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

Biophysical studies of RNA:DNA:DNA triplexes and characterization of riboswitches in cell-free transcription-translation systems

RNA research is very important since RNA molecules are involved in various gene regulatory mechanisms as well as pathways of cell physiology and disease development.1 RNAs have evolved from being considered as carriers of genetic information from DNA to proteins, with the three major types of RNA involved in protein synthesis, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).2 In addition to the RNAs involved in protein synthesis numerous regulatory non-coding RNAs (ncRNAs) have been discovered in the transcriptome. The regulatory ncRNAs are classified into small ncRNAs (sncRNAs) with transcripts less than 200 nucleotides (nt) and long non-coding RNAs (lncRNAs) with more than 200 nt.3 LncRNAs represent the most diverse and versatile class of ncRNAs that can regulate cellular functions of chromatin modification, transcription, and post-transcription through multiple mechanisms.4 They are involved in the formation of RNA:protein, RNA:RNA and RNA:DNA complexes as part of their gene regulatory mechanism.4,5 The RNA:DNA interactions can be divided into RNA:DNA heteroduplex formation, also called R-loops, and RNA:DNA:DNA triplex formation. In triplex formation, RNA binds to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding, resulting in parallel or anti-parallel triplexes, respectively. In vitro studies have confirmed the formation of RNA:DNA:DNA triplexes.6 However, the extent to which these interactions occur in cells and their effects on cellular function are still not understood, which is why these structures are so exciting to study (Chapter I RNA:DNA:DNA Triplexes). This cumulative thesis investigates several functional and regulatory important RNAs. The first project involves the improved biochemical and biophysical characterization of RNA:DNA:DNA triplex formation between lncRNAs of interest and their target genes. Triplex formation was confirmed by a series of experiments including electromobility shift assays (EMSA), thermal melting assays, circular dichroism (CD), and liquid state nuclear magnetic resonance (NMR) spectroscopy. The following is a summary of the main findings of these publications. In research article 5.1, the oxygen-sensitive HIF1α-AS1 was identified as a functionally important triplex-forming lncRNA in human endothelial cells using a combination of bioinformatics techniques, RNA/DNA pulldown, and biophysical experiments. Through RNA:DNA:DNA triplex formation, endogenous HIF1α-AS1 decreases the expression of several genes, including EPH receptor A2 (EPHA2) and adrenomedullin (ADM), by acting as an adaptor for the repressive human silencing hub (HUSH) complex, which has been studied by our collaborators in the groups of Leisegang and Brandes. 2) Triplex formation between HIF1α-AS1 and the target genes EPHA2 and ADM was investigated in biochemical and biophysical studies. The EMSA results indicated that HIF1α-AS1 forms a low mobility RNA:DNA:DNA triplex complex with the EPHA2 DNA target sequence. The CD spectrum of the triplex showed distinct features compared to the EPHA2 DNA duplex and the RNA:DNA heteroduplex. Melting curve analysis revealed a biphasic melting transition for triplexes, with a first melting point corresponding to the dissociation of the RNA strand with melting of the Hoogsteen hydrogen bonds. The second, higher melting temperature corresponds to the melting of stronger Watson-Crick base pairing. Stabilized triplexes were formed using an intramolecular EPHA2 DNA duplex hairpin construct in which both DNA strands were attached to a 5 nucleotide (nt) thymidine linker. This approach allowed improved triplex formation with lower RNA equivalents and higher melting temperatures. By NMR spectroscopy, the triplex characteristic signals were observed in the 1H NMR spectrum, the imino signals in a spectral region between 9 and 12 ppm resulting from the Hoogsteen base pairing. To elucidate the structural and sequence specific Hoogsteen base pairs 2D 1H,1H-NOESY measurements of the EPHA2 DNA duplex and the HIF1α-AS1:EPHA2 triplex were performed. The 1H,1H-NOESY spectrum of the HIF1α-AS1:EPHA2 triplex with a 10-fold excess of RNA was semi-quantitatively analyzed for changes in the DNA duplex spectrum. We discovered, strong and moderate attenuation of cross peak intensities in the imino region of the NOESY spectrum. This attenuation was proposed to result from weakening of Watson-Crick base pairing by Hoogsteen hydrogen bonding induced by RNA binding. The Hoogsteen interactions can be mapped based on the analysis of the cross peak attenuation in the NOESY spectra, which we used to generate a structural model of the RNA:DNA:DNA triplex. These biophysical results support the physiological function of HIF1α as a triplex-forming lncRNA that recruits the HUSH-epigenetic silencing complex to specific target genes such as EPHA2 and ADM, thereby silencing their gene expression through RNA:DNA:DNA triplex formation.Die Erforschung von RNAs ist von großer Bedeutung, da RNA-Moleküle an verschiedenen Mechanismen der Genregulation sowie an Signalwegen in der Zellphysiologie und bei der Entstehung von Krankheiten beteiligt sind.1 RNAs haben sich von Trägern der genetischen Information von der DNA zu Proteinen entwickelt, wobei die drei wichtigsten RNA-Typen an der