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1.
Molecules ; 27(19)2022 Sep 26.
Article in English | MEDLINE | ID: covidwho-2066274

ABSTRACT

New Ni (II) and Cu (II) complexes with pyridoxal-semicarbazone were synthesized and their structures were solved by X-ray crystallography. This analysis showed the bis-ligand octahedral structure of [Ni(PLSC-H)2]·H2O and the dimer octahedral structure of [Cu(PLSC)(SO4)(H2O)]2·2H2O. Hirshfeld surface analysis was employed to determine the most important intermolecular interactions in the crystallographic structures. The structures of both complexes were further examined using density functional theory and natural bond orbital analysis. The photocatalytic decomposition of methylene blue in the presence of both compounds was investigated. Both compounds were active toward E. coli and S. aureus, with a minimum inhibition concentration similar to that of chloramphenicol. The obtained complexes led to the formation of free radical species, as was demonstrated in an experiment with dichlorofluorescein-diacetate. It is postulated that this is the mechanistic pathway of the antibacterial and photocatalytic activities. Cyclic voltammograms of the compounds showed the peaks of the reduction of metal ions. A molecular docking study showed that the Ni(II) complex exhibited promising activity towards Janus kinase (JAK), as a potential therapy for inflammatory diseases, cancers, and immunologic disorders.


Subject(s)
Coordination Complexes , Semicarbazones , Anti-Bacterial Agents/pharmacology , Chloramphenicol , Coordination Complexes/chemistry , Crystallography, X-Ray , Escherichia coli/metabolism , Janus Kinases/metabolism , Ligands , Methylene Blue , Molecular Docking Simulation , Molecular Structure , Pyridoxal , Staphylococcus aureus/metabolism , Nickel , Copper
2.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 348-353, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-2051824

ABSTRACT

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.


Subject(s)
COVID-19 , Casein Kinase II/metabolism , rho-Associated Kinases , Acetamides , Adenosine Triphosphate , Crystallography, X-Ray , Humans , rho-Associated Kinases/genetics
3.
Acta Crystallogr D Struct Biol ; 78(Pt 6): 752-769, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1874079

ABSTRACT

In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.


Subject(s)
COVID-19 , Crystallography, X-Ray , Data Analysis , Humans , Macromolecular Substances/chemistry , SARS-CoV-2
4.
Science ; 376(6595): eabn6020, 2022 05 20.
Article in English | MEDLINE | ID: covidwho-1861569

ABSTRACT

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.


Subject(s)
Carboxypeptidases , Microtubule-Associated Proteins , Microtubules , Protein Processing, Post-Translational , Tubulin , Tyrosine , Animals , Carboxypeptidases/genetics , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubules/chemistry , Tubulin/chemistry , Tyrosine/chemistry
5.
Methods Mol Biol ; 2449: 235-261, 2022.
Article in English | MEDLINE | ID: covidwho-1826140

ABSTRACT

Like an article narrative is deemed by an editor and referees to be worthy of being a version of record on acceptance as a publication, so must the underpinning data also be scrutinized before passing it as a version of record. Indeed without the underpinning data, a study and its conclusions cannot be reproduced at any stage of evaluation, pre- or post-publication. Likewise, an independent study without its own underpinning data also cannot be reproduced let alone be considered a replicate of the first study. The PDB is a modern marvel of achievement providing an organized open access to depositor and user of the data held there opening numerous applications. Methods for modeling protein structures and for determination of structures are still improving their precision, and artifacts of the method exist. So their accuracy is realized if they are reproduced by other methods. It is on such foundations that reproducible data mining is based. Data rates are expanding considerably be they at synchrotrons, the X-ray free electron lasers (XFELs), electron cryomicroscopes (cryoEM), or at the neutron facilities. The work of a person as a referee or user with a narrative and its underpinning data may well be complemented in future by artificial intelligence with machine learning, the former for specific refereeing and the latter for the more general validation, both ideally before publication. Examples are described involving rhenium theranostics, the anti-cancer platins and the SARS-CoV-2 main protease.


Subject(s)
Artificial Intelligence , COVID-19 , Crystallography/methods , Crystallography, X-Ray , Data Mining , Humans , Macromolecular Substances/chemistry , SARS-CoV-2 , Synchrotrons
6.
Carbohydr Res ; 516: 108564, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1800172

ABSTRACT

Reaction of 2,3-O-isopropylidene-d-ribofuranosylamine with 2,4-dinitrofluorobenzene afforded the crystalline 2,3-O-isopropylidene-N-(2,4-dinitrophenyl)-ß-d-ribofuranosylamine (3) and a 1:1 crystalline complex of 2,3-O-isopropylidene-N-(2,4-dinitrophenyl-α-d-ribofuranosylamine and 2,3-O-isopropylidene-ß-d-ribofuranose; controlled acidic hydrolysis of 3 afforded N-(2,4-dinitrophenyl-α-d-ribopyranosylamine and not the expected ß-d-furanosylamine derivative. The structures of the new compounds were confirmed by NMR spectroscopy and X-ray crystallography.


