9 research outputs found
Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development
12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80â% of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR
was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG
through CRC902: âMolecular Principles of RNA-based regulation.â and through infrastructure funds (project
numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Unionâs Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Unionâs Horizon Europe Research and Innovation Programme under grant agreement number 101046203. âINSPIREDâ (MIS 5002550) project, implemented under the Action âReinforcement of the Research and Innovation Infrastructure,â funded by the Operational
Program âCompetitiveness, Entrepreneurship and Innovationâ (NSRF 2014â2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950ââSEE-DRUGâ project (purchase of UPATâs 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR
Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche MĂ©dicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020âGrant Agreement
Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortiumâs collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form
The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5â-genomic RNA elements
Abstract The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of Nâs N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5â-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domainâs flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs
Backbone chemical shift spectral assignments of SARS coronavirus-2 non-structural protein nsp9
As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication
1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a
International audienceThe non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein
Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development
SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083
Comprehensive Fragment Screening of the SARSâCoVâ2 Proteome Explores Novel Chemical Space for Drug Development
SARSâCoVâ2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including antiâvirals. Within the international Covid19âNMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMRâdetected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structureâbased drug design against the SCoV2 proteome
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortiumâs collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.This work was supported by Goethe University (Corona funds),
the DFG-funded CRC: âMolecular Principles of RNA-Based
Regulation,â DFG infrastructure funds (project numbers:
277478796, 277479031, 392682309, 452632086, 70653611), the
state of Hesse (BMRZ), the Fondazione CR Firenze (CERM),
and the IWB-EFRE-program 20007375. This project has
received funding from the European Unionâs Horizon 2020
research and innovation program under Grant Agreement No.
871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the
JQYA at Goethe through project number 2019/AS01. Work in the
lab of KV was supported by a CoRE grant from the University of
New Hampshire. The FLI is a member of the Leibniz Association
(WGL) and financially supported by the Federal Government of
Germany and the State of Thuringia. Work in the lab of RM was
supported by NIH (2R01EY021514) and NSF (DMR-2002837).
BN-B was supported by theNSF GRFP.MCwas supported byNIH
(R25 GM055246 MBRS IMSD), and MS-P was supported by the
HHMI Gilliam Fellowship. Work in the labs of KJ and KT was
supported by Latvian Council of Science Grant No. VPP-COVID
2020/1-0014. Work in the UPATâs lab was supported by the
INSPIRED (MIS 5002550) project, which is implemented under
the Action âReinforcement of the Research and Innovation
Infrastructure,â funded by the Operational Program
âCompetitiveness, Entrepreneurship and Innovationâ (NSRF
2014â2020) and cofinanced by Greece and the EU (European
Regional Development Fund) and the FP7 REGPOT CT-2011-
285950ââSEE-DRUGâ project (purchase of UPATâs 700MHz
NMR equipment). Work in the CM-G lab was supported by
the Helmholtz society. Work in the lab of ABö was supported
by the CNRS, the French National Research Agency (ANR, NMRSCoV2-
ORF8), the Fondation de la Recherche MĂ©dicale (FRM,
NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS.
Work in the lab of BM was supported by the Swiss National
Science Foundation (Grant number 200020_188711), the
GĂŒnthard Stiftung fĂŒr Physikalische Chemie, and the ETH
Zurich. Work in the labs of ABö and BM was supported by a
common grant from SNF (grant 31CA30_196256). This work was
supported by the ETHZurich, the grant ETH40 18 1, and the grant
Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble
was supported by the Agence Nationale de Recherche (France)
RA-COVID SARS2NUCLEOPROTEIN and European Research
Council Advanced Grant DynamicAssemblies. Work in the
CA lab was supported by Patto per il Sud della Regione
SicilianaâCheMISt grant (CUP G77B17000110001). Part of
this work used the platforms of the Grenoble Instruct-ERIC
center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the
Grenoble Partnership for Structural Biology (PSB), supported
by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within
the University Grenoble Alpes graduate school (Ecoles
Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-
0003). Work at the UW-Madison was supported by grant
numbers NSF MCB2031269 and NIH/NIAID AI123498. MM
is a RamĂłn y Cajal Fellow of the Spanish AEI-Ministry of
Science and Innovation (RYC2019-026574-I), and a âLa
Caixaâ Foundation (ID 100010434) Junior Leader Fellow
(LCR/BQ/PR19/11700003). Funded by project COV20/00764
fromthe Carlos III Institute of Health and the SpanishMinistry
of Science and Innovation to MMand DVL. VDJ was supported
by the Boehringer Ingelheim Fonds. Part of this work used the
resources of the Italian Center of Instruct-ERIC at the CERM/
CIRMMP infrastructure, supported by the Italian Ministry for
University and Research (FOE funding). CF was supported by
the Stiftung Polytechnische Gesellschaft. Work in the lab of
JH was supported by NSF (RAPID 2030601) and NIH
(R01GM123249).Peer reviewe