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1.
Proc Natl Acad Sci U S A ; 119(33): e2208144119, 2022 08 16.
Article in English | MEDLINE | ID: covidwho-1984601

ABSTRACT

Pattern recognition molecules (PRMs) form an important part of innate immunity, where they facilitate the response to infections and damage by triggering processes such as inflammation. The pentraxin family of soluble PRMs comprises long and short pentraxins, with the former containing unique N-terminal regions unrelated to other proteins or each other. No complete high-resolution structural information exists about long pentraxins, unlike the short pentraxins, where there is an abundance of both X-ray and cryoelectron microscopy (cryo-EM)-derived structures. This study presents a high-resolution structure of the prototypical long pentraxin, PTX3. Cryo-EM yielded a 2.5-Å map of the C-terminal pentraxin domains that revealed a radically different quaternary structure compared to other pentraxins, comprising a glycosylated D4 symmetrical octameric complex stabilized by an extensive disulfide network. The cryo-EM map indicated α-helices that extended N terminal of the pentraxin domains that were not fully resolved. AlphaFold was used to predict the remaining N-terminal structure of the octameric PTX3 complex, revealing two long tetrameric coiled coils with two hinge regions, which was validated using classification of cryo-EM two-dimensional averages. The resulting hybrid cryo-EM/AlphaFold structure allowed mapping of ligand binding sites, such as C1q and fibroblast growth factor-2, as well as rationalization of previous biochemical data. Given the relevance of PTX3 in conditions ranging from COVID-19 prognosis, cancer progression, and female infertility, this structure could be used to inform the understanding and rational design of therapies for these disorders and processes.


Subject(s)
C-Reactive Protein , Complement Activation , Serum Amyloid P-Component , Binding Sites , C-Reactive Protein/chemistry , C-Reactive Protein/immunology , COVID-19/immunology , Cryoelectron Microscopy , Female , Humans , Immunity, Innate , Ligands , Protein Conformation, alpha-Helical , Protein Domains , Serum Amyloid P-Component/chemistry
2.
Science ; 377(6607): 728-735, 2022 08 12.
Article in English | MEDLINE | ID: covidwho-1968212

ABSTRACT

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , Broadly Neutralizing Antibodies , COVID-19 , Epitopes , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Epitopes/chemistry , Epitopes/immunology , Humans , Peptides/immunology , Protein Conformation, alpha-Helical , Protein Domains , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
3.
J Mol Biol ; 434(19): 167759, 2022 10 15.
Article in English | MEDLINE | ID: covidwho-1956230

ABSTRACT

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.


Subject(s)
Cholesterol , Influenza A virus , Membrane Proteins , RNA-Binding Proteins , Cholesterol/chemistry , Humans , Influenza A virus/immunology , Membrane Proteins/chemistry , Protein Conformation, alpha-Helical , RNA-Binding Proteins/chemistry , Virus Internalization , Zika Virus/immunology
4.
Anal Chem ; 94(33): 11464-11469, 2022 08 23.
Article in English | MEDLINE | ID: covidwho-1931290

ABSTRACT

A new peptide inhibitor was designed and optimized from an α-helix-rich peptide library specifically toward the critical prion-like domain (PLD) of SARS-CoV-2. It compactly blocked the S1 protein and potently neutralized the pseudovirus which shows promising potential for prophylactic and treatment of COVID-19.


Subject(s)
COVID-19 , Prions , COVID-19/drug therapy , Humans , Peptides/chemistry , Protein Binding , Protein Conformation, alpha-Helical , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
5.
Biophys J ; 121(12): 2353-2370, 2022 06 21.
Article in English | MEDLINE | ID: covidwho-1899576

ABSTRACT

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike's stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1-20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.


