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1.
Sci Rep ; 12(1): 3860, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1799576

ABSTRACT

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.


Subject(s)
Endoribonucleases/metabolism , RNA, Viral/chemistry , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Humans , Markov Chains , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Multimerization , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Static Electricity , Viral Nonstructural Proteins/antagonists & inhibitors
2.
Acta Crystallogr D Struct Biol ; 78(Pt 3): 363-378, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1758984

ABSTRACT

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which Mpro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive Mpro a new cavity is present near the S2' subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of Mpro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/chemistry , SARS-CoV-2/chemistry , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Protein Multimerization
3.
Int J Mol Sci ; 23(6)2022 Mar 10.
Article in English | MEDLINE | ID: covidwho-1742486

ABSTRACT

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2's most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2.


Subject(s)
Coronavirus M Proteins/genetics , Genome, Viral/genetics , Mutation , Polymorphism, Single Nucleotide , SARS-CoV-2/genetics , Binding Sites/genetics , COVID-19/prevention & control , COVID-19/virology , Coronavirus M Proteins/chemistry , Coronavirus M Proteins/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Multimerization , SARS-CoV-2/physiology
4.
Nat Commun ; 13(1): 1002, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1702683

ABSTRACT

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Algorithms , Angiotensin-Converting Enzyme 2/chemistry , COVID-19/virology , Host-Pathogen Interactions , Humans , Membrane Fusion , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Multimerization , Receptors, Virus/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Virus Attachment , Virus Internalization
5.
Nat Commun ; 13(1): 742, 2022 02 08.
Article in English | MEDLINE | ID: covidwho-1684023

ABSTRACT

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.


Subject(s)
SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Humans , Immune Evasion , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Static Electricity
6.
Science ; 375(6584): 1048-1053, 2022 03 04.
Article in English | MEDLINE | ID: covidwho-1673339

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has become the dominant infective strain. We report the structures of the Omicron spike trimer on its own and in complex with angiotensin-converting enzyme 2 (ACE2) or an anti-Omicron antibody. Most Omicron mutations are located on the surface of the spike protein and change binding epitopes to many current antibodies. In the ACE2-binding site, compensating mutations strengthen receptor binding domain (RBD) binding to ACE2. Both the RBD and the apo form of the Omicron spike trimer are thermodynamically unstable. An unusual RBD-RBD interaction in the ACE2-spike complex supports the open conformation and further reinforces ACE2 binding to the spike trimer. A broad-spectrum therapeutic antibody, JMB2002, which has completed a phase 1 clinical trial, maintains neutralizing activity against Omicron. JMB2002 binds to RBD differently from other characterized antibodies and inhibits ACE2 binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites , Cryoelectron Microscopy , Epitopes , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Subunits/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics
7.
Phys Chem Chem Phys ; 24(7): 4324-4333, 2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1671657

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has been declared a global health crisis. The development of anti-SARS-CoV-2 drugs heavily depends on the systematic study of the critical biological processes of key proteins of coronavirus among which the main proteinase (Mpro) dimerization is a key step for virus maturation. Because inhibiting the Mpro dimerization can efficiently suppress virus maturation, the key residues that mediate dimerization can be treated as targets of drug and antibody developments. In this work, the structure and energy features of the Mpro dimer of SARS-CoV-2 and SARS-CoV were studied using molecular dynamics (MD) simulations. The free energy calculations using the Generalized Born (GB) model showed that the dimerization free energy of the SARS-CoV-2 Mpro dimer (-107.5 ± 10.89 kcal mol-1) is larger than that of the SARS-CoV Mpro dimer (-92.83 ± 9.81 kcal mol-1), indicating a more stable and possibly a quicker formation of the Mpro dimer of SARS-CoV-2. In addition, the energy decomposition of each residue revealed 11 key attractive residues. Furthermore, Thr285Ala weakens the steric hindrance between the two protomers of SARS-CoV-2 that can form more intimate interactions. It is interesting to find 11 repulsive residues which effectively inhibit the dimerization process. At the interface of the Mpro dimer, we detected three regions that are rich in interfacial water which stabilize the SARS-CoV-2 Mpro dimer by forming hydrogen bonds with two protomers. The key residues and rich water regions provide important targets for the future design of anti-SARS-CoV-2 drugs through inhibiting Mpro dimerization.


