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 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.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: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 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 Médicale (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-03-2020—Grant 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 consortium’s 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 structural basis of viral proteins' function in the Replication/Transcription complexes of Togaviridae and Coronaviridae viral families

Viruses are obligate intracellular parasites that infect all kingdoms of life, and many of their species have been identified as human pathogens. Their structure and organization are quite simple, consisting of their genome surrounded by a protein capsid which in some cases can be covered by a lipid or protein envelope. Despite their simplicity, from their genome are encoded numerous of proteins, essentials for viral replication. Viral proteins are categorized as structural proteins that form the viral capsid, and non-structural proteins (nsPs) that, along with host cell components, are required for viral replication. Many of their functions are exploited by the virus to escape host-defense mechanisms, a crucial step in the progression of a viral infection. Therefore, an in-depth understanding of the biological functions of viral proteins is essential for the effective prevention and treatment of viral-caused human diseases.Herein, were studied nsP3 independent domains of viral species belonging in Togaviridae and Coronaviridae viral families. Specifically, the macro domains (MDs) of MAYV, CHIKV, SARS-CoV-1, MERS-CoV and SARS-CoV-2, as well as the Alphavirus Unique Domain (AUD) of MAYV, were investigated. MDs are evolutionary conserved protein domains found in all kingdoms of life and viruses, exhibiting functional divergence. Today, MDs are considered key molecules in viral replication and viral host-immune response evasion. For the latter, it is important their function as ADP-ribosylation "erasers", a post-translational modification implicated in various cellular processes. AUD is a characteristic domain for the Alphavirus genus. It is located in the central part of nsP3, and despite its elusive biological role, its function has been linked to viral replication and successful infectivity.Molecular and cellular biology, biochemistry, and biophysical analysis methods and techniques were applied to study MD and AUD architecture and function. The overall goal was the investigation of the structure-function relationship of these domains for the identification of crucial characteristics that could be further exploited in drug design. The derived molecules can contribute to the elucidation of their functions and/or to the inhibition of their activity.For this reason, the respective polypeptides were expressed and isolated in high yield and purity to archive: their secondary structure characterization, the study of their dynamic properties on the ps-ns time scale, and their interactions with other molecules in solution via NMR spectroscopy.Regarding Alphaviruses and Coronaviruses MDs, the comparative study of their structural characteristics and their interactions with the ADPr, other nucleotide analogues and nucleic acids revealed common and different features in the dynamic profile of the loops that surrounds the ADPr binding cavity that potentially can be reflected to their functions. Moreover, their enzymatic capacity towards ADP-ribosylated substrates was tested and used for the development of a biochemical protocol that can be used for the discovery of novel compounds that can potentially act as MD inhibitors.For MAYV AUD’s structural characterization through NMR spectroscopy in solution, a variety of different polypeptides were designed, expressed, and examined. The significance of the zinc binding motif for the structural integrity of the molecule was emphasized, while were also investigated the function-related aspects of AUD’s RNA binding capacity.Οι ιοί αποτελούν υποχρεωτικά ενδοκυτταρικά παράσιτα, ενώ πολλά είδη τους έχουν αναγνωριστεί σήμερα ως ανθρώπινα παθογόνα. Η οργάνωσή τους εξαντλείται στην παρουσία του γενετικού τους υλικού μέσα σε ένα πρωτεϊνικής φύσης καψίδιο περιβαλλόμενο ορισμένες φορές από ένα λιποπρωτεϊνικής σύστασης περίβλημα. Παρά την απλή σύνθεσή τους, από το γονιδίωμά τους κωδικοποιείται πληθώρα πρωτεϊνών των οποίων οι λειτουργίες είναι απαραίτητες για τον πολλαπλασιασμό τους. Οι ιικές πρωτεΐνες διαχωρίζονται σε αυτές οι οποίες συνιστούν το ιικό περίβλημα (δομικές), και σε εκείνες οι οποίες επιτελούν, σε συνεργασία με μόρια του κυττάρου ξενιστή, όλες τις απαραίτητες λειτουργίες για τον επιτυχή ιικό πολλαπλασιασμό (μη δομικές-nsPs). Ακόμα, ορισμένες λειτουργίες τους «αξιοποιούνται» από τους ιούς για την αποφυγή των κυτταρικών μηχανισμών άμυνας, διαδικασία η οποία διαδραματίζει κυρίαρχο ρόλο στην εξέλιξη μίας ιικής μόλυνσης. Γίνεται έτσι αντιληπτό ότι για τον περιορισμό και την αντιμετώπιση των ιώσεων είναι σημαντική η σε βάθος κατανόηση του βιολογικού ρόλου των ιικών πρωτεϊνών.