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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.
Nat Commun ; 11(1): 4252, 2020 08 25.
Article in English | MEDLINE | ID: covidwho-741685

ABSTRACT

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Influenza A virus/drug effects , Peptides/pharmacology , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Cell Line , Endosomes/chemistry , Endosomes/drug effects , Female , Humans , Hydrogen-Ion Concentration , Influenza A virus/metabolism , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/metabolism , Peptides/chemistry , Peptides/metabolism , Peptides/therapeutic use , Protein Binding , Protein Conformation , Rhinovirus/drug effects , Rhinovirus/metabolism , Viral Load/drug effects , Virus Replication/drug effects
3.
J Virol ; 94(17)2020 08 17.
Article in English | MEDLINE | ID: covidwho-740271

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here.IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/metabolism , Sequence Deletion , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Coronavirus Infections/virology , Furin/metabolism , Genome, Viral , Host Specificity , Kinetics , Models, Molecular , Pandemics , Pneumonia, Viral/virology , Protein Conformation , Sequence Analysis , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Replication
4.
Sci Rep ; 10(1): 14214, 2020 08 26.
Article in English | MEDLINE | ID: covidwho-733506

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major public health concern. A handful of static structures now provide molecular insights into how SARS-CoV-2 and SARS-CoV interact with its host target, which is the angiotensin converting enzyme 2 (ACE2). Molecular recognition, binding and function are dynamic processes. To evaluate this, multiple 500 ns or 1 µs all-atom molecular dynamics simulations were performed to better understand the structural stability and interfacial interactions between the receptor binding domain of the spike (S) protein of SARS-CoV-2 and SARS-CoV bound to ACE2. Several contacts were observed to form, break and reform in the interface during the simulations. Our results indicate that SARS-CoV-2 and SARS-CoV utilizes unique strategies to achieve stable binding to ACE2. Several differences were observed between the residues of SARS-CoV-2 and SARS-CoV that consistently interacted with ACE2. Notably, a stable salt bridge between Lys417 of SARS-CoV-2 S protein and Asp30 of ACE2 as well as three stable hydrogen bonds between Tyr449, Gln493 and Gln498 of SARS-CoV-2 and Asp38, Glu35 and Lys353 of ACE2 were observed, which were absent in the ACE2-SARS-CoV interface. Some previously reported residues, which were suggested to enhance the binding affinity of SARS-CoV-2, were not observed to form stable interactions in these simulations. Molecular mechanics-generalized Born surface area based free energy of binding was observed to be higher for SARS-CoV-2 in all simulations. Stable binding to the host receptor is crucial for virus entry. Therefore, special consideration should be given to these stable interactions while designing potential drugs and treatment modalities to target or disrupt this interface.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , SARS Virus/physiology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Binding Sites , Conserved Sequence , Host-Pathogen Interactions , Humans , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Protein Conformation , Spike Glycoprotein, Coronavirus/chemistry
5.
J Chem Phys ; 153(7): 075101, 2020 Aug 21.
Article in English | MEDLINE | ID: covidwho-726966

ABSTRACT

In 2020, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide and caused the coronavirus disease 2019 (COVID-19). Spike (S) glycoproteins on the viral membrane bind to ACE2 receptors on the host cell membrane and initiate fusion, and S protein is currently among the primary drug target to inhibit viral entry. The S protein can be in a receptor inaccessible (closed) or accessible (open) state based on down and up positions of its receptor-binding domain (RBD), respectively. However, conformational dynamics and the transition pathway between closed to open states remain unexplored. Here, we performed all-atom molecular dynamics (MD) simulations starting from closed and open states of the S protein trimer in the presence of explicit water and ions. MD simulations showed that RBD forms a higher number of interdomain interactions and exhibits lower mobility in its down position than its up position. MD simulations starting from intermediate conformations between the open and closed states indicated that RBD switches to the up position through a semi-open intermediate that potentially reduces the free energy barrier between the closed and open states. Free energy landscapes were constructed, and a minimum energy pathway connecting the closed and open states was proposed. Because RBD-ACE2 binding is compatible with the semi-open state, but not with the closed state of the S protein, we propose that the formation of the intermediate state is a prerequisite for the host cell recognition.


