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1.
PLoS Pathog ; 17(1): e1009246, 2021 01.
Article in English | MEDLINE | ID: covidwho-1045566

ABSTRACT

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.


Subject(s)
Cell Fusion , Furin/genetics , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Animals , CRISPR-Cas Systems , Chlorocebus aethiops , Gene Knockout Techniques , HEK293 Cells , Humans , Protein Structure, Tertiary , Serine Endopeptidases , Vero Cells
2.
Sci Rep ; 11(1): 1529, 2021 01 15.
Article in English | MEDLINE | ID: covidwho-1033541

ABSTRACT

The genetic variations among individuals are one of the notable factors determining disease severity and drug response. Nowadays, COVID-19 pandemic has been adversely affecting many aspects of human life. We used the Tehran Cardio-Metabolic Genetic Study (TCGS) data that is an ongoing genetic study including the whole-genome sequencing of 1200 individuals and chip genotyping of more than 15,000 participants. Here, the effect of ACE2 variations by focusing on the receptor-binding site of SARS-CoV-2 and ACE2 cleavage by TMPRSS2 protease were investigated through simulations study. After analyzing TCGS data, 570 genetic variations on the ACE2 gene, including single nucleotide polymorphisms (SNP) and insertion/deletion (INDEL) were detected. Interestingly, two observed missense variants, K26R and S331F, which only the first one was previously reported, can reduce the receptor affinity for the viral Spike protein. Moreover, our bioinformatics simulation of 3D structures and docking of proteins explains important details of ACE2-Spike and ACE2-TMPRSS2 interactions, especially the critical role of Arg652 of ACE2 for protease function of TMPRSS2 was uncovered. As our results show that the genetic variation of ACE2 can at least influence the affinity of this receptor to its partners, we need to consider the genetic variations on ACE2 as well as other genes in the pathways that contribute to the pathogenesis of COVID-19 for designing efficient drugs and vaccines.


Subject(s)
/genetics , /pathology , /chemistry , Binding Sites , /virology , Disease Susceptibility , Gene Expression , Genotype , Humans , INDEL Mutation , Iran , Molecular Docking Simulation , Mutation, Missense , Polymorphism, Single Nucleotide , Protein Binding , Protein Structure, Tertiary , /metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Whole Genome Sequencing
3.
J Med Chem ; 64(1): 890-904, 2021 01 14.
Article in English | MEDLINE | ID: covidwho-997768

ABSTRACT

The sigma 1 receptor (S1R) is a molecular chaperone protein located in the endoplasmic reticulum and plasma membranes and has been shown to play important roles in various pathological disorders including pain and, as recently discovered, COVID-19. Employing structure- and QSAR-based drug design strategies, we rationally designed, synthesized, and biologically evaluated a series of novel triazole-based S1R antagonists. Compound 10 exhibited potent binding affinity for S1R, high selectivity over S2R and 87 other human targets, acceptable in vitro metabolic stability, slow clearance in liver microsomes, and excellent blood-brain barrier permeability in rats. Further in vivo studies in rats showed that 10 exhibited negligible acute toxicity in the rotarod test and statistically significant analgesic effects in the formalin test for acute inflammatory pain and paclitaxel-induced neuropathic pain models during cancer chemotherapy. These encouraging results promote further development of our triazole-based S1R antagonists as novel treatments for pain of different etiologies.


Subject(s)
Pain Management/methods , Receptors, sigma/antagonists & inhibitors , Triazoles/chemistry , Animals , Binding Sites , Blood-Brain Barrier/metabolism , Drug Design , Guinea Pigs , Half-Life , Humans , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Neuralgia/chemically induced , Neuralgia/drug therapy , Protein Structure, Tertiary , Quantitative Structure-Activity Relationship , Rats , Receptors, sigma/metabolism , Triazoles/metabolism , Triazoles/therapeutic use
4.
Adv Drug Deliv Rev ; 169: 100-117, 2021 02.
Article in English | MEDLINE | ID: covidwho-966180

