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1.
Commun Biol ; 4(1): 228, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1085408

ABSTRACT

SARS-CoV-2 has been mutating since it was first sequenced in early January 2020. Here, we analyze 45,494 complete SARS-CoV-2 geneome sequences in the world to understand their mutations. Among them, 12,754 sequences are from the United States. Our analysis suggests the presence of four substrains and eleven top mutations in the United States. These eleven top mutations belong to 3 disconnected groups. The first and second groups consisting of 5 and 8 concurrent mutations are prevailing, while the other group with three concurrent mutations gradually fades out. Moreover, we reveal that female immune systems are more active than those of males in responding to SARS-CoV-2 infections. One of the top mutations, 27964C > T-(S24L) on ORF8, has an unusually strong gender dependence. Based on the analysis of all mutations on the spike protein, we uncover that two of four SASR-CoV-2 substrains in the United States become potentially more infectious.


Subject(s)
/virology , Mutation/genetics , /genetics , 5' Untranslated Regions/genetics , Amino Acid Sequence , /metabolism , Evolution, Molecular , Female , Humans , Male , Models, Molecular , Nucleocapsid/metabolism , Open Reading Frames/genetics , Polymorphism, Single Nucleotide/genetics , Protein Binding , Protein Domains , Protein Folding , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics , United States
2.
PLoS One ; 16(2): e0246731, 2021.
Article in English | MEDLINE | ID: covidwho-1079371

ABSTRACT

SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.


Subject(s)
Antibodies, Neutralizing/immunology , Epitopes, B-Lymphocyte/immunology , Histocompatibility Antigens Class II/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Motifs , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Computer Simulation , Epitopes, B-Lymphocyte/chemistry , Humans , Models, Molecular , Peptides/chemistry , Peptides/immunology , Spike Glycoprotein, Coronavirus/chemistry , T-Lymphocytes/immunology
3.
Commun Biol ; 4(1): 193, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1075259

ABSTRACT

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.


Subject(s)
Antiviral Agents/pharmacology , /virology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyrrolidines/pharmacology , /enzymology , Thymine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Catalytic Domain , Crystallography, X-Ray , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Ligands , Models, Molecular , Protein Conformation , Pyrrolidines/chemistry , Pyrrolidines/pharmacokinetics , Thymine/chemistry , Thymine/pharmacokinetics , Uridine/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
4.
Nat Commun ; 12(1): 848, 2021 02 08.
Article in English | MEDLINE | ID: covidwho-1069106

ABSTRACT

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.


Subject(s)
/metabolism , Mutation , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization , Binding, Competitive , /virology , Humans , Models, Molecular , Pandemics , Protein Binding , Protein Domains , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
5.
Biophys J ; 120(3): 504-516, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1064898

ABSTRACT

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of Mpro (MproC) locks Mpro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric Mpro in the early-stage viral replication of both viruses.


Subject(s)
Models, Molecular , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , /enzymology , Protein Domains , Protein Folding
6.
Sci Signal ; 14(665)2021 01 12.
Article in English | MEDLINE | ID: covidwho-1066811

ABSTRACT

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin ß3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 µ2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin ß3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.


Subject(s)
/physiology , Integrin beta3/physiology , Receptors, Virus/physiology , /pathogenicity , Virus Internalization , Amino Acid Sequence , /genetics , Autophagy/physiology , Endocytosis/physiology , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , Integrin beta3/chemistry , Integrin beta3/genetics , Models, Molecular , Pandemics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/physiology , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Receptors, Virus/chemistry , Receptors, Virus/genetics , /genetics
7.
Sci Rep ; 11(1): 3238, 2021 02 05.
Article in English | MEDLINE | ID: covidwho-1065946

