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

Characterization of Nd and Sm Co-Doped CeO2-Based Systems

Nd0.20SmxCe0.8-xO1.9-x = 2 (x = 0, 0.05, 0.10, 0.15, 0.20) rare-earth-co-doped ceria electrolytes were synthesized by polyol process. Acetate compounds of cerium and dopants (Nd, Sm) were used as starting materials and triethylene glycol was used as a solvent. Structural and ionic conductivity properties of the electrolyte systems were determined by applying characterization techniques such as X-ray diffraction, the Fourier transform infrared spectroscopy, scanning electron microscope, and electrochemical impedance spectroscopy. The results of X-ray diffraction indicated that a single-phase fluorite structure formed at the relatively low calcination temperature of 600 degrees C. So, the samples were calcined at 600 degrees C for 4 h and then sintered at 1400 degrees C for 6 h to obtain dense ceramics (between 85 and 90%). The two-probe ac impedance spectroscopy was used to study the total ionic conductivity of doped and co-doped ceria samples. The results of the impedance spectroscopy indicate that the Nd0.20Sm0.05Ce0.75O1.875 composition exhibited highest ionic conductivity value, 3.60 x 10(-2) S cm(-1) at 800 degrees C

Synthesis and Characterization of Ferromagnetic Nickel Nanoparticles

Nickel nanoparticles on mass production scale have been prepared using a modified polyol process with Ni(CH3COO)(2)a <...4H(2)O, NaOH, 1,2 propandiol and hydrazinium hydroxide (N(2)H(4)auH(2)O). A mixture of face centered cubic (fcc) metallic nickel nanoparticles with 12 nm diameter were obtained. We have experimentally studied the structure of nanoparticle by X-ray and Scanning Electron Microscopy (SEM, EDS). The magnetic properties of the prepared Ni film have been studied by Ferromagnetic Resonance (FMR) technique, Vibrating Sample Magnetometer (VSM) and Vector Network Analyzer (VNA). The effective g-factor and magnetic anisotropy constant were determined as g (eff)=2.25 and K (eff)=85 Oe, respectively

Synthesis and characterization of CuFe2O4 nanorods synthesized by polyol route

Uniform, high quality, CuFe2O4 nanorods with high aspect ratios were synthesized by a surfactant-free single step polyol process at 220 degrees C. The structure of the product was characterized by XRD and FT-IR, and the morphology of the product was analyzed by SEM. The results showed that the as-prepared nanorods have a uniform cross-section and with average diameter of similar to 100 nm and aspect ratio in the range of 13-52. X-ray line profile fitting resulted in crystallite size of 15 nm, which reveals the polycrystalline nature of these nanorods. Magnetic characterization of product was performed by EPR and VSM techniques and the results show that the CuFe2O4 nanorods are ferromagnetic. The line width of the resonance lines in FMR is about 1.8 kOe which may originate from different resonance fields of randomly distributed nanocrystals which have different orientation of magnetic easy axes. (C) 2009 Elsevier B.V. All rights reserved

The effect of neutralizing agent on the synthesis and characterization of Mn3O4 nanoparticles

We report on the synthesis of Mn3O4 nanoparticles via a two-step hydrothermal route by using Mn(CH3COO)(2) as the only starting material and TMAOH and NaOH as hydrolysing agents. XRD and FT-IR analyses confirmed the composition and structure of Mn3O4. TEM images showed that spheroid Mn3O4 nanostructures obtained by this method have average particle size of 6 and 14 nm for NaOH and TMAOH hydrolyzed samples respectively. Particle size analysis indicated a strong aggregation of nanoparticles and exhibited bi-modal distribution with average size of aggregates as similar to 250 nm and 1.1 mu m for both samples. Zeta potential analysis revealed adsorbed TMAOH species on the surface of Mn3O4 nanoparticles hydrolyzed using TMAOH. ESR analyses resulted in broader lines and smaller g values than bulk Mn3O4 nanoparticles, probably due to the exchange-coupled system with unlike spins such as canted spin at surface of high-surface-disordered nanoparticles

1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication