10 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
NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs
The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1âD34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical ÎČ2-ÎČ3-ÎČ5 interfaces. Evidently, the 3âČ-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation
NMR spectroscopy of large functional RNAs: from sample preparation to lowâgamma detection
NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotopeâlabeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclearâdetected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotopeâlabeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemoâenzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclearâdetected NMR experiments including 13Câdetected experiments for ribose assignment and amino groups, the CNâspin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15Nâdetected bandâselective excitation short transient transverseârelaxationâoptimized spectroscopy (BESTâTROSY) experiment.
Basic Protocol 1: Preparation of isotopeâlabeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RPâHPLC purification
Alternate Protocol 1: Purification of isotopeâlabeled RNA from in vitro transcription with preparative PAGE
Alternate Protocol 2: Purification of isotopeâlabeled RNA samples from in vitro transcription via centrifugal concentration
Support Protocol 1: Preparation of DNA template from plasmid
Support Protocol 2: Preparation of PCR DNA as template
Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP)
Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP)
Basic Protocol 2: Preparation of siteâspecific labeled RNAs using a chemoâenzymatic synthesis
Support Protocol 5: Synthesis of modified nucleoside 3âČ,5âČâbisphosphates
Support Protocol 6: Preparation of T4 RNA Ligase 2
Support Protocol 7: Setup of NMR spectrometer for heteronuclearâdetected NMR experiments
Support Protocol 8: IPAP and DIPAP for homonuclear decoupling
Basic Protocol 3: 13Câdetected 3D (H)CCâTOCSY, (H)CPC, and (H)CPCâCCHâTOCSY experiments for ribose assignment
Basic Protocol 4: 13Câdetected 2D CNâspin filter HSQC experiment
Basic Protocol 5: 13Câdetected C(N)HâHDQC experiment for the detection of amino groups
Support Protocol 9: 13Câdetected CNâHSQC experiment for amino groups
Basic Protocol 6: 13Câdetected âaminoââNOESY experiment
Basic Protocol 7: 15Nâdetected BESTâTROSY experimen
Endocannabinoids, endocannabinoid-like compounds and cortisone in head hair of health care workers as markers of stress and resilience during the early COVID-19 pandemic
Abstract The pandemic caused by SARS-CoV-2 impacted health systems globally, creating increased workload and mental stress upon health care workers (HCW). During the first pandemic wave (March to May 2020) in southern Germany, we investigated the impact of stress and the resilience to stress in HCW by measuring changes in hair concentrations of endocannabinoids, endocannabinoid-like compounds and cortisone. HCW (nâ=â178) recruited from multiple occupation and worksites in the LMU-University-Hospital in Munich were interviewed at four interval visits to evaluate mental stress associated with the COVID-19 pandemic. A strand of hair of up to 6âcm in length was sampled once in May 2020, which enabled retrospective individual stress hormone quantifications during that aforementioned time period. Perceived anxiety and impact on mental health were demonstrated to be higher at the beginning of the COVID-19 pandemic and decreased significantly thereafter. Resilience was stable over time, but noted to be lower in women than in men. The concentrations of the endocannabinoid anandamide (AEA) and the structural congeners N-palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA) and N-stearoylethanolamide (SEA) were noted to have decreased significantly over the course of the pandemic. In contrast, the endocannabinoid 2-arachidonoylglycerol (2-AG) levels increased significantly and were found to be higher in nurses, laboratory staff and hospital administration than in physicians. PEA was significantly higher in subjects with a higher resilience but lower in subjects with anxiety. SEA was also noted to be reduced in subjects with anxiety. Nurses had significantly higher cortisone levels than physicians, while female subjects had significant lower cortisone levels than males. Hair samples provided temporal and measurable objective psychophysiological-hormonal information. The hair endocannabinoids/endocannabinoid-like compounds and cortisone correlated to each other and to professions, age and sex quite differentially, relative to specific periods of the COVID-19 pandemic
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
Exploring the druggability of conserved RNA regulatory elements in the SARS-CoV-2 genome
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30â000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2
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