ABSTRACT

The rampant spread of COVID-19, an infectious disease caused by SARS-CoV-2, all over the world has led to over millions of deaths, and devastated the social, financial and political entities around the world. Without an existing effective medical therapy, vaccines are urgently needed to avoid the spread of this disease. In this study, we propose an in silico deep learning approach for prediction and design of a multi-epitope vaccine (DeepVacPred). By combining the in silico immunoinformatics and deep neural network strategies, the DeepVacPred computational framework directly predicts 26 potential vaccine subunits from the available SARS-CoV-2 spike protein sequence. We further use in silico methods to investigate the linear B-cell epitopes, Cytotoxic T Lymphocytes (CTL) epitopes, Helper T Lymphocytes (HTL) epitopes in the 26 subunit candidates and identify the best 11 of them to construct a multi-epitope vaccine for SARS-CoV-2 virus. The human population coverage, antigenicity, allergenicity, toxicity, physicochemical properties and secondary structure of the designed vaccine are evaluated via state-of-the-art bioinformatic approaches, showing good quality of the designed vaccine. The 3D structure of the designed vaccine is predicted, refined and validated by in silico tools. Finally, we optimize and insert the codon sequence into a plasmid to ensure the cloning and expression efficiency. In conclusion, this proposed artificial intelligence (AI) based vaccine discovery framework accelerates the vaccine design process and constructs a 694aa multi-epitope vaccine containing 16 B-cell epitopes, 82 CTL epitopes and 89 HTL epitopes, which is promising to fight the SARS-CoV-2 viral infection and can be further evaluated in clinical studies. Moreover, we trace the RNA mutations of the SARS-CoV-2 and ensure that the designed vaccine can tackle the recent RNA mutations of the virus.


Subject(s)
Deep Learning , Spike Glycoprotein, Coronavirus/immunology , Allergens , /adverse effects , /immunology , Codon Usage , Computational Biology , Drug Design , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Humans , Immunogenicity, Vaccine , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Conformation , RNA, Viral , /genetics , Solubility , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Subunit/chemistry , Vaccines, Subunit/immunology
8.
Sci Rep ; 11(1): 1156, 2021 01 13.
Article in English | MEDLINE | ID: covidwho-1065939

ABSTRACT

Several viruses of the corona family interact, via their spike (S) proteins, with human cellular receptors. Spike proteins of SARS-CoV-1 and SARS-CoV-2 virions, being structurally related but not identical, mediate attachment to the human angiotensin-converting enzyme 2 (hACE2) receptor in similar but non-identical ways. Molecular-level understanding of interactions between spike proteins and hACE2 can aid strategies for blocking attachment of SARS-CoV-1, a potentially reemerging health threat, to human cells. We have identified dominant molecular-level interactions, some attractive and some repulsive, between the receptor binding domain of SARS-CoV-1 spike proteins (S-RBD) and hACE2. We performed fragment-based quantum-biochemical calculations which directly relate biomolecular structure to the hACE2...S-RBD interaction energy. Consistent with X-ray crystallography and cryo-EM, the interaction energy between hACE2 and S-RBD ([Formula: see text]26 kcal/mol) corresponds to a net intermolecular attraction which is significantly enhanced by inclusion of dispersion van der Waals forces. Protein fragments at the hACE2...S-RBD interface, that dominate host-virus attraction, have been identified together with their constituent amino acid residues. Two hACE2 fragments which include residues (GLU37, ASP38, TYR41, GLN42) and (GLU329, LYS353, GLY354), respectively, as well as three S-RBD fragments which include residues (TYR436), (ARG426) and (THR487, GLY488, TYR491), respectively, have been identified as primary attractors at the hACE2...S-RBD interface.


Subject(s)
/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Humans , Models, Molecular , Protein Binding , Protein Conformation , Thermodynamics
9.
Nat Struct Mol Biol ; 28(2): 202-209, 2021 02.
Article in English | MEDLINE | ID: covidwho-1065920

ABSTRACT

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS­CoV­2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS­CoV­2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.


Subject(s)
/chemistry , Antiviral Agents/chemistry , /drug therapy , /genetics , Antiviral Agents/therapeutic use , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Engineering , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , /physiology
10.
Nat Commun ; 12(1): 837, 2021 02 05.
Article in English | MEDLINE | ID: covidwho-1065863

ABSTRACT

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.