ABSTRACT

To address the COVID-19 pandemic, there has been an unprecedented global effort to advance potent neutralizing mAbs against SARS-CoV-2 as therapeutics. However, historical efforts to advance antiviral monoclonal antibodies (mAbs) for the treatment of other respiratory infections have been met with categorical failures in the clinic. By investigating the mechanism by which SARS-CoV-2 and similar viruses spread within the lung, along with available biodistribution data for systemically injected mAb, we highlight the challenges faced by current antiviral mAbs for COVID-19. We summarize some of the leading mAbs currently in development, and present the evidence supporting inhaled delivery of antiviral mAb as an early intervention against COVID-19 that could prevent important pulmonary morbidities associated with the infection.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antiviral Agents/therapeutic use , Immunologic Factors/therapeutic use , /drug effects , /antagonists & inhibitors , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , /metabolism , Humans , Immunization, Passive , Immunologic Factors/chemistry , Immunologic Factors/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , /metabolism , Virus Shedding/drug effects , Virus Shedding/physiology
5.
PLoS One ; 15(11): e0240345, 2020.
Article in English | MEDLINE | ID: covidwho-917985

ABSTRACT

In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variants sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 68 recurrent mutations, including ten hotspot non-synonymous mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed that certain genotypes are specific to geographic locations. We also identified co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. The phylogentic analysis identified two major Clades C1 and C2 harboring mutations L3606F and G614D, respectively and both emerging for the first time in China. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets. We have also created an inclusive unified database (http://covid-19.medbiotech.ma) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.


Subject(s)
Betacoronavirus/genetics , Genetic Variation , Genome, Viral , Betacoronavirus/classification , Betacoronavirus/isolation & purification , China , Coronavirus Infections/pathology , Coronavirus Infections/virology , Evolution, Molecular , Humans , Pandemics , Phylogeny , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , Protein Structure, Tertiary , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/chemistry , Viral Proteins/genetics
6.
Molecules ; 25(21)2020 Oct 29.
Article in English | MEDLINE | ID: covidwho-902610

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), which caused novel corona virus disease-2019 (COVID-19) pandemic, necessitated a global demand for studies related to genes and enzymes of SARS-CoV2. SARS-CoV2 infection depends on the host cell Angiotensin-Converting Enzyme-2 (ACE2) and Transmembrane Serine Protease-2 (TMPRSS2), where the virus uses ACE2 for entry and TMPRSS2 for S protein priming. The TMPRSS2 gene encodes a Transmembrane Protease Serine-2 protein (TMPS2) that belongs to the serine protease family. There is no crystal structure available for TMPS2, therefore, a homology model was required to establish a putative 3D structure for the enzyme. A homology model was constructed using SWISS-MODEL and evaluations were performed through Ramachandran plots, Verify 3D and Protein Statistical Analysis (ProSA). Molecular dynamics simulations were employed to investigate the stability of the constructed model. Docking of TMPS2 inhibitors, camostat, nafamostat, gabexate, and sivelestat, using Molecular Operating Environment (MOE) software, into the constructed model was performed and the protein-ligand complexes were subjected to MD simulations and computational binding affinity calculations. These in silico studies determined the tertiary structure of TMPS2 amino acid sequence and predicted how ligands bind to the model, which is important for drug development for the prevention and treatment of COVID-19.


Subject(s)
Betacoronavirus/drug effects , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Gabexate/analogs & derivatives , Gabexate/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Guanidines/pharmacology , Humans , Ligands , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Pneumonia, Viral/drug therapy , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Serine Endopeptidases/metabolism , Sulfonamides/pharmacology
7.
PLoS One ; 15(10): e0241172, 2020.
Article in English | MEDLINE | ID: covidwho-890193

