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1.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-624792

ABSTRACT

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Subject(s)
Actinobacteria/genetics , Genome, Bacterial , Protein Interaction Domains and Motifs/drug effects , Small Molecule Libraries/pharmacology , Tacrolimus Binding Protein 1A/chemistry , Tacrolimus Binding Protein 1A/metabolism , Actinobacteria/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Calcineurin/genetics , Calcineurin/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Evolution, Molecular , HEK293 Cells , Humans , Models, Molecular , Protein Conformation , Sequence Homology , Sirolimus/chemistry , Sirolimus/metabolism , Small Molecule Libraries/chemistry , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
2.
Molecules ; 25(17)2020 Aug 28.
Article in English | MEDLINE | ID: covidwho-740497

ABSTRACT

A pandemic caused by the novel coronavirus (SARS-CoV-2 or COVID-19) began in December 2019 in Wuhan, China, and the number of newly reported cases continues to increase. More than 19.7 million cases have been reported globally and about 728,000 have died as of this writing (10 August 2020). Recently, it has been confirmed that the SARS-CoV-2 main protease (Mpro) enzyme is responsible not only for viral reproduction but also impedes host immune responses. The Mpro provides a highly favorable pharmacological target for the discovery and design of inhibitors. Currently, no specific therapies are available, and investigations into the treatment of COVID-19 are lacking. Therefore, herein, we analyzed the bioactive phytocompounds isolated by gas chromatography-mass spectroscopy (GC-MS) from Tinospora crispa as potential COVID-19 Mpro inhibitors, using molecular docking study. Our analyses unveiled that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules, with three of them exerting biological activity and warranting further optimization and drug development to combat COVID-19.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/chemistry , Phytochemicals/chemistry , Protease Inhibitors/chemistry , Tinospora/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/classification , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Catalytic Domain , Coronavirus Infections/drug therapy , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Drug Discovery , Gas Chromatography-Mass Spectrometry , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Pandemics , Phytochemicals/classification , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Pneumonia, Viral/drug therapy , Protease Inhibitors/classification , Protease Inhibitors/isolation & purification , Protease Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Substrate Specificity , Thermodynamics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
3.
Molecules ; 25(17)2020 Aug 27.
Article in English | MEDLINE | ID: covidwho-737517

ABSTRACT

Three types of new coronaviruses (CoVs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and corona-virus disease 2019 (COVID-19). Neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. Each of the 3C-like (3CL) proteases of the three CoVs is essential for the proliferation of the CoVs, and an inhibitor of the 3CL protease (3CLpro) is thought to be an ideal therapeutic agent against SARS, MERS, or COVID-19. Among these, SARS-CoV is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. This article briefly reviews a series of studies on SARS-CoV, focusing on the development of inhibitors for the SARS-CoV 3CLpro based on molecular interactions with the 3CL protease. Our recent approach, based on the structure-based rational design of a novel scaffold for SARS-CoV 3CLpro inhibitor, is also included. The achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including SARS-CoV-2.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Protease Inhibitors/chemistry , SARS Virus/pathogenicity , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/classification , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Catalytic Domain , Coronavirus Infections/drug therapy , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Kinetics , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/drug therapy , Protease Inhibitors/classification , Protease Inhibitors/therapeutic use , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS Virus/genetics , SARS Virus/metabolism , Severe Acute Respiratory Syndrome/drug therapy , Substrate Specificity , Thermodynamics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
4.
Nat Commun ; 11(1): 4303, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-733523

ABSTRACT

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Betacoronavirus/metabolism , Chlorocebus aethiops , Epitope Mapping , Epitopes , Humans , Peptide Library , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
5.
Molecules ; 25(17)2020 Aug 23.
Article in English | MEDLINE | ID: covidwho-727434

ABSTRACT

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the Informational spectrum method applied for small molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Cysteine Endopeptidases/chemistry , Mezlocillin/chemistry , Protease Inhibitors/chemistry , Raltegravir Potassium/chemistry , Viral Nonstructural Proteins/chemistry , Allosteric Site , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Betacoronavirus/pathogenicity , Catalytic Domain , Coronavirus Infections/drug therapy , Coronavirus Infections/enzymology , Coronavirus Infections/virology , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Drug Repositioning , Gene Expression , High-Throughput Screening Assays , Humans , Mezlocillin/pharmacology , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/enzymology , Pneumonia, Viral/virology , Protease Inhibitors/pharmacology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Raltegravir Potassium/pharmacology , Thermodynamics , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
6.
Molecules ; 25(17)2020 Aug 22.
Article in English | MEDLINE | ID: covidwho-727433