Subject(s)
Coronavirus 3C Proteases/chemistry , SARS-CoV-2/enzymology , COVID-19 , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Protein Multimerization
8.
Lancet ; 399(10323): 461-472, 2022 01 29.
Article in English | MEDLINE | ID: covidwho-1641748

ABSTRACT

BACKGROUND: A range of safe and effective vaccines against SARS CoV 2 are needed to address the COVID 19 pandemic. We aimed to assess the safety and efficacy of the COVID-19 vaccine SCB-2019. METHODS: This ongoing phase 2 and 3 double-blind, placebo-controlled trial was done in adults aged 18 years and older who were in good health or with a stable chronic health condition, at 31 sites in five countries (Belgium, Brazil, Colombia, Philippines, and South Africa). The participants were randomly assigned 1:1 using a centralised internet randomisation system to receive two 0·5 mL intramuscular doses of SCB-2019 (30 µg, adjuvanted with 1·50 mg CpG-1018 and 0·75 mg alum) or placebo (0·9% sodium chloride for injection supplied in 10 mL ampoules) 21 days apart. All study staff and participants were masked, but vaccine administrators were not. Primary endpoints were vaccine efficacy, measured by RT-PCR-confirmed COVID-19 of any severity with onset from 14 days after the second dose in baseline SARS-CoV-2 seronegative participants (the per-protocol population), and the safety and solicited local and systemic adverse events in the phase 2 subset. This study is registered on EudraCT (2020-004272-17) and ClinicalTrials.gov (NCT04672395). FINDINGS: 30 174 participants were enrolled from March 24, 2021, until the cutoff date of Aug 10, 2021, of whom 30 128 received their first assigned vaccine (n=15 064) or a placebo injection (n=15 064). The per-protocol population consisted of 12 355 baseline SARS-CoV-2-naive participants (6251 vaccinees and 6104 placebo recipients). Most exclusions (13 389 [44·4%]) were because of seropositivity at baseline. There were 207 confirmed per-protocol cases of COVID-19 at 14 days after the second dose, 52 vaccinees versus 155 placebo recipients, and an overall vaccine efficacy against any severity COVID-19 of 67·2% (95·72% CI 54·3-76·8), 83·7% (97·86% CI 55·9-95·4) against moderate-to-severe COVID-19, and 100% (97·86% CI 25·3-100·0) against severe COVID-19. All COVID-19 cases were due to virus variants; vaccine efficacy against any severity COVID-19 due to the three predominant variants was 78·7% (95% CI 57·3-90·4) for delta, 91·8% (44·9-99·8) for gamma, and 58·6% (13·3-81·5) for mu. No safety issues emerged in the follow-up period for the efficacy analysis (median of 82 days [IQR 63-103]). The vaccine elicited higher rates of mainly mild-to-moderate injection site pain than the placebo after the first (35·7% [287 of 803] vs 10·3% [81 of 786]) and second (26·9% [189 of 702] vs 7·4% [52 of 699]) doses, but the rates of other solicited local and systemic adverse events were similar between the groups. INTERPRETATION: Two doses of SCB-2019 vaccine plus CpG and alum provides notable protection against the entire severity spectrum of COVID-19 caused by circulating SAR-CoV-2 viruses, including the predominating delta variant. FUNDING: Clover Biopharmaceuticals and the Coalition for Epidemic Preparedness Innovations.


Subject(s)
Adjuvants, Immunologic/therapeutic use , COVID-19 Vaccines/therapeutic use , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/therapeutic use , Adolescent , Adult , Aged , Alum Compounds/therapeutic use , Belgium , Brazil , Colombia , Double-Blind Method , Female , Humans , Male , Middle Aged , Oligodeoxyribonucleotides/therapeutic use , Philippines , Protein Multimerization , Recombinant Proteins/therapeutic use , Risk , SARS-CoV-2 , South Africa , Young Adult
9.
Nat Commun ; 13(1): 155, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1616979

ABSTRACT

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


Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Bispecific/metabolism , COVID-19/virology , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mice, Transgenic , Neutralization Tests/methods , Protein Binding , Protein Conformation , Protein Multimerization/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
10.
Cell ; 185(4): 603-613.e15, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1588149

ABSTRACT

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.