ABSTRACT

The novel coronavirus 2019 (COVID-19) global pandemic has drastically affected the world economy, raised public anxiety, and placed a substantial psychological burden on the governments and healthcare professionals by affecting over 4.7 million people worldwide. As a preventive measure to minimise the risk of community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in India, a nationwide lockdown was imposed initially for 21 days to limit the movement of 1.3 billion people. These restrictions continue in most areas, with a conditional relaxation occurring in a few Indian states. In an attempt to assess the emerging mutants of SARS-CoV-2 and determine their spread in India, we analysed 112 complete genomes of SARS-CoV-2 in a time-lapse manner. We found 72 distinct SARS-CoV-2 haplotypes, defined by 143 polymorphic sites and high haplotype diversity, suggesting that this virus possesses a high evolutionary potential. We also demonstrated that early introduction of SARS-CoV-2 into India was from China, Italy and Iran and observed signs of community spread of the virus following its rapid demographic expansion since its first outbreak in the country. Additionally, we identified 18 mutations in the SARS-CoV-2 spike glycoprotein and a few selected mutations showed to increase stability, binding affinity, and molecular flexibility in the overall tertiary structure of the protein that may facilitate interaction between the receptor binding domain (RBD) of spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor. The study provides a pragmatic view of haplotype-dependent spread of SARS-CoV-2 in India which could be important in tailoring the pharmacologic treatments to be more effective for those infected with the most common haplotypes. The findings based on the time-lapse sentinel surveillance of SARS-CoV-2 will aid in the development of a real-time practical framework to tackle the ongoing, fast-evolving epidemic challenges in the country.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Sentinel Surveillance , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Genome, Viral/genetics , Haplotypes , Humans , India/epidemiology , Molecular Docking Simulation , Mutation , Pandemics/prevention & control , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , Polymorphism, Genetic , Protein Structure, Tertiary , Quarantine/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Sci Rep ; 10(1): 17699, 2020 10 19.
Article in English | MEDLINE | ID: covidwho-880703

ABSTRACT

Angiotensin converting enzyme 2 (ACE2) (EC:3.4.17.23) is a transmembrane protein which is considered as a receptor for spike protein binding of novel coronavirus (SARS-CoV2). Since no specific medication is available to treat COVID-19, designing of new drug is important and essential. In this regard, in silico method plays an important role, as it is rapid and cost effective compared to the trial and error methods using experimental studies. Natural products are safe and easily available to treat coronavirus affected patients, in the present alarming situation. In this paper five phytochemicals, which belong to flavonoid and anthraquinone subclass, have been selected as small molecules in molecular docking study of spike protein of SARS-CoV2 with its human receptor ACE2 molecule. Their molecular binding sites on spike protein bound structure with its receptor have been analyzed. From this analysis, hesperidin, emodin and chrysin are selected as competent natural products from both Indian and Chinese medicinal plants, to treat COVID-19. Among them, the phytochemical hesperidin can bind with ACE2 protein and bound structure of ACE2 protein and spike protein of SARS-CoV2 noncompetitively. The binding sites of ACE2 protein for spike protein and hesperidin, are located in different parts of ACE2 protein. Ligand spike protein causes conformational change in three-dimensional structure of protein ACE2, which is confirmed by molecular docking and molecular dynamics studies. This compound modulates the binding energy of bound structure of ACE2 and spike protein. This result indicates that due to presence of hesperidin, the bound structure of ACE2 and spike protein fragment becomes unstable. As a result, this natural product can impart antiviral activity in SARS CoV2 infection. The antiviral activity of these five natural compounds are further experimentally validated with QSAR study.


Subject(s)
Betacoronavirus/metabolism , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Amino Acid Sequence , Anthraquinones/chemistry , Anthraquinones/metabolism , Betacoronavirus/isolation & purification , Binding Sites , Coronavirus Infections/pathology , Coronavirus Infections/virology , Emodin/chemistry , Emodin/metabolism , Humans , Molecular Docking Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Spike Glycoprotein, Coronavirus/chemistry
9.
J Phys Chem Lett ; 11(21): 9144-9151, 2020 Nov 05.
Article in English | MEDLINE | ID: covidwho-867355

ABSTRACT

The raging COVID-19 pandemic caused by SARS-CoV-2 has infected tens of millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV-2 helicase (Nsp13), which is critical for viral replication and the most conserved nonstructural protein within the coronavirus family. Using homology modeling that couples published electron-density with molecular dynamics (MD)-based structural refinements, we generated structural models of the SARS-CoV-2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ∼970 000 chemical compounds against the ATP-binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP-binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/enzymology , RNA Helicases/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Betacoronavirus/isolation & purification , Binding Sites , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Structure, Tertiary , RNA Helicases/chemistry , RNA Helicases/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
10.
Science ; 370(6517): 725-730, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-787982

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.


Subject(s)
Linoleic Acid/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Animals , Betacoronavirus , Binding Sites , Chlorocebus aethiops , Cryoelectron Microscopy , Humans , Middle East Respiratory Syndrome Coronavirus , Models, Molecular , Peptidyl-Dipeptidase A/metabolism , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , SARS Virus , Spike Glycoprotein, Coronavirus/ultrastructure , Vero Cells
11.
Sci Data ; 7(1): 309, 2020 09 16.
Article in English | MEDLINE | ID: covidwho-772950

ABSTRACT

Emergence of coronaviruses poses a threat to global health and economy. The current outbreak of SARS-CoV-2 has infected more than 28,000,000 people and killed more than 915,000. To date, there is no treatment for coronavirus infections, making the development of therapies to prevent future epidemics of paramount importance. To this end, we collected information regarding naturally-occurring variants of the Angiotensin-converting enzyme 2 (ACE2), an epithelial receptor that both SARS-CoV and SARS-CoV-2 use to enter the host cells. We built 242 structural models of variants of human ACE2 bound to the receptor binding domain (RBD) of the SARS-CoV-2 surface spike glycoprotein (S protein) and refined their interfaces with HADDOCK. Our dataset includes 140 variants of human ACE2 representing missense mutations found in genome-wide studies, 39 mutants with reported effects on the recognition of the RBD, and 63 predictions after computational alanine scanning mutagenesis of ACE2-RBD interface residues. This dataset will help accelerate the design of therapeutics against SARS-CoV-2, as well as contribute to prevention of possible future coronaviruses outbreaks.


Subject(s)
Drug Design , Peptidyl-Dipeptidase A/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Betacoronavirus , Binding Sites , Coronavirus Infections , Humans , Models, Molecular , Pandemics , Pneumonia, Viral , Protein Binding , Protein Structure, Tertiary , Receptors, Virus/chemistry
12.
Sci Rep ; 10(1): 14991, 2020 09 14.
Article in English | MEDLINE | ID: covidwho-766137

ABSTRACT

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the innate and adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the notable exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight of the S trimer (17% for the HEK293 glycoform) they shield approximately 40% of the protein surface.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Polysaccharides/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Adaptive Immunity , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antigen-Antibody Complex , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Binding Sites , Coronavirus Infections/immunology , Coronavirus Infections/virology , Glycosylation , HEK293 Cells , HLA Antigens/metabolism , Humans , Immunity, Innate , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
13.
PLoS One ; 15(9): e0238089, 2020.
Article in English | MEDLINE | ID: covidwho-751013

ABSTRACT

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Subject(s)
Coronavirus Infections/pathology , Epitopes/chemistry , Pneumonia, Viral/pathology , Amino Acid Sequence , Antibodies, Viral/blood , Antibodies, Viral/immunology , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Epitope Mapping , Epitopes/blood , Epitopes/immunology , Furin/metabolism , Humans , Pandemics , Peptide Nucleic Acids/chemistry , Peptides/chemistry , Pneumonia, Viral/virology , Protein Array Analysis , Protein Structure, Tertiary , Proteolysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
14.
EBioMedicine ; 59: 102980, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-733876

ABSTRACT

BACKGROUND: Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits. METHODS: We have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model. FINDING: We have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds. INTERPRETATION: Our finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19). FUNDING: Project funding was supported by the ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Organoselenium Compounds/therapeutic use , Superoxide Dismutase-1/metabolism , Amyotrophic Lateral Sclerosis/mortality , Amyotrophic Lateral Sclerosis/pathology , Animals , Azoles/chemistry , Azoles/metabolism , Azoles/therapeutic use , Betacoronavirus/metabolism , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Disease Models, Animal , Enzyme Stability , Mice , Mice, Transgenic , Molecular Dynamics Simulation , Neuroprotective Agents/chemistry , Neuroprotective Agents/metabolism , Neuroprotective Agents/therapeutic use , Organoselenium Compounds/chemistry , Organoselenium Compounds/metabolism , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Superoxide Dismutase-1/genetics , Survival Rate , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism
15.
Anal Chem ; 92(16): 11297-11304, 2020 08 18.
Article in English | MEDLINE | ID: covidwho-733551

ABSTRACT

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.


