Binding adaptation of GS 441524 diversifies macro domains and downregulate SARS CoV 2 de MARylation capacity

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host cell homoeostasis. One of these mechanisms is ADP ribosylation, a fundamental post translational modification PTM characterized by the addition of ADP ribose ADPr on substrates. Poly ADP ribose polymerases PARPs are implicated in this process and they perform ADP ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains MDs are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro D type class because of their properties to recognize and revert the ADP ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA dependent RNA polymerase RdRp . Herein, GS 441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS 441524 selectively modifies SARS CoV 2 MD de MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD GS 441524 complexes, using solution NMR and X ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS CoV 2 M

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

results of the German Conn´s Registry

Primärer Hyperaldosteronismus (PA) hat nachteilige Auswirkungen auf die Nierenfunktion, die aufgrund ihrer Ausprägung nicht allein durch die erhöhten Blutdruckwerte erklärbar sind. Bisher ist die Datenlage hinsichtlich primärem Hyperaldosteronismus und Nierenfunktion bzw. deren Entwicklung unter verschiedenen Therapiearten jedoch noch dürftig. Diese prospektive, multizentrische Studie des Deutschen Conn-Registers umfasste 29 Patienten mit neu diagnostiziertem PA vor und ein Jahr nach Beginn der Behandlung, sowie eine zweite Kohorte von 119 Patienten, die 5,3 und 6,8 Jahre nach Beginn der Behandlung untersucht wurden. Es wurden die glomeruläre Filtrationsrate (GFR), der Albumin-Kreatinin-Quotient im Spontanurin und andere biochemische und klinische Parameter erhoben. In einer größeren Querschnittsstudie wurde die Nierenfunktion abhängig von der Art der Behandlung ausgewertet (Adrenalektomie (ADX, n = 86), Spironolacton (n = 65) und Eplerenon (n = 18)). Die GFR und der Albumin-Kreatinin-Quotient sanken bei den neu diagnostizierten Patienten nach Beginn der Behandlung deutlich ab. In Kohorte 2 verblieben die GFR und der Albumin-Kreatinin-Quotient während des Beobachtungszeitraumes stabil, die Blutdruckwerte waren gut eingestellt. In der Querschnittsanalyse der Gesamtkohorte wurden keine Unterschiede bzgl. GFR und Albumin-Kreatinin- Quotient zwischen den PA Patienten mit unterschiedlichen Behandlungsschemata gesehen. Allerdings zeigten sich unter Eplerenonbehandlung niedrigere Kaliumwerte und eine höhere Anzahl benötigter Antihypertensiva. Zusammenfassend lässt sich sagen, dass die beobachtete Beeinträchtigung der Nierenfunktion und die Albuminurie der Patienten durch Beginn einer spezifischen Behandlung reversibel waren. Die konservative Therapie mit Spironolacton oder Eplerenon scheint bei ausreichend hoher Dosierung ebenso effektiv zu sein wie die Durchführung einer Adrenalektomie was die Blutdruckeinstellung sowie die Verbesserung der Nierenfunktion betrifft.Primary aldosteronism (PA) has deleterious effects on kidney function independent of blood pressure levels. Up to now, data on effectiveness of different PA therapies regarding renal function are scarce. This prospective multi-center study of the German Conn’s Registry included 29 patients with newly diagnosed PA evaluated before and 1 year after treatment initiation, and a second cohort including 119 patients who were evaluated 5.3 and 6.8 years after treatment initiation. Glomerular filtration rate (GFR), spot urine albumin excretion/urinary creatinine (UAE/Ucrea) ratio and other biochemical and clinical parameters were measured. In a larger cross-sectional cohort, renal function was evaluated depending on the type of treatment (adrenalectomy (ADX; n=86), spironolactone (n=65) or eplerenone (n=18)). GFR and UAE/Ucrea ratio significantly decreased in newly diagnosed PA patients after treatment initiation. In the second cohort, GFR and UAE/Ucrea ratio did not change during study period, and blood pressure was well controlled. In the larger cross-sectional cohort, no differences were seen in GFR and UAE/Ucrea ratio between PA patients on different treatment regimens. However, eplerenone treatment showed lower potassium levels and higher number of required antihypertensive medications. In conclusion, renal dysfunction with elevated albuminuria was seen in PA patients and was reversible after treatment initiation. Medical therapies with spironolactone or eplerenone seem to be as effective as ADX regarding renal function and blood pressure; however, sufficient daily doses need to be given

Study of the soils and survey of the microrelief in the western part of the Forest of Soignes (Belgium) : with special attention to the uprooting of trees and the suitability for tree plantations /

Proefschrift voorgelegd ter verkrijging van van het diploma van licentiaat in de bodemkund