’, activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and µS1/S2 severely curtail “pH-independent” viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2.

COVID 19 – Eye Issues

2020 ◽  
Vol 5 (Special) ◽  
Keyword(s):  
Host Cell ◽  
Target Cell ◽  
Cell Receptor ◽  
Human Tissues ◽  
Type Ii ◽  
Cell Infection ◽  
Host Cell Receptor ◽  
Infected Cells ◽  
Cellular Entry

The coronavirus illness (COVID-19) is caused by a new recombinant SARS-CoV (SARS-CoV) virus (SARS-CoV-2). Target cell infection by SARS-CoV is mediated by the prickly protein of the coronavirus and host cell receptor, enzyme 2 converting angiotensin (ACE2) [3]. Similarly, a recent study suggests that cellular entry by SARS-CoV-2 is dependent on both ACE2 as well as type II transmembrane axial protease (TMPRSS2) [4]. This means that detection of ACE2 and PRSS2 expression in human tissues can predict potential infected cells and their respective effects in COVID-19 patients [1].

scholarly journals The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

2021 ◽  
Vol 22 (6) ◽  
pp. 2926
Author(s):  
Dinendra L. Abeyawardhane ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Kristen M. Varney ◽  
Richard R. Rustandi ◽  
...  
Keyword(s):  
Host Cell ◽  
The Elderly ◽  
Cell Receptor ◽  
Host Cells ◽  
Binary Toxin ◽  
Clostridioides Difficile ◽  
Host Cell Receptor ◽  
Oligomeric Assembly ◽  
Nosocomial Disease ◽  
Binary Toxins

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

scholarly journals HIV-1 Envelope Conformation, Allostery, and Dynamics

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 852
Author(s):  
Ashley Lauren Bennett ◽  
Rory Henderson
Keyword(s):  
Host Cell ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Critical Role ◽  
Cell Receptor ◽  
Broadly Neutralizing Antibodies ◽  
Structural Mapping ◽  
Host Cell Receptor ◽  
Immunogen Design ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.

scholarly journals Ehrlichia chaffeensis and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Omid Teymournejad ◽  
Mingqun Lin ◽  
Yasuko Rikihisa
Keyword(s):  
Host Cell ◽  
Cell Receptor ◽  
Ros Generation ◽  
Ehrlichia Chaffeensis ◽  
Biological Processes ◽  
Wild Type ◽  
New Approach ◽  
Host Cell Receptor ◽  
Life Threatening ◽  
Human Monocytic Ehrlichiosis

ABSTRACT The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X−/−, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X−/− mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis. Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes.

Adherence of Legionella pneumophila to U-937 cells, guinea-pig alveolar macrophages, and MRC-5 cells by a novel, complement-independent binding mechanism

1994 ◽  
Vol 40 (10) ◽  
pp. 865-872 ◽  
Author(s):  
Frank C. Gibson III ◽  
Arthur O. Tzianabos ◽  
Frank G. Rodgers
Keyword(s):  
Monoclonal Antibodies ◽  
Guinea Pig ◽  
Host Cell ◽  
Alveolar Macrophages ◽  
Legionella Pneumophila ◽  
Cell Receptor ◽  
Host Cells ◽  
Order Kinetic ◽  
Complement Receptors ◽  
Host Cell Receptor

In the absence of serum, Legionella pneumophila demonstrated wash-resistant adherence to U-937 cells, primary guinea-pig alveolar macrophages, and MRC-5 cells. Neither complement nor antibody was required for binding. The dynamics of adherence following inoculation of L. pneumophila at increasing 10-fold multiplicities of infection to each of the three host cell types resulted in a first-order kinetic relationship of binding, indicative of one bacterial adhesin molecule recognized by one host cell receptor moiety. Host cell receptor saturation studies showed that depending on the cell type, 2–8% of the bacterial inoculum adhered to cells under these nonopsonic conditions. Preliminary adhesin and receptor characterization studies were preformed to define the chemical composition of the binding structures on both the organism and the three different host cell surfaces. The adherence phenomenon was investigated using competitive binding assays in the presence of putative adhesin analogs as well as following treatments modifying the microbial and host cell surface membranes. Attachment was evaluated both by viable bacterial cell colony counts and by indirect immunofluorescent assay. With the exception of aldehyde treatments, the various membrane-modifying regimes and the presence of the adhesin analogs were shown to have no effect on organism or host cell viability. Data suggested that the L. pneumophila adhesin responsible for opsonin-independent binding to these host cells was a protein structure with lectin-like properties. Furthermore, this protein would appear to be intimately associated with carbohydrate or lipid structures located on the bacterial outer membrane. The receptor moiety present on all host cells responsible for binding L. pneumophila had properties consistent with a carbohydrate or complex saccharide structure. To evaluate the role of complement receptors as the structures necessary for L. pneumophila infection of macrophages, a battery of monoclonal antibodies were used to block the complement receptor (CR) types 1 (CD35), CR3 (CD 18, CD11b), and CR4 (CD18, CD11c). Blocking studies with CR-specific monoclonal antibodies indicated that CR1 and the integrin receptors CR3 and CR4 were not involved in the opsonin-independent binding of L. pneumophila to macrophage-like cells.Key words: Legionella, opsonin-independent attachment, bacterial adherence, complement receptors, adhesion–receptor interactions.

Sign in / Sign up

Export Citation Format

Share Document