Subject(s)
Microscopy, Atomic Force/methods , Nanostructures/chemistry , Spectrum Analysis, Raman/methods , Virion/chemistry , Animals , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , Capsid/chemistry , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cryoelectron Microscopy , Discriminant Analysis , Herpesvirus 1, Human/physiology , Humans , Influenza A Virus, H1N1 Subtype/physiology , Least-Squares Analysis , Levivirus/metabolism , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Structure, Tertiary , Vero Cells
16.
Eur J Pharmacol ; 885: 173496, 2020 Oct 15.
Article in English | MEDLINE | ID: covidwho-726509

ABSTRACT

The rapid breakout of the coronavirus disease of 2019 (COVID-19) has been declared pandemic with serious global concern due to high morbidity and mortality. As we enter the phase beyond limitations there is an urgent need for explicit treatment against COVID-19. To face this immediate global challenge, drug development from scratch is a lengthy process and unrealistic to conquer this battle. Drug repurposing is an emerging and practical approach where existing drugs, safe for humans, are redeployed to fight this harder to treat disease. A number of multi clinical studies have repurposed combined cocktail (remdesivir + chloroquine and favipiravir + chloroquine) to be effective against COVID-19. However, the exact mechanistic aspect has not yet been revealed. In the present study, we have tried to decipher the mechanistic aspects of existing medicines at the viral entry and replication stage via the structural viroinformatics approach. Here we implied the molecular docking and dynamic simulations with emphasis on the unique structural properties of host receptor angiotensin-converting enzyme 2 (ACE2), SARS-CoV2 spike protein and RNA dependent RNA polymerase enzyme (RdRp) of the SARS-CoV2. Deep structural analysis of target molecules exposed key binding residues and structural twists involved in binding with important pharmacophore features of existing drugs [(7-chloro-N-[5-(diethylamino)pentan-2-yl]quinolin-4-amine (chloroquine),N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide) (SSAA09E2), 2-ethylbutyl (2S)-2-{[(S)-{[(2R,3S,4R,5R)-5-{4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl}-5-cyano-3 (remdesivir) and 6-Fluor-3-oxo-3,4-dihydro-2-pyrazincarboxamid (favipiravir)]. It is evident from this structural informatics study that combo of chloroquine + SSAA09E2 with remdesivir or favipiravir could significantly restrain the virus at the entry and replication stage. Thus, drug repurposition is an attractive approach with reduced time and cost to treat COVID-19, we don't have enough time as the whole world is lockdown and we are in urgent need of an obvious therapeutics' measures.


Subject(s)
Computational Biology , Coronavirus Infections/drug therapy , Drug Repositioning , Pneumonia, Viral/drug therapy , Amino Acid Sequence , Coronavirus Infections/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Targeted Therapy , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/metabolism , Protein Structure, Tertiary , /metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
17.
Phys Chem Chem Phys ; 22(34): 19069-19079, 2020 Sep 08.
Article in English | MEDLINE | ID: covidwho-722683

ABSTRACT

A dynamical approach is proposed to discriminate between reactive (rES) and nonreactive (nES) enzyme-substrate complexes taking the SARS-CoV-2 main protease (Mpro) as an important example. Molecular dynamics simulations with the quantum mechanics/molecular mechanics potentials (QM(DFT)/MM-MD) followed by the electron density analysis are employed to evaluate geometry and electronic properties of the enzyme with different substrates along MD trajectories. We demonstrate that mapping the Laplacian of the electron density and the electron localization function provides easily visible images of the substrate activation that allow one to distinguish rES and nES. The computed fractions of reactive enzyme-substrate complexes along MD trajectories well correlate with the findings of recent experimental studies on the substrate specificity of Mpro. The results of our simulations demonstrate the role of the theory level used in QM subsystems for a proper description of the nucleophilic attack of the catalytic cysteine residue in Mpro. The activation of the carbonyl group of a substrate is correctly characterized with the hybrid DFT functional PBE0, whereas the use of a GGA-type PBE functional, that lacks the admixture of the Hartree-Fock exchange fails to describe activation.


Subject(s)
Betacoronavirus/enzymology , Cysteine Endopeptidases/metabolism , Viral Nonstructural Proteins/metabolism , Betacoronavirus/isolation & purification , Catalytic Domain , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cysteine/chemistry , Cysteine/metabolism , Density Functional Theory , Electrons , Humans , Molecular Dynamics Simulation , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Structure, Tertiary , Substrate Specificity
18.
Front Immunol ; 11: 1784, 2020.
Article in English | MEDLINE | ID: covidwho-719731