Subject(s)
COVID-19 , Tropocollagen , Cryoelectron Microscopy , Finite Element Analysis , Humans , Protein Conformation, alpha-Helical , SARS-CoV-2 , Tropocollagen/chemistry
6.
Protein Sci ; 31(6): e4355, 2022 06.
Article in English | MEDLINE | ID: covidwho-1881449

ABSTRACT

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alpha-helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high-affinity binding to the spike protein with apparent Kd values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/genetics , Defensins , Humans , Ligands , Membrane Glycoproteins/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Conformation, alpha-Helical , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope Proteins/chemistry
7.
J Mol Model ; 28(5): 128, 2022 Apr 24.
Article in English | MEDLINE | ID: covidwho-1802772

ABSTRACT

In COVID-19 infection, the SARS-CoV-2 spike protein S1 interacts to the ACE2 receptor of human host, instigating the viral infection. To examine the competitive inhibitor efficacy of broad spectrum alpha helical AMPs extracted from frog skin, a comparative study of intermolecular interactions between viral S1 and AMPs was performed relative to S1-ACE2p interactions. The ACE2 binding region with S1 was extracted as ACE2p from the complex for ease of computation. Surprisingly, the Spike-Dermaseptin-S9 complex had more intermolecular interactions than the other peptide complexes and importantly, the S1-ACE2p complex. We observed how atomic displacements in docked complexes impacted structural integrity of a receptor-binding domain in S1 through conformational sampling analysis. Notably, this geometry-based sampling approach confers the robust interactions that endure in S1-Dermaseptin-S9 complex, demonstrating its conformational transition. Additionally, QM calculations revealed that the global hardness to resist chemical perturbations was found more in Dermaseptin-S9 compared to ACE2p. Moreover, the conventional MD through PCA and the torsional angle analyses indicated that Dermaseptin-S9 altered the conformations of S1 considerably. Our analysis further revealed the high structural stability of S1-Dermaseptin-S9 complex and particularly, the trajectory analysis of the secondary structural elements established the alpha helical conformations to be retained in S1-Dermaseptin-S9 complex, as substantiated by SMD results. In conclusion, the functional dynamics proved to be significant for viral Spike S1 and Dermaseptin-S9 peptide when compared to ACE2p complex. Hence, Dermaseptin-S9 peptide inhibitor could be a strong candidate for therapeutic scaffold to prevent infection of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2 , Antimicrobial Cationic Peptides , COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/therapeutic use , Anura/metabolism , COVID-19/drug therapy , COVID-19/prevention & control , Humans , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
8.
Cell Chem Biol ; 29(2): 215-225.e5, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1664751

ABSTRACT

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.


Subject(s)
Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Amino Acid Sequence , Anticoagulants/chemistry , Anticoagulants/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Pairing , Binding Sites , COVID-19/blood , COVID-19/drug therapy , Cell Membrane/chemistry , Cell Membrane/metabolism , Factor V/chemistry , Factor V/genetics , Factor V/metabolism , Factor Va/chemistry , Factor Va/genetics , Factor Va/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Humans , Immune Sera/chemistry , Immune Sera/metabolism , Models, Molecular , Nucleic Acid Conformation , Protamines , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , SELEX Aptamer Technique , Substrate Specificity
9.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1650946

ABSTRACT

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.


Subject(s)
Amino Alcohols/pharmacology , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/pharmacology , Phenyl Ethers/pharmacology , Receptors, Virus/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Sulfhydryl Compounds/pharmacology , Allosteric Regulation , Amino Alcohols/chemistry , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Line , Disulfides/antagonists & inhibitors , Disulfides/chemistry , Disulfides/metabolism , Dose-Response Relationship, Drug , Humans , Molecular Docking Simulation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Oxidation-Reduction , Phenyl Ethers/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Sulfhydryl Compounds/chemistry
10.
Biosci Rep ; 41(12)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1592575

ABSTRACT

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Endotoxins/pharmacology , Peptide Fragments/pharmacology , Spike Glycoprotein, Coronavirus/pharmacology , Alphacoronavirus , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , CD13 Antigens/metabolism , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cricetulus , Endotoxins/toxicity , Hemolysis/drug effects , Humans , Molecular Docking Simulation , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Conformation, alpha-Helical , Sheep, Domestic , Spike Glycoprotein, Coronavirus/toxicity , Structure-Activity Relationship
11.
Int J Biol Macromol ; 197: 68-76, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1587673

