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

Novel β-Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus

The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel α/β-fold formed by a mixed parallel/antiparallel six-stranded β-barrel, an α-helix covering one opening of the barrel, and a 3(10)-helix alongside the barrel. We further characterized the full-length 179-residue protein and show that the polypeptide segments of residues 1 to 12 and 129 to 179 are flexibly disordered. The structure is analyzed in a search for possible correlations with the recently reported activity of nsp1 in the degradation of mRNA

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

Highly efficient production of a Thermomyces lanuginosus IOC-4145 β-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 2(3) factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications

Characterization of BEX3.

<p>(A) SDS-PAGE of BEX3 before and after the addition of 4 or 7 M of urea to the sample loading buffer. The molecular weight marker (MW) is shown in kDa for selected proteins. * and ** indicate monomer and dimer of BEX3. (B) Association and dissociation of BEX3 from p75^NTR_DD in surface plasmon resonance studies. Individual sensorgrams were superimposed to show the interactions between BEX3 and p75^NTR_DD at the indicated concentrations. The calculated affinity for the BEX3/p75^NTR_DD-interaction was 0.55 μM. The BEX3 sample has been characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137916#pone.0137916.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137916#pone.0137916.s002" target="_blank">S2</a> Figs.</p

Structure of BEX3 probed by fluorescence.

<p>(A) Tryptophan intrinsic emission fluorescence spectra of 50 μM BEX3 were collected at the excitation wavelength of 280 nm. Center of spectral mass is indicated in nm. (B) Bis-ANS emission fluorescence spectra of 2 μM BEX3 were collected at the excitation wavelength of 360 nm. In both panels, the spectra were collected for the proteins in the absence (solid line) or presence (dotted line) of 7 M urea. The data were fit to a coarse lowess curve using GraphPad Prism v.6.</p

Biophysical Studies on BEX3, the p75^NTR-Associated Cell Death Executor, Reveal a High-Order Oligomer with Partially Folded Regions

<div><p>BEX3 (Brain Expressed X–linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55–120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our <i>in vitro</i> data.</p></div

Amino acid sequence of BEX3 highlighting its structural elements.

<p>PTN K: amino acid segments resistant to proteinase K. The residues that were predicted to be recognizable by proteinase K by the Peptide Cutter program (<a href="http://web.expasy.org/peptide_cutter" target="_blank">http://web.expasy.org/peptide_cutter</a>) are indicated in red. Δδ NMR: represent residues with significant chemical shift perturbation by urea. Dashes represent values that were not determined. SSP: Residues with > 10% <u>s</u>econdary <u>s</u>tructure <u>p</u>ropensity calculated from C^α, C^β and H^α chemical shifts. <i>R</i>_<i>2</i><i>/R</i>_<i>1</i>: NMR relaxation indicating residues with below-average conformational flexibility. PRED: Secondary structure prediction from amino acid sequence. CCR: <u>C</u>oiled-<u>c</u>oil p<u>r</u>ediction from amino acid sequence.</p