Στην παρούσα εργασία μελετήθηκαν ανεξάρτητες επικράτειες της nsP3 συγκεκριμένων ιικών ειδών, των οικογενειών Togaviridae και Coronaviridae. Ειδικότερα αντικείμενο μελέτης αποτέλεσαν οι macro επικράτειες (macro domains-MDs) των ιών MAYV, CHIKV, SARS-CoV-1, MERS-CoV και SARS-CoV-2 και η Alphavirus Unique Domain (AUD) του MAYV. Οι MDs αποτελούν μία εξελικτικά συντηρημένη οικογένεια δομών. Εντοπίζονται σε όλες τις μορφές ζωής και στους ιούς, εμφανίζοντας λειτουργικές διαφοροποιήσεις. Σήμερα έχει αποδειχθεί η συμμετοχή τους στον αποτελεσματικό πολλαπλασιασμό των ιών και στην καταπολέμηση της ανοσολογικής απόκρισης του ξενιστή. Καθοριστικής σημασίας για την τελευταία διαδικασία είναι η ικανότητά τους να «διαγράφουν» την ADP-ριβοζυλίωση, μία μεταφραστική τροποποίηση εμπλεκόμενη σε ποικίλες κυτταρικές διεργασίες. Η AUD είναι χαρακτηριστική επικράτεια η οποία εντοπίζεται μόνο στο γένος Alphavirus. Έπεται αλληλουχικά της MD και η αποσαφήνιση του βιολογικού της ρόλου βρίσκεται ακόμα υπό διερεύνηση, έχοντας συνδεθεί με τον πολλαπλασιασμό και τη μολυσματικότητα των ιών.Οι επικράτειες αυτές μελετήθηκαν ως προς την αρχιτεκτονική, τη λειτουργία τους και τις αλληλεπιδράσεις τους με άλλα μόρια εφαρμόζοντας μεθόδους και τεχνικές μοριακής και κυτταρικής βιολογίας, πρωτεϊνικής χημείας, βιοχημείας και βιοφυσικών αναλύσεων. Απώτερος στόχος ήταν η διερεύνηση της σχέσης δομής-δράσης τους για την εξαγωγή συμπερασμάτων τα οποία μπορούν να αξιοποιηθούν στο σχεδιασμό βιοδραστικών εκλεκτικών μορίων-προσδετών. Τα μόρια αυτά, δυνητικά, μπορούν να συνεισφέρουν στη σε βάθος κατανόηση ή και αναστολή της λειτουργίας τους.Για το λόγο αυτόν, στην παρούσα διατριβή, εκφράστηκαν και απομονώθηκαν επιτυχώς σε υψηλή απόδοση και με υψηλή καθαρότητα τα πολυπεπτίδια τα οποία αντιστοιχούν στις συγκεκριμένες ιικές επικράτειες και μελετήθηκαν τα διαμορφωτικά χαρακτηριστικά τους, η δυναμική τους σε κλίμακα χρόνου ps-ns, και οι αλληλεπιδράσεις τους με άλλα μόρια, μέσω τις NMR φασματοσκοπίας.Για τις MDs των Alphaviruses και Coronaviruses πραγματοποιήθηκε ενδελεχής συγκριτική μελέτη των δομικών τους χαρακτηριστικών αλλά και των αλληλεπιδράσεων τους με τον φυσικό προσδέτη, ADPr, άλλα νουκλεοτιδικά ανάλογα και νουκλεϊκά οξέα, υποδεικνύοντας τις ομοιότητες και τις διαφορές στη δυναμική συμπεριφορά των βρόγχων οι οποίοι δημιουργούν την κοιλότητα δέσμευσης του φυσικού προσδέτη. Ακόμα, ελέγχθηκε η ικανότητα απo-ADP-ριβοζυλίωσης υποστρωμάτων, ιδιότητα η οποία αξιοποιήθηκε στην ανάπτυξη ενός βιοχημικού πρωτοκόλλου αξιολόγησης μορίων για την ανασταλτική τους δράση ως προς την υδρολυτική ικανότητα των MDs. Τέλος, χαρακτηρίστηκε η ένωση GS- 441524 ως ειδικός αναστολέας της ενζυμικής δράσης της MD του SARS-CoV-2.Όσον αφορά στην AUD του MAYV, στην παρούσα μελέτη παρασκευάστηκαν και εξετάστηκαν διαφορετικού μήκους πολυπεπτίδια για τον δομικό χαρακτηρισμό της επικράτειας σε διάλυμα μέσω της NMR φασματοσκοπίας. Προσδιορίστηκε η σημασία του μεταλλικού κέντρου στη δομική ακεραιότητά του πολυπεπτίδιού και εξετάστηκε η ικανότητα πρόσδεσης νουκλεϊκών οξέων, ιδιότητα συνδεδεμένη με της λειτουργία της AUD

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

International audienceMayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical "macro" (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD-ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP-r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure-function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions

NMR study of non-structural proteins-part I: H-1, C-13, N-15 backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV)

International audienceno abstrac

NMR study of non-structural proteins-part II: H-1, C-13, N-15 backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)

International audienceno abstrac

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

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 consortium’s 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: “Molecular Principles of RNA-Based Regulation,” 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 Union’s 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 UPAT’s lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–“SEE-DRUG” project (purchase of UPAT’s 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 Médicale (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 Günthard Stiftung für 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 Siciliana–CheMISt 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 Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” 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