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1.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1621335

ABSTRACT

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Endosomes/metabolism , SARS-CoV-2 , Sorting Nexins/chemistry , COVID-19/virology , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Crystallography, X-Ray , Cytosol/metabolism , Endocytosis , Gene Expression Profiling , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Lentivirus , Lysosomes/metabolism , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Domains , Sorting Nexins/metabolism , Virus Internalization
2.
Nat Commun ; 13(1): 222, 2022 01 11.
Article in English | MEDLINE | ID: covidwho-1621242

ABSTRACT

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.


Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Animals , COVID-19/virology , Cell Line , Cryoelectron Microscopy , Evolution, Molecular , Furin/metabolism , Humans , Linoleic Acid/metabolism , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Virus Internalization
3.
Nat Commun ; 13(1): 19, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1616981

ABSTRACT

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.


Subject(s)
COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/immunology , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , COVID-19/virology , Epitopes, T-Lymphocyte/metabolism , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/metabolism , Humans , Immunodominant Epitopes/immunology , Immunodominant Epitopes/metabolism , Jurkat Cells , K562 Cells , Peptides/chemistry , Peptides/immunology , Peptides/metabolism , Protein Binding , Protein Conformation , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Surface Plasmon Resonance/methods
4.
Nat Commun ; 13(1): 155, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1616979

ABSTRACT

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Bispecific/metabolism , COVID-19/virology , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mice, Transgenic , Neutralization Tests/methods , Protein Binding , Protein Conformation , Protein Multimerization/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
5.
Biochem Biophys Res Commun ; 590: 34-41, 2022 01 29.
Article in English | MEDLINE | ID: covidwho-1588232

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to more than 270 million infections and 5.3 million of deaths worldwide. Several major variants of SARS-CoV-2 have emerged and posed challenges in controlling the pandemic. The recently occurred Omicron variant raised serious concerns about reducing the efficacy of vaccines and neutralization antibodies due to its vast mutations. We have modelled the complex structure of the human ACE2 protein and the receptor binding domain (RBD) of Omicron Spike protein (S-protein), and conducted atomistic molecular dynamics simulations to study the binding interactions. The analysis shows that the Omicron RBD binds more strongly to the human ACE2 protein than the original strain. The mutations at the ACE2-RBD interface enhance the tight binding by increasing hydrogen bonding interaction and enlarging buried solvent accessible surface area.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , Host-Pathogen Interactions , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
6.
J Med Chem ; 64(8): 4991-5000, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1574766

ABSTRACT

The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Oligopeptides/chemistry , Binding Sites , Coronavirus 3C Proteases/metabolism , Crystallography/methods , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Models, Molecular , Neutrons , Oligopeptides/metabolism , Protein Conformation , Protons
7.
Viruses ; 13(12)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1572669

ABSTRACT

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants' evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.


Subject(s)
Furin/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Genetic Fitness , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Phylogeny , Protein Binding , Protein Conformation , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry
8.
Mol Immunol ; 141: 287-296, 2022 01.
Article in English | MEDLINE | ID: covidwho-1559780

ABSTRACT

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19 Serological Testing , COVID-19/diagnosis , Cell Surface Display Techniques , Epitopes/immunology , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Single-Chain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/blood , Antibody Specificity , Baculoviridae , COVID-19/prevention & control , Escherichia coli , Female , Genetic Vectors , Mice , Mice, Inbred BALB C , Models, Molecular , Peptide Library , Protein Conformation , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Single-Chain Antibodies/biosynthesis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
9.
Nat Struct Mol Biol ; 28(7): 614-625, 2021 07.
Article in English | MEDLINE | ID: covidwho-1550333

ABSTRACT

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.


Subject(s)
Adenosine Triphosphate/chemistry , Protein Folding , Proteostasis/physiology , Signal Transduction/physiology , Valosin Containing Protein/metabolism , Acetanilides/pharmacology , Animals , Benzothiazoles/pharmacology , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/antagonists & inhibitors
10.
PLoS One ; 16(11): e0258645, 2021.
Article in English | MEDLINE | ID: covidwho-1518355

ABSTRACT

All approved coronavirus disease 2019 (COVID-19) vaccines in current use are safe, effective, and reduce the risk of severe illness. Although data on the immunological presentation of patients with COVID-19 is limited, increasing experimental evidence supports the significant contribution of B and T cells towards the resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Despite the availability of several COVID-19 vaccines with high efficacy, more effective vaccines are still needed to protect against the new variants of SARS-CoV-2. Employing a comprehensive immunoinformatic prediction algorithm and leveraging the genetic closeness with SARS-CoV, we have predicted potential immune epitopes in the structural proteins of SARS-CoV-2. The S and N proteins of SARS-CoV-2 and SARS-CoVs are main targets of antibody detection and have motivated us to design four multi-epitope vaccines which were based on our predicted B- and T-cell epitopes of SARS-CoV-2 structural proteins. The cardinal epitopes selected for the vaccine constructs are predicted to possess antigenic, non-allergenic, and cytokine-inducing properties. Additionally, some of the predicted epitopes have been experimentally validated in published papers. Furthermore, we used the C-ImmSim server to predict effective immune responses induced by the epitope-based vaccines. Taken together, the immune epitopes predicted in this study provide a platform for future experimental validations which may facilitate the development of effective vaccine candidates and epitope-based serological diagnostic assays.


Subject(s)
Computational Biology , Epitope Mapping , SARS-CoV-2/immunology , Viral Structural Proteins/immunology , Amino Acid Sequence , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Databases as Topic , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Models, Molecular , Protein Conformation , Reproducibility of Results , Viral Structural Proteins/chemistry
11.
Sci Rep ; 11(1): 22042, 2021 11 11.
Article in English | MEDLINE | ID: covidwho-1510622

ABSTRACT

The mutation of SARS-CoV-2 influences viral function as residue replacements affect both physiochemical properties and folding conformations. Although a large amount of data on SARS-CoV-2 is available, the investigation of how viral functions change in response to mutations is hampered by a lack of effective structural analysis. Here, we exploit the advances of protein structure fingerprint technology to study the folding conformational changes induced by mutations. With integration of both protein sequences and folding conformations, the structures are aligned for SARS-CoV to SARS-CoV-2, including Alpha variant (lineage B.1.1.7) and Delta variant (lineage B.1.617.2). The results showed that the virus evolution with change in mutational positions and physicochemical properties increased the affinity between spike protein and ACE2, which plays a critical role in coronavirus entry into human cells. Additionally, these structural variations impact vaccine effectiveness and drug function over the course of SARS-CoV-2 evolution. The analysis of structural variations revealed how the coronavirus has gradually evolved in both structure and function and how the SARS-CoV-2 variants have contributed to more severe acute disease worldwide.


Subject(s)
COVID-19/virology , Evolution, Molecular , Mutation , SARS-CoV-2/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Interaction Maps , Protein Multimerization , SARS Virus/chemistry , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
12.
Angew Chem Int Ed Engl ; 60(48): 25428-25435, 2021 11 22.
Article in English | MEDLINE | ID: covidwho-1490696

ABSTRACT

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/chemistry , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Microbial Sensitivity Tests , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Vero Cells
13.
Int J Mol Sci ; 22(21)2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1488616

ABSTRACT

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.


Subject(s)
COVID-19/drug therapy , Cathepsin L/drug effects , Coronavirus 3C Proteases/drug effects , Leupeptins/chemistry , Leupeptins/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Catalytic Domain/drug effects , Cathepsin L/chemistry , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Discovery , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptidomimetics , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Virus Replication/drug effects , X-Ray Diffraction
14.
Int J Mol Sci ; 22(21)2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1488615

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received global attention due to the serious threat it poses to public health. Since the outbreak in December 2019, millions of people have been affected and its rapid global spread has led to an upsurge in the search for treatment. To discover hit compounds that can be used alone or in combination with repositioned drugs, we first analyzed the pharmacokinetic and toxicological properties of natural products from Brazil's semiarid region. After, we analyzed the site prediction and druggability of the SARS-CoV-2 main protease (Mpro), followed by docking and molecular dynamics simulation. The best SARS-CoV-2 Mpro complexes revealed that other sites were accessed, confirming that our approach could be employed as a suitable starting protocol for ligand prioritization, reinforcing the importance of catalytic cysteine-histidine residues and providing new structural data that could increase the antiviral development mainly against SARS-CoV-2. Here, we selected 10 molecules that could be in vitro assayed in response to COVID-19. Two compounds (b01 and b02) suggest a better potential for interaction with SARS-CoV-2 Mpro and could be further studied.