Subject(s)
Betacoronavirus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Binding Sites , Hydrogen Bonding , Models, Chemical , Molecular Dynamics Simulation , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Principal Component Analysis , Protein Binding , Protein Conformation , Protein Domains , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics
6.
Sci Rep ; 10(1): 14031, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-724696

ABSTRACT

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10-25 January) 2020. We also applied structural bioinformatics to assess the potential impact of D614G on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/epidemiology , Evolution, Molecular , Pneumonia, Viral/epidemiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Betacoronavirus/pathogenicity , Child , Child, Preschool , Computer Simulation , Coronavirus Infections/virology , Female , Genome, Viral/genetics , Humans , Infant , Male , Middle Aged , Mutation, Missense , Pandemics , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Pneumonia, Viral/virology , Protein Conformation , Spike Glycoprotein, Coronavirus/metabolism , Virulence/genetics , Young Adult
7.
Sci Rep ; 10(1): 13866, 2020 08 17.
Article in English | MEDLINE | ID: covidwho-720849

ABSTRACT

The Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The virus has rapidly spread in humans, causing the ongoing Coronavirus pandemic. Recent studies have shown that, similarly to SARS-CoV, SARS-CoV-2 utilises the Spike glycoprotein on the envelope to recognise and bind the human receptor ACE2. This event initiates the fusion of viral and host cell membranes and then the viral entry into the host cell. Despite several ongoing clinical studies, there are currently no approved vaccines or drugs that specifically target SARS-CoV-2. Until an effective vaccine is available, repurposing FDA approved drugs could significantly shorten the time and reduce the cost compared to de novo drug discovery. In this study we attempted to overcome the limitation of in silico virtual screening by applying a robust in silico drug repurposing strategy. We combined and integrated docking simulations, with molecular dynamics (MD), Supervised MD (SuMD) and Steered MD (SMD) simulations to identify a Spike protein - ACE2 interaction inhibitor. Our data showed that Simeprevir and Lumacaftor bind the receptor-binding domain of the Spike protein with high affinity and prevent ACE2 interaction.


Subject(s)
Betacoronavirus/drug effects , Computational Biology/methods , Coronavirus Infections/metabolism , Drug Discovery/methods , Drug Repositioning/methods , Pneumonia, Viral/metabolism , Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Betacoronavirus/chemistry , Binding Sites , Coronavirus Infections/virology , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding/drug effects , Protein Conformation , Protein Domains/drug effects , Protein Interaction Maps/drug effects , Simeprevir/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism
8.
PLoS One ; 15(8): e0237300, 2020.
Article in English | MEDLINE | ID: covidwho-710429

ABSTRACT

The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H+ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct "core" structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Betacoronavirus/isolation & purification , Computer Simulation , Coronavirus Infections/virology , Humans , Hydrogen , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Pneumonia, Viral/virology , Point Mutation , Protein Conformation , Structural Homology, Protein , Vaccines, Attenuated , Vaccines, Inactivated , Viral Envelope Proteins/immunology , Viral Vaccines , Water/chemistry
9.
J Enzyme Inhib Med Chem ; 35(1): 1539-1544, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-694175

ABSTRACT

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.


Subject(s)
Coronavirus Infections/drug therapy , Flavonoids/chemistry , Pneumonia, Viral/drug therapy , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Antiviral Agents/chemistry , Betacoronavirus , Drug Design , Fluorescence Resonance Energy Transfer , Humans , Molecular Docking Simulation , Pandemics , Protease Inhibitors/chemistry , Protein Binding , Protein Conformation , Spectrophotometry , Tryptophan/chemistry
10.
Structure ; 28(8): 874-878, 2020 08 04.
Article in English | MEDLINE | ID: covidwho-692175

ABSTRACT

During global pandemics, the spread of information needs to be faster than the spread of the virus in order to ensure the health and safety of human populations worldwide. In our current crisis, the demand for SARS-CoV-2 drugs and vaccines highlights the importance of biological targets and their three-dimensional shape. In particular, structural biology as a field was poised to quickly respond to crises due to previous experience and expertise and because of its early adoption of open access practices.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Cysteine Endopeptidases/chemistry , Databases, Protein , Humans , Models, Molecular , Molecular Biology , Protein Conformation , RNA Replicase/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry
12.
Int J Mol Sci ; 21(15)2020 Jul 28.
Article in English | MEDLINE | ID: covidwho-680191

ABSTRACT

Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.