ABSTRACT

COVID-19 has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. The incubation period for the virus is ~1-14 days and all age groups may be susceptible to a fatality rate of about 5.9%. COVID-19 is caused by a novel single-stranded, positive (+) sense RNA beta coronavirus. The development of a vaccine for SARS-CoV-2 is an urgent need worldwide. Immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. In this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of COVID-19. The epitopes were computed by using B cells, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) base on the proteins of SARS-CoV-2. A vaccine was devised by fusing together the B cell, HTL, and CTL epitopes with linkers. To enhance the immunogenicity, the ß-defensin (45 mer) amino acid sequence, and pan-HLA DR binding epitopes (13aa) were adjoined to the N-terminal of the vaccine with the help of the EAAAK linker. To enable the intracellular delivery of the modeled vaccine, a TAT sequence (11aa) was appended to C-terminal. Linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. The secondary and three-dimensional (3D) structure of the final vaccine was then predicted. Furthermore, the complex between the final vaccine and immune receptors (toll-like receptor-3 (TLR-3), major histocompatibility complex (MHC-I), and MHC-II) were evaluated by molecular docking. Lastly, to confirm the expression of the designed vaccine, the mRNA of the vaccine was enhanced with the aid of the Java Codon Adaptation Tool, and the secondary structure was generated from Mfold. Then we performed in silico cloning. The final vaccine requires experimental validation to determine its safety and efficacy in controlling SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/chemistry , Computational Biology/methods , Coronavirus Infections/prevention & control , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Proteins/immunology , Viral Vaccines/immunology , Amino Acid Sequence , Coronavirus Infections/virology , HLA-DR Antigens/immunology , Humans , Immunogenicity, Vaccine , Molecular Docking Simulation , Pneumonia, Viral/virology , Protein Folding , Protein Structure, Tertiary , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Subunit/immunology , beta-Defensins/immunology
19.
Int J Mol Sci ; 21(16)2020 Aug 13.
Article in English | MEDLINE | ID: covidwho-717745

ABSTRACT

While SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as the receptor for cell entry, it is important to examine other potential interactions between the virus and other cell receptors. Based on the clinical observation of low prevalence of smoking among hospitalized COVID-19 patients, we examined and identified a "toxin-like" amino acid (aa) sequence in the Receptor Binding Domain of the Spike Glycoprotein of SARS-CoV-2 (aa 375-390), which is homologous to a sequence of the Neurotoxin homolog NL1, one of the many snake venom toxins that are known to interact with nicotinic acetylcholine receptors (nAChRs). We present the 3D structural location of this "toxin-like" sequence on the Spike Glycoprotein and the superposition of the modelled structure of the Neurotoxin homolog NL1 and the SARS-CoV-2 Spike Glycoprotein. We also performed computational molecular modelling and docking experiments using 3D structures of the SARS-CoV-2 Spike Glycoprotein and the extracellular domain of the nAChR α9 subunit. We identified a main interaction between the aa 381-386 of the SARS-CoV-2 Spike Glycoprotein and the aa 189-192 of the extracellular domain of the nAChR α9 subunit, a region which forms the core of the "toxin-binding site" of the nAChRs. The mode of interaction is very similar to the interaction between the α9 nAChR and α-bungarotoxin. A similar interaction was observed between the pentameric α7 AChR chimera and SARS-CoV-2 Spike Glycoprotein. The findings raise the possibility that SARS-CoV-2 may interact with nAChRs, supporting the hypothesis of dysregulation of the nicotinic cholinergic system being implicated in the pathophysiology of COVID-19. Nicotine and other nicotinic cholinergic agonists may protect nAChRs and thus have therapeutic value in COVID-19 patients.


Subject(s)
Betacoronavirus/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence/genetics , Computational Biology , Coronavirus Infections/physiopathology , Humans , Molecular Docking Simulation , Neurotoxins/genetics , Neurotoxins/metabolism , Pandemics , Pneumonia, Viral/physiopathology , Protein Structure, Tertiary/genetics , Sequence Alignment , Snake Venoms/genetics
20.
PLoS One ; 15(8): e0237295, 2020.
Article in English | MEDLINE | ID: covidwho-695314

ABSTRACT

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.


Subject(s)
Betacoronavirus/metabolism , Molecular Dynamics Simulation , Peptidyl-Dipeptidase A/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Algorithms , Betacoronavirus/isolation & purification , Binding Sites , Coronavirus Infections/pathology , Coronavirus Infections/virology , Glycosylation , Humans , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
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