Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 36
Filter
1.
Mar Drugs ; 20(3)2022 Feb 28.
Article in English | MEDLINE | ID: covidwho-1715534

ABSTRACT

Several natural products recovered from a marine-derived Aspergillus niger were tested for their inhibitory activity against SARS CoV-2 in vitro. Aurasperone A (3) was found to inhibit SARS CoV-2 efficiently (IC50 = 12.25 µM) with comparable activity with the positive control remdesivir (IC50 = 10.11 µM). Aurasperone A exerted minimal cytotoxicity on Vero E6 cells (CC50 = 32.36 mM, SI = 2641.5) and it was found to be much safer than remdesivir (CC50 = 415.22 µM, SI = 41.07). To putatively highlight its molecular target, aurasperone A was subjected to molecular docking against several key-viral protein targets followed by a series of molecular dynamics-based in silico experiments that suggested Mpro to be its primary viral protein target. More potent anti-SARS CoV-2 Mpro inhibitors can be developed according to our findings presented in the present investigation.


Subject(s)
Antiviral Agents/pharmacology , Chromones/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/isolation & purification , Aspergillus niger/chemistry , Chlorocebus aethiops , Chromones/isolation & purification , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Molecular Docking Simulation , Protease Inhibitors/isolation & purification , RNA Helicases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
2.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
3.
PLoS One ; 16(10): e0258856, 2021.
Article in English | MEDLINE | ID: covidwho-1542176

ABSTRACT

Hypoxia is a common pathway to the progression of end-stage kidney disease. Retinoic acid-inducible gene I (RIG-I) encodes an RNA helicase that recognizes viruses including SARS-CoV2, which is responsible for the production of interferon (IFN)-α/ß to prevent the spread of viral infection. Recently, RIG-I activation was found under hypoxic conditions, and klotho deficiency was shown to intensify the activation of RIG-I in mouse brains. However, the roles of these functions in renal inflammation remain elusive. Here, for in vitro study, the expression of RIG-I and IFN-α/ß was examined in normal rat kidney (NRK)-52E cells incubated under hypoxic conditions (1% O2). Next, siRNA targeting RIG-I or scramble siRNA was transfected into NRK52E cells to examine the expression of RIG-I and IFN-α/ß under hypoxic conditions. We also investigated the expression levels of RIG-I and IFN-α/ß in 33 human kidney biopsy samples diagnosed with IgA nephropathy. For in vivo study, we induced renal hypoxia by clamping the renal artery for 10 min in wild-type mice (WT mice) and Klotho-knockout mice (Kl-/- mice). Incubation under hypoxic conditions increased the expression of RIG-I and IFN-α/ß in NRK52E cells. Their upregulation was inhibited in NRK52E cells transfected with siRNA targeting RIG-I. In patients with IgA nephropathy, immunohistochemical staining of renal biopsy samples revealed that the expression of RIG-I was correlated with that of IFN-α/ß (r = 0.57, P<0.001, and r = 0.81, P<0.001, respectively). The expression levels of RIG-I and IFN-α/ß were upregulated in kidneys of hypoxic WT mice and further upregulation was observed in hypoxic Kl-/- mice. These findings suggest that hypoxia induces the expression of IFN-α/ß through the upregulation of RIG-I, and that klotho deficiency intensifies this hypoxia-induced expression in kidneys.


Subject(s)
Glucuronidase/metabolism , Hypoxia/metabolism , Interferon-alpha/metabolism , Kidney/metabolism , RNA Helicases/metabolism , Up-Regulation , Animals , Glucuronidase/genetics , Hypoxia/genetics , Mice , Mice, Knockout , RNA, Small Interfering , Rats
4.
Nat Commun ; 12(1): 6761, 2021 11 19.
Article in English | MEDLINE | ID: covidwho-1526072

ABSTRACT

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.


Subject(s)
Integration Host Factors/metabolism , SARS-CoV-2/metabolism , Adaptor Proteins, Signal Transducing/metabolism , DNA Helicases/metabolism , Humans , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/metabolism , Virus Replication/physiology
5.
FEBS Lett ; 595(23): 2872-2896, 2021 12.
Article in English | MEDLINE | ID: covidwho-1516705

ABSTRACT

The current work investigated SARS-CoV-2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC-based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress-stimulated NCAP to undergo liquid-liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , /metabolism , A549 Cells , Adaptor Proteins, Signal Transducing/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , DNA Helicases/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , eIF-2 Kinase/metabolism
6.
mBio ; 12(5): e0131621, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1406604