Proteinsynthese beteiligt sind: Boten-RNA (mRNA), Transfer-RNA (tRNA) und ribosomale RNA (rRNA).2 Neben den an der Proteinsynthese beteiligten RNAs wurden im Transkriptom zahlreiche regulatorische nicht-kodierende RNAs (ncRNAs) entdeckt. Die regulatorischen ncRNAs werden unterteilt in kurze ncRNAs (sncRNAs) mit Transkripten kürzer als 200 Nukleotide (nt) und lange ncRNAs (lncRNAs) mit Transkripten länger als 200 nt.3 LncRNAs stellen die vielfältigste und vielseitigste Klasse von ncRNAs dar, die durch verschiedene Mechanismen zelluläre Funktionen der Chromatinmodifikation, Transkription und Posttranskription regulieren können.4 Sie sind an der Bildung von RNA:Protein-, RNA:RNA- und RNA:DNA-Komplexen als Teil ihrer Genregulationsmechanismen beteiligt.4,5 Die RNA:DNA Interaktionen können in die Bildung von RNA:DNA Heteroduplexen, auch R-Schleifen genannt und die Bildung von RNA:DNA:DNA Triplexen unterteilt werden. Bei der Triplexbildung bindet die RNA über Hoogsteen oder inverse Hoogsteen Wasserstoffbrücken an die große Furche der doppelsträngigen DNA, was zu parallelen oder anti-parallelen Triplexen führt. In vitro Studien haben die Bildung von RNA:DNA:DNA Triplexen bestätigt. 6 Inwieweit diese Wechselwirkungen in Zellen auftreten und sich auf die Zellfunktionen auswirken, ist noch nicht geklärt, weshalb diese Strukturen für die Forschung von großem Interesse sind (Kapitel I RNA:DNA:DNA Triplexe). In dieser kumulativen Dissertation werden verschiedene funktionell und regulatorisch wichtige RNAs untersucht. Das erste Projekt umfasst die verbesserte biochemische und biophysikalische Charakterisierung von RNA:DNA:DNA Triplexen. Die Triplexbildung wurde durch eine Reihe von Experimenten bestätigt, darunter Gelelektrophorese Untersuchungen (electromobility shift assay, EMSA), thermische Schmelzanalyse, Circulardichroismus- (CD) und Kernspinresonanzspektroskopie (NMR). In den folgenden Abschnitten werden die wichtigsten Ergebnisse dieser Veröffentlichungen zusammengefasst. In der Publikation 5.1 wurde das sauerstoffsensitive HIF1α-AS1 als funktionell wichtige Triplex-bildende lncRNA in humanen Endothelzellen durch eine Kombination von bioinformatischen Techniken, RNA/DNA-Pulldown und biophysikalischen Experimenten identifiziert. Durch RNA:DNA:DNA Triplexbildung reduziert endogenes HIF1α-AS1 die Expression mehrerer Gene, einschließlich des EPH- Rezeptors 2 (EPHA2) und Adrenomedullin (ADM), indem es als Adaptor für den repressiven human Silencing Hub (HUSH)-Komplex fungiert, der von den Arbeitsgruppen Leisegang und Brandes untersucht wurde. Die Triplexbildung zwischen HIF1α-AS1 und den Zielgenen EPHA2 und ADM wurde biochemisch und biophysikalisch untersucht. Die EMSA-Ergebnisse zeigten, dass HIF1α-AS1 einen RNA:DNA:DNA Triplex-Komplex mit reduzierter Mobilität mit der EPHA2 DNA-Zielsequenz bildet. Das CD Spektrum des Triplexes zeigte deutliche Unterschiede im Vergleich zur EPHA2 Duplex DNA und zum RNA:DNA Heteroduplex. Die Schmelzkurvenanalyse ergab einen zweiphasigen Schmelzübergang für Triplexe, wobei der erste Schmelzpunkt der Dissoziation des RNA-Stranges mit dem Schmelzen der Hoogsteen Wasserstoffbrücken entspricht. Der zweite, höhere Schmelzpunkt entspricht der stärkeren Watson-Crick Basenpaarung. Stabilisierte Triplexe wurden mit Hilfe eines EPHA2 intramolekularen Haarnadel DNA Duplex Konstrukts gebildet, bei dem beide DNA-Stränge an einen 5 Nukleotide langen Thymidin-Linker gebunden waren. Dieser Ansatz ermöglichte eine verbesserte Triplexbildung mit geringeren RNA-Äquivalenten und höheren Schmelztemperaturen. Mittels NMR Spektroskopie wurden charakteristische Triplex Signale im 1H-NMR Spektrum beobachtet, wobei die Imino Signale im Spektralbereich zwischen 9 und 12 ppm lagen, was auf die Hoogsteen Basenpaarung zurückzuführen ist. Zur Aufklärung der strukturellen und sequenzspezifischen Hoogsteen Basenpaare wurden 2D-1H,1H-NOESY Messungen des EPHA2 DNA Duplexes und des HIF1α-AS1:EPHA2 Triplexes im 10-fachen Überschuss durchgeführt. Im Imino Bereich des NOESY Spektrums wurde eine starke und eine mäßige Abschwächung der Intensitäten der Kreuzpeaks beobachtet. Als Ursache wurde die Abschwächung der Watson-Crick Basenpaarung durch die Hoogsteen Bindung vermutet, die durch die RNA-Bindung induziert wird. Die Hoogsteen Wechselwirkungen können durch die Analyse der Abschwächung der Kreuzpeaks in den NOESY Spektren dargestellt werden, was wiederum zur Erstellung eines Strukturmodells des RNA:DNA:DNA Triplexes verwendet wurde. Diese biophysikalischen Ergebnisse unterstützen die physiologische Funktion von HIF1α-AS1 als Triplex-bildende lncRNA, die den HUSH-epigenetischen Suppressor-Komplex an spezifische Zielgene wie EPHA2 und ADM rekrutiert und dadurch deren Genexpression durch RNA:DNA:DNA Triplexbildung stilllegt

Switching at the ribosome: riboswitches need rProteins as modulators to regulate translation

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation

Fendrr synergizes with Wnt signalling to regulate fibrosis related genes during lung development via its RNA:dsDNA triplex element

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis

1H, 13C, 15N and 31P chemical shift assignment for stem-loop 4 from the 5'-UTR of SARS-CoV-2

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy

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

1H, 13C and 15N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive H, C and N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy