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1.
Nat Struct Mol Biol ; 28(7): 614-625, 2021 07.
Article in English | MEDLINE | ID: covidwho-1550333

ABSTRACT

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.


Subject(s)
Adenosine Triphosphate/chemistry , Protein Folding , Proteostasis/physiology , Signal Transduction/physiology , Valosin Containing Protein/metabolism , Acetanilides/pharmacology , Animals , Benzothiazoles/pharmacology , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/antagonists & inhibitors
3.
Nat Commun ; 12(1): 4848, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1354102

ABSTRACT

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Subject(s)
Methyltransferases/chemistry , RNA Helicases/chemistry , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Models, Molecular , Phosphates/chemistry , Phosphates/metabolism , Protein Conformation , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/enzymology , Structure-Activity Relationship , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
4.
Int J Mol Sci ; 21(19)2020 Sep 27.
Article in English | MEDLINE | ID: covidwho-1299427

ABSTRACT

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.


Subject(s)
Enzyme Inhibitors/chemistry , Membrane Proteins , Molecular Docking Simulation , Nucleotidyltransferases , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Drug Evaluation, Preclinical , Fluorescence Polarization , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry
5.
Biochem Biophys Res Commun ; 541: 50-55, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1030847

ABSTRACT

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


Subject(s)
Adenosine Triphosphate/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Liquid-Liquid Extraction , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Adenosine Triphosphate/chemistry , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Motifs/genetics , SARS-CoV-2/chemistry
6.
J Phys Chem Lett ; 11(21): 9144-9151, 2020 Nov 05.
Article in English | MEDLINE | ID: covidwho-867355

ABSTRACT

The raging COVID-19 pandemic caused by SARS-CoV-2 has infected tens of millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV-2 helicase (Nsp13), which is critical for viral replication and the most conserved nonstructural protein within the coronavirus family. Using homology modeling that couples published electron-density with molecular dynamics (MD)-based structural refinements, we generated structural models of the SARS-CoV-2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ∼970 000 chemical compounds against the ATP-binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP-binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/enzymology , RNA Helicases/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Structure, Tertiary , RNA Helicases/chemistry , RNA Helicases/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
7.
Antiviral Res ; 182: 104899, 2020 10.
Article in English | MEDLINE | ID: covidwho-694239

ABSTRACT

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Flavivirus/drug effects , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/pharmacology , Antiviral Agents/chemistry , Betacoronavirus/drug effects , Betacoronavirus/enzymology , COVID-19 , Flavivirus/enzymology , Humans , Inhibitory Concentration 50 , Pandemics , RNA Viruses/drug effects , RNA Viruses/enzymology , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2
8.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Article in English | MEDLINE | ID: covidwho-611394

ABSTRACT

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/metabolism , Betacoronavirus/enzymology , Enzyme Inhibitors/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Amino Acid Sequence , Antiviral Agents/chemistry , Binding Sites , Coronavirus RNA-Dependent RNA Polymerase , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Binding , RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2 , Thermodynamics , Viral Nonstructural Proteins/chemistry
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