Subject(s)
Betacoronavirus/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Betacoronavirus/chemistry , Catalytic Domain , Humans , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Multimerization
13.
J Comput Chem ; 41(24): 2158-2161, 2020 09 15.
Article in English | MEDLINE | ID: covidwho-671260

ABSTRACT

D614G spike glycoprotein (sgp) mutation in rapidly spreading severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) is associated with enhanced fitness and higher transmissibility in new cases of COVID-19 but the underlying mechanism is unknown. Here, using atomistic simulation, a plausible mechanism has been delineated. In G614 sgp but not wild type, increased D(G)614-T859 Cα-distance within 65 ns is interpreted as S1/S2 protomer dissociation. Overall, ACE2-binding, post-fusion core, open-state and sub-optimal antibody-binding conformations were preferentially sampled by the G614 mutant, but not wild type. Furthermore, in the wild type, only one of the three sgp chains has optimal communication route between residue 614 and the receptor-binding domain (RBD); whereas, two of the three chains communicated directly in G614 mutant. These data provide evidence that D614G sgp mutant is more available for receptor binding, cellular invasion and reduced antibody interaction; thus, providing framework for enhanced fitness and higher transmissibility in D614G SARS-COV-2 mutant.


Subject(s)
Betacoronavirus/metabolism , Computer Simulation , Coronavirus Infections/virology , Models, Chemical , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Binding Sites , Humans , Models, Molecular , Mutation , Pandemics , Protein Binding , Protein Conformation , Protein Domains
14.
Int J Mol Sci ; 21(14)2020 Jul 21.
Article in English | MEDLINE | ID: covidwho-670417

ABSTRACT

(1) Background: Virtual screening studies on the therapeutically relevant proteins of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) require a detailed characterization of their druggable binding sites, and, more generally, a convenient pocket mapping represents a key step for structure-based in silico studies; (2) Methods: Along with a careful literature search on SARS-CoV-2 protein targets, the study presents a novel strategy for pocket mapping based on the combination of pocket (as performed by the well-known FPocket tool) and docking searches (as performed by PLANTS or AutoDock/Vina engines); such an approach is implemented by the Pockets 2.0 plug-in for the VEGA ZZ suite of programs; (3) Results: The literature analysis allowed the identification of 16 promising binding cavities within the SARS-CoV-2 proteins and the here proposed approach was able to recognize them showing performances clearly better than those reached by the sole pocket detection; and (4) Conclusions: Even though the presented strategy should require more extended validations, this proved successful in precisely characterizing a set of SARS-CoV-2 druggable binding pockets including both orthosteric and allosteric sites, which are clearly amenable for virtual screening campaigns and drug repurposing studies. All results generated by the study and the Pockets 2.0 plug-in are available for download.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Viral Proteins/chemistry , Binding Sites/drug effects , Drug Repositioning , Humans , Molecular Docking Simulation , Pandemics , Protein Binding/drug effects , Protein Conformation
15.
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
16.
Nat Commun ; 11(1): 3618, 2020 07 17.
Article in English | MEDLINE | ID: covidwho-651635

ABSTRACT

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human-infecting coronaviruses is still lacking. We used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post-fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Motifs , Coronavirus/chemistry , Coronavirus/classification , Coronavirus Infections/virology , Cryoelectron Microscopy , Humans , Membrane Fusion , Models, Molecular , Pandemics , Pneumonia, Viral/virology , Protein Conformation , Protein Multimerization , Virus Internalization
17.
Eur J Pharm Sci ; 153: 105465, 2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-639701