ABSTRACT

Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/metabolism , RNA Helicases/metabolism , RNA Helicases/pharmacology , Animals , Blotting, Western , Cell Line, Tumor , Chlorocebus aethiops , Fluorescent Antibody Technique , Humans , Immunoprecipitation , RNA, Viral/metabolism , Vero Cells , Virus Replication/drug effects
7.
Int J Biol Macromol ; 190: 636-648, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1401500

ABSTRACT

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , COVID-19/drug therapy , Coronavirus Nucleocapsid Proteins/chemistry , DNA Helicases/chemistry , Decitabine/chemistry , Imatinib Mesylate/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Poly-ADP-Ribose Binding Proteins/chemistry , RNA Helicases/chemistry , RNA Recognition Motif Proteins/chemistry , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , DNA Helicases/antagonists & inhibitors , DNA Helicases/metabolism , Decitabine/pharmacology , Drug Delivery Systems , Genomics , Imatinib Mesylate/pharmacology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA Recognition Motif Proteins/antagonists & inhibitors , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism
8.
Nat Commun ; 12(1): 502, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1387327

ABSTRACT

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Matrix Proteins/metabolism , COVID-19/genetics , COVID-19/metabolism , Cell Membrane/virology , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , Protein Domains , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Recognition Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics
9.
Cell ; 184(1): 184-193.e10, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1385213

ABSTRACT

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cryoelectron Microscopy , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Viral Replicase Complex Proteins/chemistry , Amino Acid Sequence , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/enzymology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Methyltransferases/metabolism , Models, Molecular , RNA Helicases/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/enzymology , Sequence Alignment , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication
10.
Nat Commun ; 11(1): 5874, 2020 11 18.
Article in English | MEDLINE | ID: covidwho-1387320

ABSTRACT

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Virus Replication , Betacoronavirus/genetics , Betacoronavirus/metabolism , Binding Sites , Cryoelectron Microscopy , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Structure-Activity Relationship , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
11.
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
12.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1292181

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , RNA Helicases/antagonists & inhibitors , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Chlorocebus aethiops , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , RNA Helicases/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Suramin/pharmacology , Vero Cells , Viral Nonstructural Proteins/metabolism
13.
Molecules ; 26(13)2021 Jun 22.
Article in English | MEDLINE | ID: covidwho-1288958

ABSTRACT

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Subject(s)
Antiviral Agents/pharmacology , Fluorescence Resonance Energy Transfer/methods , High-Throughput Screening Assays/methods , Protease Inhibitors/pharmacology , RNA Viruses/enzymology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Drug Evaluation, Preclinical , Encephalitis Viruses, Tick-Borne/enzymology , Fluorescent Dyes/chemistry , Humans , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , SARS-CoV-2/enzymology , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
14.
Science ; 373(6551): 236-241, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1266364

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Subject(s)
Coenzymes/metabolism , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cyclic N-Oxides/pharmacology , Iron/metabolism , SARS-CoV-2/drug effects , Sulfur/metabolism , Amino Acid Motifs , Animals , Antiviral Agents/pharmacology , Binding Sites , Catalytic Domain , Chlorocebus aethiops , Coenzymes/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/pharmacology , Iron/chemistry , Protein Domains , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Spin Labels , Sulfur/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Zinc/metabolism
15.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1254144

ABSTRACT

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
16.
Sci Rep ; 11(1): 10290, 2021 05 13.
Article in English | MEDLINE | ID: covidwho-1228274

ABSTRACT

As the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic engulfs millions worldwide, the quest for vaccines or drugs against the virus continues. The helicase protein of SARS-CoV-2 represents an attractive target for drug discovery since inhibition of helicase activity can suppress viral replication. Using in silico approaches, we have identified drugs that interact with SARS-CoV-2 helicase based on the presence of amino acid arrangements matching binding sites of drugs in previously annotated protein structures. The drugs exhibiting an RMSD of ≤ 3.0 Å were further analyzed using molecular docking, molecular dynamics (MD) simulation, and post-MD analyses. Using these approaches, we found 12 drugs that showed strong interactions with SARS-CoV-2 helicase amino acids. The analyses were performed using the recently available SARS-CoV-2 helicase structure (PDB ID: 5RL6). Based on the MM-GBSA approach, out of the 12 drugs, two drugs, namely posaconazole and grazoprevir, showed the most favorable binding energy, - 54.8 and - 49.1 kcal/mol, respectively. Furthermore, of the amino acids found conserved among all human coronaviruses, 10/11 and 10/12 were targeted by, respectively, grazoprevir and posaconazole. These residues are part of the crucial DEAD-like helicase C and DEXXQc_Upf1-like/ DEAD-like helicase domains. Strong interactions of posaconazole and grazoprevir with conserved amino acids indicate that the drugs can be potent against SARS-CoV-2. Since the amino acids are conserved among the human coronaviruses, the virus is unlikely to develop resistance mutations against these drugs. Since these drugs are already in use, they may be immediately repurposed for SARS-CoV-2 therapy.


Subject(s)
Amides/pharmacology , Carbamates/pharmacology , Cyclopropanes/pharmacology , Drug Repositioning , Enzyme Inhibitors/pharmacology , Quinoxalines/pharmacology , RNA Helicases/antagonists & inhibitors , SARS-CoV-2/enzymology , Sulfonamides/pharmacology , Triazoles/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Domains/drug effects , RNA Helicases/chemistry , RNA Helicases/metabolism , SARS-CoV-2/drug effects , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Viral Proteins/metabolism
17.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1196910

ABSTRACT

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
18.
Molecules ; 26(5)2021 Mar 07.
Article in English | MEDLINE | ID: covidwho-1136523

ABSTRACT

With the emergence and global spread of the COVID-19 pandemic, the scientific community worldwide has focused on search for new therapeutic strategies against this disease. One such critical approach is targeting proteins such as helicases that regulate most of the SARS-CoV-2 RNA metabolism. The purpose of the current study was to predict a library of phytochemicals derived from diverse plant families with high binding affinity to SARS-CoV-2 helicase (Nsp13) enzyme. High throughput virtual screening of the Medicinal Plant Database for Drug Design (MPD3) database was performed on SARS-CoV-2 helicase using AutoDock Vina. Nilotinib, with a docking value of -9.6 kcal/mol, was chosen as a reference molecule. A compound (PubChem CID: 110143421, ZINC database ID: ZINC257223845, eMolecules: 43290531) was screened as the best binder (binding energy of -10.2 kcal/mol on average) to the enzyme by using repeated docking runs in the screening process. On inspection, the compound was disclosed to show different binding sites of the triangular pockets collectively formed by Rec1A, Rec2A, and 1B domains and a stalk domain at the base. The molecule is often bound to the ATP binding site (referred to as binding site 2) of the helicase enzyme. The compound was further discovered to fulfill drug-likeness and lead-likeness criteria, have good physicochemical and pharmacokinetics properties, and to be non-toxic. Molecular dynamic simulation analysis of the control/lead compound complexes demonstrated the formation of stable complexes with good intermolecular binding affinity. Lastly, affirmation of the docking simulation studies was accomplished by estimating the binding free energy by MMPB/GBSA technique. Taken together, these findings present further in silco investigation of plant-derived lead compounds to effectively address COVID-19.


Subject(s)
Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , Antiviral Agents/toxicity , Binding Sites , Biological Availability , COVID-19/drug therapy , Computational Biology/methods , Databases, Chemical , Drug Design , Humans , Hydrogen Bonding , Methyltransferases/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Phytochemicals/chemistry , Phytochemicals/metabolism , Plants, Medicinal/chemistry , Protein Binding , Protein Domains/drug effects , Pyrimidines/chemistry , Pyrimidines/metabolism , Pyrimidines/pharmacokinetics , Pyrimidines/toxicity , RNA Helicases/chemistry , Structure-Activity Relationship , Thermodynamics , Viral Nonstructural Proteins/chemistry
19.
PLoS One ; 16(2): e0246181, 2021.
Article in English | MEDLINE | ID: covidwho-1088753

ABSTRACT

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Allosteric Site , Binding Sites , COVID-19/drug therapy , COVID-19/metabolism , Computational Biology/methods , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Models, Molecular , Molecular Targeted Therapy , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects
20.
Cell Rep ; 34(2): 108628, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-1036973

ABSTRACT

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.


Subject(s)
Immunity, Innate , Interferon-Induced Helicase, IFIH1/metabolism , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Cell Line , Epithelial Cells/cytology , Epithelial Cells/immunology , Epithelial Cells/virology , Humans , Induced Pluripotent Stem Cells/cytology , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Interferons/genetics , Interferons/metabolism , RNA Helicases/metabolism , RNA Interference , RNA, Double-Stranded/metabolism , RNA, Small Interfering/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Signal Transduction , Transcription Factor RelA/metabolism , Virus Replication
SELECTION OF CITATIONS
SEARCH DETAIL