Subject(s)
Ribose , Amino Sugars , Crystallography, X-Ray , Hydrolysis , Magnetic Resonance Spectroscopy , Ribose/analogs & derivatives
7.
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
8.
Sci Rep ; 12(1): 2505, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1747189

ABSTRACT

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Binding Sites , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Docking Simulation , Nitriles/chemistry , Nitriles/metabolism , Nitriles/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Virus Replication/drug effects
9.
Int J Mol Sci ; 23(5)2022 Feb 28.
Article in English | MEDLINE | ID: covidwho-1736947

ABSTRACT

N-(4-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)selanyl)phenyl)acetamide (5), C19H15NO3Se, was prepared in two steps from 4,4'-diselanediyldianiline (3) via reduction and subsequent nucleophilic reaction with 2-methyl-3-bromo-1,4-naphthalenedione, followed by acetylation with acetic anhydride. The cytotoxicity was estimated against 158N and 158JP oligodendrocytes and the redox profile was also evaluated using different in vitro assays. The technique of single-crystal X-ray diffraction is used to confirm the structure of compound 5. The enantiopure 5 crystallizes in space group P21 with Flack parameter 0.017 (8), exhibiting a chiral layered absolute structure. Molecular structural studies showed that the crystal structure is foremost stabilized by N-H···O and relatively weak C-H···O contacts between molecules, and additionally stabilized by weak C-H···π and Se···N interactions. Hirshfeld surface analysis is used to quantitatively investigate the noncovalent interactions that stabilize crystal packing. Framework energy diagrams were used to graphically represent the stabilizing interaction energies for crystal packing. The analysis of the energy framework shows that the interactions energies of and C-H···π and C-O···π are primarily dispersive and are the crystal's main important forces. Density functional theory (DFT) calculations were used to determine the compound's stability, chemical reactivity, and other parameters by determining the HOMO-LUMO energy differences. The determination of its optimized surface of the molecular electrostatic potential (MEP) was also carried out. This study was conducted to demonstrate both the electron-rich and electron-poor sites.


Subject(s)
Halogens , Hydrogen , Acetamides , Crystallography, X-Ray , Density Functional Theory , Hydrogen Bonding
10.
Acta Crystallogr C Struct Chem ; 78(Pt 4): 231-239, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1730787

ABSTRACT

The structure of a trinuclear zinc complex, hexakis(µ2-2-anilinobenzoato)diaquatrizinc(II), [Zn2(C13H10NO2)6(H2O)2] or (NPA)6Zn3(H2O)2 (NPA is 2-anilinobenzoate or N-phenylanthranilate), is reported. The complex crystallizes in the triclinic space group P-1 and the central ZnII atom is located on an inversion center. The NPA ligand is found to coordinate via the carboxylate O atoms with unique C-O bond lengths that support an unequal distribution of resonance over the carboxylate fragment. The axial H2O ligands form hydrogen bonds with neighboring molecules that stabilize the supramolecular system in rigid straight chains, with an angle of 180° along the c axis. π stacking is the primary stabilization along the a and b axes, resulting in a highly ordered supramolecular structure. Docking studies show that this unique supramolecular structure of a trinuclear zinc complex has potential for binding to the main protease (Mpro) in SARS-CoV-2 in a different location from Remdesivir, but with a similar binding strength.


Subject(s)
COVID-19 , Zinc , Crystallography, X-Ray , Humans , Hydrogen Bonding , Ligands , SARS-CoV-2 , Zinc/chemistry , ortho-Aminobenzoates
11.
J Med Chem ; 65(4): 2880-2904, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1705973

ABSTRACT

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Peptidomimetics/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Chlorocebus aethiops , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Glutamine/chemistry , Glutamine/pharmacology , Humans , Ketones/chemistry , Ketones/pharmacology , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Peptidomimetics/chemistry , SARS-CoV-2/enzymology , Vero Cells , Virus Replication/drug effects
12.
FASEB J ; 36(3): e22199, 2022 03.
Article in English | MEDLINE | ID: covidwho-1684809

ABSTRACT

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence.


Subject(s)
COVID-19/virology , Models, Molecular , Protein Domains , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Protein Stability , Protein Structure, Secondary , Scattering, Small Angle , Temperature , X-Ray Diffraction
13.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: covidwho-1684239

ABSTRACT

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.


