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1.
Sci Signal ; 14(689)2021 06 29.
Article in English | MEDLINE | ID: covidwho-1406596

ABSTRACT

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Subject(s)
COVID-19/virology , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Humans , Manganese/metabolism , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nucleic Acid Conformation , RNA Caps/chemistry , RNA Caps/genetics , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , Signal Transduction , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
2.
Viruses ; 13(8)2021 07 29.
Article in English | MEDLINE | ID: covidwho-1335227

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Subject(s)
COVID-19/virology , Methyltransferases/metabolism , RNA Caps/genetics , RNA, Viral/genetics , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Antibodies, Monoclonal/analysis , Humans , Methyltransferases/analysis , Methyltransferases/genetics , Protein Transport , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/analysis , Viral Regulatory and Accessory Proteins/genetics
3.
Nat Commun ; 12(1): 3287, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1253936

ABSTRACT

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Subject(s)
Magnesium/chemistry , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Amino Acid Sequence , Binding Sites , Biocatalysis , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Viral , Humans , Magnesium/metabolism , Methylation , Methyltransferases , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Caps/chemistry , RNA Caps/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics
4.
SLAS Discov ; 26(6): 757-765, 2021 07.
Article in English | MEDLINE | ID: covidwho-1194439

ABSTRACT

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Subject(s)
Adenosine/analogs & derivatives , High-Throughput Screening Assays , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/chemistry , Adenosine/pharmacology , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Methylation , Methyltransferases , Models, Molecular , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Tritium , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism
5.
SLAS Discov ; 26(6): 749-756, 2021 07.
Article in English | MEDLINE | ID: covidwho-1136206

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , Nitro Compounds/pharmacology , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Drug Repositioning , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Mass Spectrometry/methods , Methylation , Nitro Compounds/chemistry , Prescription Drugs/chemistry , Prescription Drugs/pharmacology , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thiazoles/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
6.
SLAS Discov ; 26(5): 620-627, 2021 06.
Article in English | MEDLINE | ID: covidwho-1021348

ABSTRACT

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.


Subject(s)
Antiviral Agents/pharmacology , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/pharmacology , Binding, Competitive , COVID-19/drug therapy , COVID-19/virology , Enzyme Inhibitors/pharmacology , Fluorescence Polarization , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Humans , Methyltransferases , Protein Binding , RNA Caps/antagonists & inhibitors , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Virus Replication
7.
ACS Nano ; 14(6): 7617-7627, 2020 06 23.
Article in English | MEDLINE | ID: covidwho-647565

ABSTRACT

The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective "naked-eye" detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA-DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/µL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual "naked-eye" detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.


Subject(s)
Betacoronavirus/genetics , Biosensing Techniques/methods , Coronavirus Infections/diagnosis , Metal Nanoparticles , Nucleocapsid Proteins/genetics , Oligonucleotides, Antisense/genetics , Pneumonia, Viral/diagnosis , Base Sequence , Betacoronavirus/isolation & purification , COVID-19 , Colorimetry/methods , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Genes, Viral , Gold , Humans , Metal Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Nanotechnology/methods , Pandemics , Phosphoproteins , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA Caps/genetics , RNA, Viral/genetics , SARS-CoV-2 , Surface Plasmon Resonance/methods
8.
Eur J Med Chem ; 201: 112557, 2020 Sep 01.
Article in English | MEDLINE | ID: covidwho-597389

ABSTRACT

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.


Subject(s)
Coronavirus Infections/drug therapy , Exoribonucleases/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , Nucleosides/chemistry , Pneumonia, Viral/drug therapy , RNA Caps/metabolism , S-Adenosylmethionine/analogs & derivatives , S-Adenosylmethionine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Adenine/chemistry , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Exoribonucleases/metabolism , Humans , Methylation , Methyltransferases/metabolism , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , RNA Caps/chemistry , RNA Caps/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
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