Subject(s)
Biological Products/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/drug effects , Drug Design , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Drug Discovery/methods , Drug Repositioning , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Conformation , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/drug effects
15.
Int J Mol Sci ; 22(21)2021 Oct 27.
Article in English | MEDLINE | ID: covidwho-1488610

ABSTRACT

The angiotensin-converting enzyme 2 (ACE2) is the receptor used by SARS-CoV and SARS-CoV-2 coronaviruses to attach to cells via the receptor-binding domain (RBD) of their viral spike protein. Since the start of the COVID-19 pandemic, several structures of protein complexes involving ACE2 and RBD as well as monoclonal antibodies and nanobodies have become available. We have leveraged the structural data to design peptides to target the interaction between the RBD of SARS-CoV-2 and ACE2 and SARS-CoV and ACE2, as contrasting exemplar, as well as the dimerization surface of ACE2 monomers. The peptides were modelled using our original method: PiPreD that uses native elements of the interaction between the targeted protein and cognate partner(s) that are subsequently included in the designed peptides. These peptides recapitulate stretches of residues present in the native interface plus novel and highly diverse conformations surrogating key interactions at the interface. To facilitate the access to this information we have created a freely available and dedicated web-based repository, PepI-Covid19 database, providing convenient access to this wealth of information to the scientific community with the view of maximizing its potential impact in the development of novel therapeutic and diagnostic agents.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Host-Pathogen Interactions/drug effects , Peptides/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Databases, Factual , Humans , Models, Molecular , Peptide Library , Peptides/chemistry , Protein Conformation , Protein Domains , Protein Engineering , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry
17.
J Virol ; 95(16): e0018721, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1486048

ABSTRACT

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/chemistry , Host-Pathogen Interactions/drug effects , Porcine epidemic diarrhea virus/drug effects , Quercetin/analogs & derivatives , Tumor Suppressor Protein p53/chemistry , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Binding Sites , Cell Line , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Molecular Docking Simulation , Nuclear Localization Signals , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Quercetin/chemistry , Quercetin/pharmacology , S Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/genetics , Signal Transduction , Swine , Swine Diseases/drug therapy , Swine Diseases/genetics , Swine Diseases/metabolism , Swine Diseases/virology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vero Cells , Virus Replication/drug effects
18.
Biomed Res Int ; 2021: 9982729, 2021.
Article in English | MEDLINE | ID: covidwho-1476892

ABSTRACT

The human transmembrane protease serine 2 (TMPRSS2) protein plays an important role in prostate cancer progression. It also facilitates viral entry into target cells by proteolytically cleaving and activating the S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the current study, we used different available tools like SIFT, PolyPhen2.0, PROVEAN, SNAP2, PMut, MutPred2, I-Mutant Suite, MUpro, iStable, ConSurf, ModPred, SwissModel, PROCHECK, Verify3D, and TM-align to identify the most deleterious variants and to explore possible effects on the TMPRSS2 stability, structure, and function. The six missense variants tested were evaluated to have deleterious effects on the protein by SIFT, PolyPhen2.0, PROVEAN, SNAP2, and PMut. Additionally, V160M, G181R, R240C, P335L, G432A, and D435Y variants showed a decrease in stability by at least 2 servers; G181R, G432A, and D435Y are highly conserved and identified posttranslational modifications sites (PTMs) for proteolytic cleavage and ADP-ribosylation using ConSurf and ModPred servers. The 3D structure of TMPRSS2 native and mutants was generated using 7 meq as a template from the SwissModeller group, refined by ModRefiner, and validated using the Ramachandran plot. Hence, this paper can be advantageous to understand the association between these missense variants rs12329760, rs781089181, rs762108701, rs1185182900, rs570454392, and rs867186402 and susceptibility to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Mutation, Missense , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Binding Sites , Computational Biology/methods , Evolution, Molecular , Genetic Predisposition to Disease , Humans , Models, Molecular , Phylogeny , Polymorphism, Single Nucleotide , Protein Conformation , Protein Stability , Serine Endopeptidases/metabolism
19.
Int J Biol Macromol ; 187: 976-987, 2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1474606

ABSTRACT

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.


Subject(s)
3C Viral Proteases/chemistry , 3C Viral Proteases/metabolism , Ampelopsis/chemistry , Antiviral Agents/pharmacology , Flavonoids/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Binding Sites/drug effects , Cysteine/metabolism , Flavonoids/chemistry , Flavonols/chemistry , Flavonols/pharmacology , Mass Spectrometry , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/chemistry , Plant Extracts/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding/drug effects , Protein Conformation/drug effects , SARS-CoV-2/drug effects
20.
Molecules ; 26(19)2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1463768

ABSTRACT

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.


Subject(s)
Choanoflagellata/chemistry , Choanoflagellata/metabolism , Computational Biology/methods , PDZ Domains , Protein Binding , Amino Acid Sequence , Evolution, Molecular , Humans , Phylogeny , Protein Conformation , Signal Transduction , Software , Substrate Specificity
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