Subject(s)
Exoribonucleases/chemistry , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Domains , RNA, Viral/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Binding Sites/genetics , COVID-19/virology , Catalytic Domain , Crystallography, X-Ray , Exoribonucleases/genetics , Exoribonucleases/metabolism , Humans , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Mutation, Missense , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
14.
Nature ; 602(7896): 343-348, 2022 02.
Article in English | MEDLINE | ID: covidwho-1671588

ABSTRACT

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.


Subject(s)
Carbapenems/biosynthesis , Methyltransferases/chemistry , Methyltransferases/metabolism , S-Adenosylmethionine/metabolism , Streptomyces/enzymology , Thienamycins/biosynthesis , Vitamin B 12/metabolism , Binding Sites , Biocatalysis , Coenzymes/metabolism , Crystallography, X-Ray , Kinetics , Methylation , Models, Molecular , Protein Binding , Protein Domains , Streptomyces/metabolism , beta-Lactamase Inhibitors/metabolism , beta-Lactamases/chemistry , beta-Lactamases/metabolism
15.
J Nat Prod ; 85(2): 327-336, 2022 02 25.
Article in English | MEDLINE | ID: covidwho-1655431

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 5 million deaths worldwide to date. Due to the limited therapeutic options so far available, target-based virtual screening with LC/MS support was applied to identify the novel and high-content compounds 1-4 with inhibitory effects on SARS-CoV-2 in Vero E6 cells from the plant Dryopteris wallichiana. These compounds were also evaluated against SARS-CoV-2 in Calu-3 cells and showed unambiguous inhibitory activity. The inhibition assay of targets showed that compounds 3 and 4 mainly inhibited SARS-CoV-2 3CLpro, with effective Kd values. Through docking and molecular dynamics modeling, the binding site is described, providing a comprehensive understanding of 3CLpro and interactions for 3, including hydrogen bonds, hydrophobic bonds, and the spatial occupation of the B ring. Compounds 3 and 4 represent new, potential lead compounds for the development of anti-SARS-CoV-2 drugs. This study has led to the development of a target-based virtual screening method for exploring the potency of natural products and for identifying natural bioactive compounds for possible COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , Biological Products/pharmacology , Microbial Sensitivity Tests/methods , Phloroglucinol/pharmacology , SARS-CoV-2/drug effects , Terpenes/pharmacology , Chromatography, High Pressure Liquid , Chromatography, Liquid , Crystallography, X-Ray , Drug Delivery Systems , Dryopteris/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Docking Simulation , Molecular Structure , Virtual Reality
16.
Science ; 375(6578): eabl6251, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1650842

ABSTRACT

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Immune Evasion , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , BNT162 Vaccine/immunology , Betacoronavirus/immunology , COVID-19/immunology , COVID-19/virology , Cross Reactions , Cryoelectron Microscopy , Crystallography, X-Ray , Epitopes , Evolution, Molecular , Humans , Models, Molecular , Mutation , Polysaccharides/analysis , Protein Binding , Protein Domains , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Pseudotyping
17.
Science ; 375(6583): 864-868, 2022 02 25.
Article in English | MEDLINE | ID: covidwho-1650843

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , Immune Evasion , Receptors, Coronavirus/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Antigenic Drift and Shift , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/immunology , Broadly Neutralizing Antibodies/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains/genetics , Protein Interaction Domains and Motifs/genetics , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
18.
STAR Protoc ; 3(1): 101158, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1650422

ABSTRACT

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).


Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/genetics , Crystallization , Crystallography, X-Ray , Humans
19.
Sci Rep ; 12(1): 1260, 2022 01 24.
Article in English | MEDLINE | ID: covidwho-1648095

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus, responsible for outbreaks of a severe respiratory illness in humans with a fatality rate of 30%. Currently, there are no vaccines or United States food and drug administration (FDA)-approved therapeutics for humans. The spike protein displayed on the surface of MERS-CoV functions in the attachment and fusion of virions to host cellular membranes and is the target of the host antibody response. Here, we provide a molecular method for neutralizing MERS-CoV through potent antibody-mediated targeting of the receptor-binding subdomain (RBD) of the spike protein. The structural characterization of the neutralizing antibody (KNIH90-F1) complexed with RBD using X-ray crystallography revealed three critical epitopes (D509, R511, and E513) in the RBD region of the spike protein. Further investigation of MERS-CoV mutants that escaped neutralization by the antibody supported the identification of these epitopes in the RBD region. The neutralizing activity of this antibody is solely provided by these specific molecular structures. This work should contribute to the development of vaccines or therapeutic antibodies for MERS-CoV.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Crystallography, X-Ray , Humans , Protein Domains
20.
J Virol ; 96(1): e0125321, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1639525

ABSTRACT

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , Catalytic Domain , Coronavirus/classification , Coronavirus/enzymology , Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray , Molecular Docking Simulation , Naphthoquinones/chemistry , Protein Binding
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