ABSTRACT

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Amyloidosis/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Protein Domains/physiology , Amyloidosis/genetics , Coronavirus 3C Proteases/genetics , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Kinetics , Models, Molecular , Molecular Dynamics Simulation , Mutation , Polymerization , Protein Conformation, alpha-Helical , Protein Domains/genetics , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature
12.
Infect Genet Evol ; 97: 105153, 2022 01.
Article in English | MEDLINE | ID: covidwho-1521407

ABSTRACT

Amid the ongoing COVID-19 pandemic, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences were downloaded from the GISAID database, aligned, and the variants identified. 437,006 unique viral genomes were analyzed. Polymorphisms in the protein sequence were investigated and examined longitudinally to identify sequence and strain variants appearing between January 5th, 2020 and January 16th, 2021. A structural analysis was also performed to investigate mutations in the receptor binding domain and the N-terminal domain of the spike protein. Within the spike protein, there were 766 unique mutations observed in the N-terminal domain and 360 in the receptor binding domain. Four residues that directly contact ACE2 were mutated in more than 100 sequences, including positions K417, Y453, S494, and N501. Within the furin cleavage site of the spike protein, a high degree of conservation was observed, but the P681H mutation was observed in 10.47% of sequences analyzed. Within the RNA dependent RNA polymerase complex proteins, 327 unique mutations were observed in Nsp8, 166 unique mutations were observed in Nsp7, and 1157 unique mutations were observed in Nsp12. Only 4 sequences analyzed contained mutations in the 9 residues that directly interact with the therapeutic Remdesivir, suggesting limited mutations in drug interacting residues. The identification of new variants emphasizes the need for further study on the effects of the mutations and the implications of increased prevalence, particularly for vaccine or therapeutic efficacy.


Subject(s)
COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Africa/epidemiology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Asia/epidemiology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Databases, Factual , Epidemiological Monitoring , Europe/epidemiology , Evolution, Molecular , Furin/genetics , Furin/metabolism , Gene Expression , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United States/epidemiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
13.
J Mol Model ; 27(11): 323, 2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1525539

ABSTRACT

The world has face the COVID-19 pandemic which has already caused millions of death. Due to the urgency in fighting the virus, we study five residues of free amino acids present in the structure of the SARS-CoV-2 spike protein (S). We investigated the spontaneous interaction between amino acids and silver ions (Ag+), considering these ions as a virucide chemical agent for SARS-CoV-2. The amino acid-Ag+ systems were investigated in a gaseous medium and a simulated water environment was described with a continuum model (PCM) the calculations were performed within the framework of density functional theory (DFT). Calculations related to the occupied orbitals of higher energy showed that Ag+ has a tendency to interact with the nitrile groups (-NH). The negative values of the Gibbs free energies show that the interaction process between amino acids-Ag+ in both media occurs spontaneously. There is a decrease in Gibbs free energy from the amino acid-Ag+ interactions immersed in a water solvation simulator.


Subject(s)
Amino Acids/chemistry , Antiviral Agents/chemistry , Density Functional Theory , Silver/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acids/metabolism , Antiviral Agents/metabolism , Binding Sites , Cations, Monovalent , Gene Expression , Humans , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , Silver/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Static Electricity , Thermodynamics
14.
Sci Rep ; 11(1): 22288, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1517638

