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1.
IUCrJ ; 7(Pt 5): 814-824, 2020 Sep 01.
Article in English | MEDLINE | ID: covidwho-1546123

ABSTRACT

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07-2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

2.
Methods Enzymol ; 661: 407-431, 2021.
Article in English | MEDLINE | ID: covidwho-1439812

ABSTRACT

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.


Subject(s)
COVID-19 , Nucleic Acids , Severe Acute Respiratory Syndrome , DNA Repair , Humans , Molecular Docking Simulation , SARS-CoV-2
3.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1385180

ABSTRACT

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Models, Molecular , RNA, Viral/genetics , Scattering, Small Angle , Viral Nonstructural Proteins , Virus Replication , X-Ray Diffraction
4.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1316407

ABSTRACT

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Models, Molecular , RNA, Viral/genetics , Scattering, Small Angle , Viral Nonstructural Proteins , Virus Replication , X-Ray Diffraction
5.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1223143

ABSTRACT

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Subject(s)
RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , Crystallography , Methylation , Methyltransferases/chemistry , Methyltransferases/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/metabolism , RNA Caps/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , S-Adenosylhomocysteine/chemistry , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Synchrotrons , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism
6.
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 , COVID-19/drug therapy , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/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
7.
Nat Commun ; 12(1): 743, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1061105

ABSTRACT

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.


Subject(s)
Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Humans , Mutation , Polyproteins/metabolism , Substrate Specificity , Virus Replication/drug effects
8.
Protein Sci ; 29(7): 1596-1605, 2020 07.
Article in English | MEDLINE | ID: covidwho-71902

ABSTRACT

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.


Subject(s)
Betacoronavirus/chemistry , Endoribonucleases/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Oligonucleotides/chemistry , SARS Virus/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Betacoronavirus/genetics , Betacoronavirus/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Endoribonucleases/genetics , Endoribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Models, Molecular , Oligonucleotides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
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