Subject(s)
/prevention & control , Evolution, Molecular , Spike Glycoprotein, Coronavirus/genetics , /metabolism , Animals , Binding, Competitive , /virology , Cryoelectron Microscopy , Humans , Models, Molecular , Pandemics , Protein Binding , Protein Domains , /physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
11.
Nat Commun ; 12(1): 141, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1065862

ABSTRACT

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


Subject(s)
CD13 Antigens/metabolism , Coronavirus 229E, Human/physiology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Cell Line, Tumor , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Conformation, alpha-Helical , Protein Multimerization/physiology , Protein Structure, Quaternary , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
12.
J Am Chem Soc ; 142(52): 21883-21890, 2020 12 30.
Article in English | MEDLINE | ID: covidwho-1065801

ABSTRACT

The SARS coronavirus 2 (SARS-CoV-2) main protease (Mpro) is an attractive broad-spectrum antiviral drug target. Despite the enormous progress in structure elucidation, the Mpro's structure-function relationship remains poorly understood. Recently, a peptidomimetic inhibitor has entered clinical trial; however, small-molecule orally available antiviral drugs have yet to be developed. Intrigued by a long-standing controversy regarding the existence of an inactive state, we explored the proton-coupled dynamics of the Mpros of SARS-CoV-2 and the closely related SARS-CoV using a newly developed continuous constant pH molecular dynamics (MD) method and microsecond fixed-charge all-atom MD simulations. Our data supports a general base mechanism for Mpro's proteolytic function. The simulations revealed that protonation of His172 alters a conserved interaction network that upholds the oxyanion loop, leading to a partial collapse of the conserved S1 pocket, consistent with the first and controversial crystal structure of SARS-CoV Mpro determined at pH 6. Interestingly, a natural flavonoid binds SARS-CoV-2 Mpro in the close proximity to a conserved cysteine (Cys44), which is hyper-reactive according to the CpHMD titration. This finding offers an exciting new opportunity for small-molecule targeted covalent inhibitor design. Our work represents a first step toward the mechanistic understanding of the proton-coupled structure-dynamics-function relationship of CoV Mpros; the proposed strategy of designing small-molecule covalent inhibitors may help accelerate the development of orally available broad-spectrum antiviral drugs to stop the current pandemic and prevent future outbreaks.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , /drug effects , /enzymology , Binding Sites , Cysteine/chemistry , Drug Design , Humans , Hydrogen-Ion Concentration , Models, Molecular , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology , Protein Conformation , Protons , Small Molecule Libraries , Structure-Activity Relationship
13.
Molecules ; 26(4)2021 Feb 04.
Article in English | MEDLINE | ID: covidwho-1063418

ABSTRACT

The 3CL-Protease appears to be a very promising medicinal target to develop anti-SARS-CoV-2 agents. The availability of resolved structures allows structure-based computational approaches to be carried out even though the lack of known inhibitors prevents a proper validation of the performed simulations. The innovative idea of the study is to exploit known inhibitors of SARS-CoV 3CL-Pro as a training set to perform and validate multiple virtual screening campaigns. Docking simulations using four different programs (Fred, Glide, LiGen, and PLANTS) were performed investigating the role of both multiple binding modes (by binding space) and multiple isomers/states (by developing the corresponding isomeric space). The computed docking scores were used to develop consensus models, which allow an in-depth comparison of the resulting performances. On average, the reached performances revealed the different sensitivity to isomeric differences and multiple binding modes between the four docking engines. In detail, Glide and LiGen are the tools that best benefit from isomeric and binding space, respectively, while Fred is the most insensitive program. The obtained results emphasize the fruitful role of combining various docking tools to optimize the predictive performances. Taken together, the performed simulations allowed the rational development of highly performing virtual screening workflows, which could be further optimized by considering different 3CL-Pro structures and, more importantly, by including true SARS-CoV-2 3CL-Pro inhibitors (as learning set) when available.


Subject(s)
/virology , /enzymology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , /antagonists & inhibitors , Drug Design , Drug Evaluation, Preclinical/methods , Drug Repositioning/methods , Humans , Models, Molecular , Molecular Docking Simulation/methods , Peptide Hydrolases/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation
14.
Nat Commun ; 12(1): 636, 2021 01 27.
Article in English | MEDLINE | ID: covidwho-1049963

ABSTRACT

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.


Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/ultrastructure , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/ultrastructure , Amino Acid Sequence , Catalytic Domain , Cryoelectron Microscopy , Endoribonucleases/metabolism , Models, Chemical , Models, Molecular , Uridine Triphosphate/metabolism , Viral Nonstructural Proteins/metabolism
15.
J Enzyme Inhib Med Chem ; 36(1): 497-503, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1045926

ABSTRACT

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CLpro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro with IC50's of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC50's of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.


Subject(s)
Antiviral Agents/pharmacology , /antagonists & inhibitors , Flavanones/pharmacology , Plant Extracts/pharmacology , Protease Inhibitors/pharmacology , Virus Replication/drug effects , Animals , /virology , Chlorocebus aethiops , Drug Discovery , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Models, Molecular , Vero Cells
16.
Viruses ; 13(2)2021 01 25.
Article in English | MEDLINE | ID: covidwho-1045365

ABSTRACT

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.


Subject(s)
Antiviral Agents/chemistry , Protease Inhibitors/chemistry , /enzymology , Amino Acid Sequence , Antiviral Agents/metabolism , Catalytic Domain , /metabolism , Crystallography, X-Ray , Drug Discovery , Inhibitory Concentration 50 , Models, Molecular , Protease Inhibitors/metabolism , Protein Binding , SARS Virus/enzymology
17.
Nat Commun ; 12(1): 469, 2021 01 20.
Article in English | MEDLINE | ID: covidwho-1039642

ABSTRACT

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.


Subject(s)
Antibodies, Monoclonal/pharmacology , /drug effects , /metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Chlorocebus aethiops , Disease Models, Animal , Female , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Models, Molecular , Mutation , Protein Binding , /immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
18.
Nat Commun ; 12(1): 488, 2021 01 20.
Article in English | MEDLINE | ID: covidwho-1039641

ABSTRACT

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC50 of 2.1 µM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLproC111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , /chemistry , /drug effects , /metabolism , /genetics , Cytokines/metabolism , Drug Discovery , Drug Interactions , HEK293 Cells , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Models, Molecular , Pandemics , Protein Conformation , /metabolism , Ubiquitins/metabolism
19.
J Zhejiang Univ Sci B ; 22(1): 21-30, 2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1032346

ABSTRACT

Adenosine diphosphate (ADP)-ribosylation is a unique post-translational modification that regulates many biological processes, such as DNA damage repair. During DNA repair, ADP-ribosylation needs to be reversed by ADP-ribosylhydrolases. A group of ADP-ribosylhydrolases have a catalytic domain, namely the macrodomain, which is conserved in evolution from prokaryotes to humans. Not all macrodomains remove ADP-ribosylation. One set of macrodomains loses enzymatic activity and only binds to ADP-ribose (ADPR). Here, we summarize the biological functions of these macrodomains in DNA damage repair and compare the structure of enzymatically active and inactive macrodomains. Moreover, small molecular inhibitors have been developed that target macrodomains to suppress DNA damage repair and tumor growth. Macrodomain proteins are also expressed in pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these domains may not be directly involved in DNA damage repair in the hosts or pathogens. Instead, they play key roles in pathogen replication. Thus, by targeting macrodomains it may be possible to treat pathogen-induced diseases, such as coronavirus disease 2019 (COVID-19).


Subject(s)
/metabolism , DNA Repair/physiology , N-Glycosyl Hydrolases/metabolism , ADP-Ribosylation , Evolution, Molecular , Humans , Models, Biological , Models, Molecular , N-Glycosyl Hydrolases/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Protein Domains , /pathogenicity
20.
Biochem Biophys Res Commun ; 541: 50-55, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1030847

ABSTRACT

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.


Subject(s)
Adenosine Triphosphate/metabolism , Liquid-Liquid Extraction , RNA, Viral/metabolism , /metabolism , Adenosine Triphosphate/chemistry , Binding Sites , /genetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Motifs/genetics , /chemistry
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