ABSTRACT

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Cathepsin L/chemistry , Phytochemicals/chemistry , Protease Inhibitors/chemistry , Receptors, Virus/chemistry , Serine Endopeptidases/chemistry , Amino Acid Sequence , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Betacoronavirus/pathogenicity , Binding Sites , Cathepsin L/antagonists & inhibitors , Cathepsin L/genetics , Cathepsin L/metabolism , Coronavirus Infections/drug therapy , Coronavirus Infections/enzymology , Coronavirus Infections/virology , Coumarins/chemistry , Coumarins/isolation & purification , Coumarins/pharmacology , Gene Expression , High-Throughput Screening Assays , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , India , Molecular Docking Simulation , Molecular Dynamics Simulation , Monosaccharides/chemistry , Monosaccharides/isolation & purification , Monosaccharides/pharmacology , Pandemics , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Plants, Medicinal/chemistry , Pneumonia, Viral/drug therapy , Pneumonia, Viral/enzymology , Pneumonia, Viral/virology , Protease Inhibitors/isolation & purification , Protease Inhibitors/pharmacology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Quinazolines/chemistry , Quinazolines/isolation & purification , Quinazolines/pharmacology , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Thermodynamics , Virus Internalization/drug effects
7.
J Transl Med ; 18(1): 321, 2020 08 24.
Article in English | MEDLINE | ID: covidwho-727282

ABSTRACT

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Subject(s)
Betacoronavirus/physiology , Mutation, Missense , Peptidyl-Dipeptidase A/genetics , Protein Interaction Domains and Motifs/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Betacoronavirus/metabolism , Binding Sites/genetics , Coronavirus Infections/ethnology , Coronavirus Infections/genetics , Coronavirus Infections/virology , DNA Mutational Analysis/methods , Databases, Genetic , Genetic Predisposition to Disease/ethnology , Genetic Variation , Geography , Humans , Models, Molecular , Molecular Docking Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/ethnology , Pneumonia, Viral/genetics , Pneumonia, Viral/virology , Polymorphism, Single Nucleotide , Protein Binding , Protein Structure, Secondary/genetics , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
8.
Nat Commun ; 11(1): 4198, 2020 08 21.
Article in English | MEDLINE | ID: covidwho-724360

ABSTRACT

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Immunoglobulin A/immunology , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Chlorocebus aethiops , Cross Reactions , Epitopes , HEK293 Cells , Humans , Immunoglobulin A/metabolism , Immunoglobulin A, Secretory/immunology , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Interaction Domains and Motifs , SARS Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
9.
Science ; 369(6508): 1261-1265, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-697063

ABSTRACT

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Subject(s)
Betacoronavirus/metabolism , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Engineering , Receptors, Virus/genetics , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Binding Sites , Binding, Competitive , Cell Line , Humans , Models, Molecular , Mutagenesis , Mutation , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry
11.
Int J Antimicrob Agents ; 56(3): 106119, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-690298

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a highly transmissible viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical trials have reported improved outcomes resulting from an effective reduction or absence of viral load when patients were treated with chloroquine (CQ) or hydroxychloroquine (HCQ). In addition, the effects of these drugs were improved by simultaneous administration of azithromycin (AZM). The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein binds to the cell surface angiotensin-converting enzyme 2 (ACE2) receptor, allowing virus entry and replication in host cells. The viral main protease (Mpro) and host cathepsin L (CTSL) are among the proteolytic systems involved in SARS-CoV-2 S protein activation. Hence, molecular docking studies were performed to test the binding performance of these three drugs against four targets. The findings showed AZM affinity scores (ΔG) with strong interactions with ACE2, CTSL, Mpro and RBD. CQ affinity scores showed three low-energy results (less negative) with ACE2, CTSL and RBD, and a firm bond score with Mpro. For HCQ, two results (ACE2 and Mpro) were firmly bound to the receptors, however CTSL and RBD showed low interaction energies. The differences in better interactions and affinity between HCQ and CQ with ACE2 and Mpro were probably due to structural differences between the drugs. On other hand, AZM not only showed more negative (better) values in affinity, but also in the number of interactions in all targets. Nevertheless, further studies are needed to investigate the antiviral properties of these drugs against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Azithromycin/chemistry , Betacoronavirus/chemistry , Cathepsin L/chemistry , Chloroquine/chemistry , Cysteine Endopeptidases/chemistry , Hydroxychloroquine/chemistry , Peptidyl-Dipeptidase A/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Motifs , Antiviral Agents/chemistry , Azithromycin/pharmacology , Betacoronavirus/metabolism , Binding Sites , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cysteine Endopeptidases/metabolism , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Hydroxychloroquine/pharmacology , Molecular Docking Simulation , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Virus Attachment/drug effects
12.
J Med Virol ; 92(6): 584-588, 2020 06.
Article in English | MEDLINE | ID: covidwho-685102