Subject(s)
COVID-19/immunology , COVID-19/virology , Immunity/immunology , SARS-CoV-2/immunology , T Follicular Helper Cells/immunology , Vaccination , Vaccines, Synthetic/immunology , /immunology , Adult , B-Lymphocytes/immunology , COVID-19/blood , Clone Cells , Cohort Studies , Cytokines/metabolism , Female , Germinal Center/immunology , HLA-DP beta-Chains/immunology , Humans , Immunodominant Epitopes/immunology , Jurkat Cells , Lymph Nodes/metabolism , Male , Middle Aged , Peptides/chemistry , Peptides/metabolism , Protein Multimerization , Receptors, Antigen, T-Cell/metabolism
11.
J Phys Chem Lett ; 12(51): 12249-12255, 2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1586057

ABSTRACT

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/physiology , Viral Matrix Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/chemistry , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Protein Multimerization , Protein Transport , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/chemistry
12.
Nat Commun ; 12(1): 3172, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1550281

ABSTRACT

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Subject(s)
Nerve Tissue Proteins/ultrastructure , Neuropilin-1/ultrastructure , Receptors, Cell Surface/ultrastructure , Semaphorin-3A/ultrastructure , Animals , COS Cells , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neuropilin-1/genetics , Neuropilin-1/isolation & purification , Neuropilin-1/metabolism , Protein Binding/genetics , Protein Domains/genetics , Protein Multimerization/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Semaphorin-3A/genetics , Semaphorin-3A/isolation & purification , Semaphorin-3A/metabolism
13.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Article in English | MEDLINE | ID: covidwho-1517667

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in ß-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.


Subject(s)
SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Motifs , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Glycosylation , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
14.
Sci Rep ; 11(1): 22042, 2021 11 11.
Article in English | MEDLINE | ID: covidwho-1510622

ABSTRACT

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


Subject(s)
COVID-19/virology , Evolution, Molecular , Mutation , SARS-CoV-2/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Interaction Maps , Protein Multimerization , SARS Virus/chemistry , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
15.
Angew Chem Int Ed Engl ; 60(48): 25428-25435, 2021 11 22.
Article in English | MEDLINE | ID: covidwho-1490696

ABSTRACT

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


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/chemistry , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Microbial Sensitivity Tests , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Vero Cells
16.
Science ; 374(6573): 1353-1360, 2021 Dec 10.
Article in English | MEDLINE | ID: covidwho-1483980

ABSTRACT

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.


Subject(s)
Immune Evasion , Membrane Fusion , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antibody Affinity , Antigens, Viral/immunology , Cell Line , Epitopes/immunology , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein Multimerization , Receptors, Coronavirus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology
17.
Molecules ; 26(19)2021 Oct 07.
Article in English | MEDLINE | ID: covidwho-1463771

ABSTRACT

3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Flavonoids/pharmacology , Glycosides/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Chlorocebus aethiops , Coronavirus 3C Proteases/metabolism , Flavonoids/chemistry , Glycosides/chemistry , Humans , Molecular Docking Simulation , Polyphenols/chemistry , Polyphenols/pharmacology , Protease Inhibitors/chemistry , Protein Multimerization/drug effects , SARS-CoV-2/metabolism , Vero Cells
18.
Eur J Med Chem ; 221: 113494, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1446590

ABSTRACT

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50 = 19.5 µM) and anti-IAV activity (EC50 = 16 µM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 µM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.


Subject(s)
Antiviral Agents/pharmacology , Protein Multimerization/drug effects , Pyrimidines/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Triazoles/pharmacology , Viral Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Chlorocebus aethiops , Dogs , Drug Design , HEK293 Cells , Humans , Influenza A virus/drug effects , Madin Darby Canine Kidney Cells , Microbial Sensitivity Tests , Pyrimidines/chemical synthesis , SARS-CoV-2/drug effects , Triazoles/chemical synthesis , Vero Cells
19.
Nat Commun ; 12(1): 5652, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440473

ABSTRACT

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/therapy , Immunization, Passive/methods , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Antibodies, Viral/administration & dosage , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , Binding Sites/genetics , Binding Sites/immunology , Broadly Neutralizing Antibodies/administration & dosage , Broadly Neutralizing Antibodies/isolation & purification , Broadly Neutralizing Antibodies/metabolism , CHO Cells , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cricetulus , Epitopes/immunology , HEK293 Cells , Humans , Mice , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Neutralization Tests , Pandemics/prevention & control , Protein Multimerization , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
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