ABSTRACT

COVID-19, is a disease resulting from the SARS-CoV-2 global pandemic. Due to the current global emergency and the length of time required to develop specific antiviral agent(s) and a vaccine for SARS-CoV-2, the world health organization (WHO) adopted the strategy of repurposing existing medications to treat COVID-19. Iron oxide nanoparticles (IONPs) were previously approved by the US food and drug administration (FDA) for anemia treatment and studies have also demonstrated its antiviral activity in vitro. Therefore, we performed a docking study to explore the interaction of IONPs (Fe2O3 and Fe3O4) with the spike protein receptor binding domain (S1-RBD) of SARS-CoV-2 that is required for virus attachment to the host cell receptors. A similar docking analysis was also performed with hepatitis C virus (HCV) glycoproteins E1 and E2. These studies revealed that both Fe2O3 and Fe3O4 interacted efficiently with the SARS-CoV-2 S1-RBD and to HCV glycoproteins, E1 and E2. Fe3O4 formed a more stable complex with S1-RBD whereas Fe2O3 favored HCV E1 and E2. These interactions of IONPs are expected to be associated with viral proteins conformational changes and hence, viral inactivation. Therefore, we recommend FDA-approved-IONPs to proceed for COVID-19 treatment clinical trials.


Subject(s)
Coronavirus Infections/drug therapy , Ferric Compounds/therapeutic use , Metal Nanoparticles/therapeutic use , Molecular Docking Simulation , Pneumonia, Viral/drug therapy , Drug Approval , Drug Repositioning , Humans , Pandemics , Protein Conformation , Spike Glycoprotein, Coronavirus/drug effects , United States , United States Food and Drug Administration , Viral Envelope Proteins/drug effects , Viral Envelope Proteins/metabolism
18.
Microbes Infect ; 22(4-5): 221-225, 2020.
Article in English | MEDLINE | ID: covidwho-627037

ABSTRACT

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.


Subject(s)
Betacoronavirus/metabolism , Birds/metabolism , Coronavirus Infections/virology , Mammals/metabolism , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/virology , Amino Acid Sequence , Animals , Humans , Pandemics , Phylogeny , Protein Conformation , Species Specificity
19.
Microbes Infect ; 22(4-5): 182-187, 2020.
Article in English | MEDLINE | ID: covidwho-626674

ABSTRACT

Envelope protein of coronaviruses is a structural protein existing in both monomeric and homo-pentameric form. It has been related to a multitude of roles including virus infection, replication, dissemination and immune response stimulation. In the present study, we employed an immunoinformatic approach to investigate the major immunogenic domains of the SARS-CoV-2 envelope protein and map them among the homologue proteins of coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Also, when not available, we predicted the envelope protein structural folding and mapped SARS-CoV-2 epitopes. Envelope sequences alignment provides evidence of high sequence homology for some of the investigated virus specimens; while the structural mapping of epitopes resulted in the interesting maintenance of the structural folding and epitope sequence localization also in the envelope proteins scoring a lower alignment score. In line with the One-Health approach, our evidences provide a molecular structural rationale for a potential role of taxonomically related coronaviruses in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.


Subject(s)
Betacoronavirus/metabolism , Computational Biology/methods , Coronavirus Infections/immunology , Coronavirus Infections/virology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Viral Envelope Proteins/immunology , Animals , Betacoronavirus/classification , Betacoronavirus/genetics , Betacoronavirus/immunology , Epitope Mapping , Gene Expression Regulation, Viral , Humans , Models, Molecular , One Health , Pandemics , Phylogeny , Protein Conformation , Sequence Alignment , Sequence Analysis, Protein
20.
Nat Commun ; 11(1): 3202, 2020 06 24.
Article in English | MEDLINE | ID: covidwho-616613

ABSTRACT

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.


Subject(s)
Betacoronavirus/enzymology , Cysteine Endopeptidases/chemistry , Viral Nonstructural Proteins/chemistry , Catalytic Domain , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/metabolism , Ligands , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Domains , Protein Structure, Secondary , Temperature , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
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