ABSTRACT

Numerous repositioned drugs have been sought to decrease the severity of SARS-CoV-2 infection. It is known that among its physicochemical properties, Ursodeoxycholic Acid (UDCA) has a reduction in surface tension and cholesterol solubilization, it has also been used to treat cholesterol gallstones and viral hepatitis. In this study, molecular docking was performed with the SARS-CoV-2 Spike protein and UDCA. In order to confirm this interaction, we used Molecular Dynamics (MD) in "SARS-CoV-2 Spike protein-UDCA". Using another system, we also simulated MD with six UDCA residues around the Spike protein at random, naming this "SARS-CoV-2 Spike protein-6UDCA". Finally, we evaluated the possible interaction between UDCA and different types of membranes, considering the possible membrane conformation of SARS-CoV-2, this was named "SARS-CoV-2 membrane-UDCA". In the "SARS-CoV-2 Spike protein-UDCA", we found that UDCA exhibits affinity towards the central region of the Spike protein structure of - 386.35 kcal/mol, in a region with 3 alpha helices, which comprises residues from K986 to C1032 of each monomer. MD confirmed that UDCA remains attached and occasionally forms hydrogen bonds with residues R995 and T998. In the presence of UDCA, we observed that the distances between residues atoms OG1 and CG2 of T998 in the monomers A, B, and C in the prefusion state do not change and remain at 5.93 ± 0.62 and 7.78 ± 0.51 Å, respectively, compared to the post-fusion state. Next, in "SARS-CoV-2 Spike protein-6UDCA", the three UDCA showed affinity towards different regions of the Spike protein, but only one of them remained bound to the region between the region's heptad repeat 1 and heptad repeat 2 (HR1 and HR2) for 375 ps of the trajectory. The RMSD of monomer C was the smallest of the three monomers with a value of 2.89 ± 0.32, likewise, the smallest RMSF was also of the monomer C (2.25 ± 056). In addition, in the simulation of "SARS-CoV-2 membrane-UDCA", UDCA had a higher affinity toward the virion-like membrane; where three of the four residues remained attached once they were close (5 Å, to the centre of mass) to the membrane by 30 ns. However, only one of them remained attached to the plasma-like membrane and this was in a cluster of cholesterol molecules. We have shown that UDCA interacts in two distinct regions of Spike protein sequences. In addition, UDCA tends to stay bound to the membrane, which could potentially reduce the internalization of SARS-CoV-2 in the host cell.


Subject(s)
Antiviral Agents/metabolism , Drug Repositioning/methods , Lipid Bilayers/metabolism , Molecular Docking Simulation/methods , Phospholipids/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Ursodeoxycholic Acid/metabolism , Antiviral Agents/chemistry , COVID-19/metabolism , COVID-19/virology , Humans , Hydrogen Bonding , Membrane Fusion , Molecular Dynamics Simulation , Protein Binding , Protein Conformation, alpha-Helical , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Ursodeoxycholic Acid/chemistry , Virion/metabolism
15.
Infect Genet Evol ; 97: 105128, 2022 01.
Article in English | MEDLINE | ID: covidwho-1500148

ABSTRACT

The scientific community has been releasing whole genomic sequences of SARS-CoV-2 to facilitate the investigation of molecular features and evolutionary history. We retrieved 36 genomes of 18 prevalent countries of Asia, Europe and America for genomic diversity and mutational analysis. Besides, we studied mutations in the RBD regions of Spike (S) proteins to analyze the drug efficiency against these mutations. In this research, phylogenenetic analysis, evolutionary modeling, substitution pattern analysis, molecular docking, dynamics simulation, etc. were performed. The genomic sequences showed >99% similarity with the reference sequence of China.TN93 + G was predicted as a best nucleotide substitution model. It was revealed that effective transition from the co-existing SARS genome to the SARS-CoV-2 and a noticeable positive selection in the SARS-CoV-2 genomes occurred. Moreover, three mutations in RBD domain, Val/ Phe367, Val/ Leu 382 and Ala/ Val522, were discovered in the genomes from Netherland, Bangladesh and the USA, respectively. Molecular docking and dynamics study showed RBD with mutation Val/Leu382 had the lowest binding affinity with remdesivir. In conclusion, the SARS-CoV-2 genomes are similar, but multiple degrees of transitions and transversions occurred. The mutations cause a significant conformational change, which are needed to be investigated during drug and vaccine development.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , COVID-19/epidemiology , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Amino Acid Substitution , Antiviral Agents/pharmacology , Bangladesh/epidemiology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , China/epidemiology , Evolution, Molecular , Gene Expression , Humans , Likelihood Functions , Molecular Docking Simulation , Molecular Dynamics Simulation , Netherlands/epidemiology , Phylogeny , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United States/epidemiology
16.
J Virol ; 95(16): e0061721, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1486509