ABSTRACT

Last December 2019, a new virus, named novel Coronavirus (COVID-2019) causing many cases of severe pneumonia was reported in Wuhan, China. The virus knowledge is limited and especially about COVID-2019 pathogenesis. The Open Reading Frame 1ab (ORF1ab) of COVID-2019 has been analyzed to evidence the presence of mutation caused by selective pressure on the virus. For selective pressure analysis fast-unconstrained Bayesian approximation (FUBAR) was used. Homology modelling has been performed by SwissModel and HHPred servers. The presence of transmembrane helical segments in Coronavirus ORF1ab non structural protein 2 (nsp2) and nsp3 was tested by TMHMM, MEMSAT, and MEMPACK tools. Three-dimensional structures have been analyzed and displayed using PyMOL. FUBAR analysis revealed the presence of potential sites under positive selective pressure (P < .05). Position 723 in the COVID-2019 has a serine instead a glycine residue, while at aminoacidic position 1010 a proline instead an isoleucine. Significant (P < .05) pervasive negative selection in 2416 sites (55%) was found. The positive selective pressure could account for some clinical features of this virus compared with severe acute respiratory syndrome (SARS) and Bat SARS-like CoV. The stabilizing mutation falling in the endosome-associated-protein-like domain of the nsp2 protein could account for COVID-2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID-2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen.


Subject(s)
Betacoronavirus/genetics , SARS Virus/genetics , Viral Nonstructural Proteins/chemistry , Viral Proteins/chemistry , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Female , Gene Expression , Humans , Male , Models, Molecular , Mutation , Pandemics , Pneumonia, Viral/virology , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS Virus/pathogenicity , Selection, Genetic , Structural Homology, Protein , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
13.
J Theor Biol ; 505: 110425, 2020 11 21.
Article in English | MEDLINE | ID: covidwho-680417

ABSTRACT

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/drug therapy , Drug Design , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/drug therapy , Protein Engineering/methods , Amino Acid Substitution , Betacoronavirus/genetics , Binding Sites , Directed Molecular Evolution , Humans , Pandemics , Peptidyl-Dipeptidase A/therapeutic use , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Stability , Spike Glycoprotein, Coronavirus/metabolism
14.
J Virol ; 94(15)2020 07 16.
Article in English | MEDLINE | ID: covidwho-661225

ABSTRACT

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a pandemic. Here, we used X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptors allows the definition of residues important for binding. From the 20 amino acids in ACE2 that contact S, up to 7 can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or acquisition of N-glycosylation sites located near the S interface. Of note, pigs and dogs, which are not infected or are not effectively infected and have only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with that from bat coronavirus strain RaTG13 (Bat-CoV-RaTG13) and pangolin coronavirus (Pangolin-CoV) strain hCoV-19/pangolin/Guangdong/1/2019 revealed that the latter contains only one substitution, whereas Bat-CoV-RaTG13 exhibits five. However, ACE2 of pangolin exhibits seven changes relative to human ACE2, and a similar number of substitutions is present in ACE2 of bats, raccoon dogs, and civets, suggesting that SARS-CoV-2 may not be especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-CoV-2.IMPORTANCE SARS-CoV-2 is threatening people worldwide, and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear, and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes, indicating that the species barrier might be low. Exceptions are dogs and especially pigs, which revealed relatively low ACE2 expression levels in the respiratory tract. Monitoring of animals is necessary to prevent the generation of a new coronavirus reservoir. Finally, our analysis also showed that SARS-CoV-2 may not be specifically adapted to any of its putative intermediate hosts.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment , Animals , Animals, Domestic , Betacoronavirus/metabolism , Chiroptera/virology , Coronavirus Infections/metabolism , Dogs , Glycosylation , Host-Pathogen Interactions , Humans , Models, Animal , Pandemics , Pets , Pneumonia, Viral/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Raccoons/virology , Sequence Alignment , Sequence Analysis, Protein , Swine , Viverridae/virology
15.
Science ; 369(6508): 1249-1255, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-654484

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.