ABSTRACT

The current pandemic of COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, we first analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human angiotensin-converting enzyme 2 (ACE2) receptor. Among 32,123 genomes of SARS-CoV-2 isolates (December 2019 through March 2020), 302 nonsynonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F+D614G), which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. IMPORTANCE A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious has been the research hot spot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human angiotensin-converting enzyme 2 (ACE2) receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , COVID-19/transmission , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Gene Expression , Host-Pathogen Interactions/genetics , Humans , Kinetics , Molecular Dynamics Simulation , Phenylalanine/chemistry , Phenylalanine/metabolism , Phylogeny , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics , Valine/chemistry , Valine/metabolism , Virulence , Virus Attachment
17.
J Virol ; 95(15): e0046321, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1486505

ABSTRACT

The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase), which is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2-Å resolution. The structure reveals an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C terminus of the nsp16 subunit and an additional helix in the N terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the S-adenosyl methionine (SAM) binding pocket, suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses. IMPORTANCE Some coronaviruses are dangerous pathogens, while some cause only common colds. The reasons are not understood, although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail, we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved, suggesting that development of antivirals against multiple coronaviruses is feasible.


Subject(s)
Betacoronavirus/enzymology , Methyltransferases/chemistry , Viral Proteins/chemistry , Betacoronavirus/genetics , Binding Sites , Crystallography, X-Ray , Methyltransferases/genetics , Protein Conformation, alpha-Helical , Viral Proteins/genetics
18.
Curr Top Med Chem ; 21(16): 1429-1438, 2021 Oct 25.
Article in English | MEDLINE | ID: covidwho-1468281

ABSTRACT

As a part of the efforts to quickly develop pharmaceutical treatments for COVID-19 through repurposing existing drugs, some researchers around the world have combined the recently released crystal structure of SARS-CoV-2 Mpro in complex with a covalently bonded inhibitor with virtual screening procedures employing molecular docking approaches. In this context, protease inhibitors (PIs) clinically available and currently used to treat infectious diseases, particularly viral ones, are relevant sources of promising drug candidates to inhibit the SARS-CoV-2 Mpro, a key viral enzyme involved in crucial events during its life cycle. In the present perspective, we summarized the published studies showing the promising use of HIV and HCV PIs as potential repurposing drugs against the SARS-CoV-2 Mpro.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus M Proteins/antagonists & inhibitors , Drug Repositioning , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Binding Sites , COVID-19/virology , Coronavirus M Proteins/chemistry , Coronavirus M Proteins/genetics , Coronavirus M Proteins/metabolism , Humans , Kinetics , Models, Molecular , Molecular Targeted Therapy , Protease Inhibitors/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Randomized Controlled Trials as Topic , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Thermodynamics
19.
Mol Syst Biol ; 17(9): e10079, 2021 09.
Article in English | MEDLINE | ID: covidwho-1406892

ABSTRACT

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Host-Pathogen Interactions/genetics , Protein Processing, Post-Translational , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Binding Sites , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Computational Biology/methods , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Mimicry , Neuropilin-1/chemistry , Neuropilin-1/genetics , Neuropilin-1/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Multimerization , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism , Virus Replication
20.
Nat Commun ; 12(1): 141, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1387322

ABSTRACT

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.


Subject(s)
CD13 Antigens/metabolism , Coronavirus 229E, Human/physiology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Cell Line, Tumor , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Conformation, alpha-Helical , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
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