Subject(s)
Betacoronavirus/chemistry , Immune Evasion , Immunity, Innate , Protein Biosynthesis , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Betacoronavirus/immunology , Betacoronavirus/metabolism , Betacoronavirus/physiology , Binding Sites , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cryoelectron Microscopy , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Humans , Interferon-beta/genetics , Interferon-beta/metabolism , Models, Molecular , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , Protein Structure, Secondary , RNA, Messenger/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/metabolism
16.
Antimicrob Agents Chemother ; 64(9)2020 08 20.
Article in English | MEDLINE | ID: covidwho-646490

ABSTRACT

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Cysteine Endopeptidases/chemistry , Protease Inhibitors/chemistry , Pyrrolidines/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Motifs , Animals , Antiviral Agents/pharmacology , Betacoronavirus/pathogenicity , Catalytic Domain , Chlorocebus aethiops , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Gene Expression , Molecular Docking Simulation , Protease Inhibitors/pharmacology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Pyrrolidines/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
17.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Article in English | MEDLINE | ID: covidwho-640223

ABSTRACT

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , Peptidyl-Dipeptidase A/chemistry , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Animals , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , Chiroptera/virology , Coronavirus Infections/virology , Cryoelectron Microscopy , Evolution, Molecular , Furin/chemistry , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Stability , Proteolysis , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structural Homology, Protein
18.
Biophys Chem ; 264: 106425, 2020 09.
Article in English | MEDLINE | ID: covidwho-634721

ABSTRACT

The novel SARS-CoV-2 is the etiological agent causing the Coronavirus disease 2019 (COVID-19), which continues to become an inevitable pandemic outbreak. Over a short span of time, the structures of therapeutic target proteins for SARS-CoV-2 were identified based on the homology modelled structure of similar SARS-CoV transmission of 2003. Since the onset of the disease, the research community has been looking for a potential drug lead. Out of all the known resolved structures related to SARS-CoV, Main protease (Mpro) is considered an attractive anti-viral drug target on the grounds of its role in viral replication and probable non-interactive competency to bind to any viral host protein. To the best of our knowledge, till date only one compound has been identified and tested in-vivo as a potent inhibitor of Mpro protein, addressed as N3 (PubChem Compound CID: 6323191) and is known to bind irreversibly to Mpro suppressing its activity. Using computational approach, we intend to identify a probable natural fungal metabolite to interact and inhibit Mpro. After screening various small molecules for molecular docking and dynamics simulation, we propose Pyranonigrin A, a secondary fungal metabolite to possess potent inhibitory potential against the Main protease (Mpro) expressed in SARS-CoV-2 virus.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/enzymology , Protease Inhibitors/chemistry , Pyrones/chemistry , Pyrroles/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Betacoronavirus/pathogenicity , Binding Sites , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Drug Discovery , Gene Expression , Hydrogen Bonding , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Sequence Homology, Amino Acid , Thermodynamics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
19.
Cell ; 182(3): 722-733.e11, 2020 08 06.
Article in English | MEDLINE | ID: covidwho-628738

ABSTRACT

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , SARS Virus/immunology , Universal Design , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/chemistry , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Middle East Respiratory Syndrome Coronavirus/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding , Protein Interaction Domains and Motifs/immunology , Receptors, Virus/metabolism , SARS Virus/chemistry , Sf9 Cells , Specific Pathogen-Free Organisms , Spodoptera , Transfection , Vaccination/methods , Vero Cells , Viral Vaccines
20.
Biophys Chem ; 264: 106420, 2020 09.
Article in English | MEDLINE | ID: covidwho-623862

ABSTRACT

One notable feature of the SARS-CoV-2 genome, the spike (S) protein of SARS-CoV-2 has a polybasic furin cleavage site (FCS) at its S1-S2 boundary through the insertion of 12 nucleotides encoding four amino acid residues PRRA. Quite intriguingly, this polybasic FCS is absent in coronaviruses of the same clade as SARS-CoV-2. Thus, with currently available experimental structural data for S protein, this short article presents a set of comprehensive structural characterization of the insertion of FCS into S protein, and argues against a hypothesis of the origin of SARS-CoV-2 from purposeful manipulation: (1), the inserted FCS is spatially located at a random coil loop region, mostly distantly solvent-exposed (instead of deeply buried), with no structural proximity to the other part of the S protein; (2), the insertion of FCS itself does not alter, neither stabilize nor de-stabilize, the three-dimensional structure of S; (3), the net result here is the insertion of a furin cleavage site into S protein, whose S1 and S2 subunits will still be strongly electrostatically bonded together from a structural and biophysical point of view, even if the polybasic FCS is actually cleaved by furin protease before or after viral cell entry.


Subject(s)
Betacoronavirus/chemistry , Furin/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Betacoronavirus/genetics , Gene Expression , Humans , Hydrogen Bonding , Models, Molecular , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proteolysis , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structural Homology, Protein
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