SARS-CoV-2 methyltransferase nsp10-16 in complex with natural and drug-like purine analogs for guiding structure-based drug discovery

Viviane Kremling
Sven Falke
Yaiza Fernández-García
Christiane Ehrt
Antonia Kiene
Bjarne Klopprogge
Emilie Scheer
Fabian Barthels
Philipp Middendorf
Sebastian Kühn
Stephan Günther
Matthias Rarey
Henry N Chapman
Dominik Oberthür
Janina Sprenger author has email address

Center for Free-Electron Laser Science CFEL, Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany;
Department of Virology, Bernhard-Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany;
Universität Hamburg, Center for Bioinformatics, Albert-Einstein-Ring 8-10, 22761 Hamburg, Germany
The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany;
Institute of Pharmaceutical and Biomedical Science (IPBS), Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany;
Department of Physics, Universität Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany

https://doi.org/10.7554/eLife.98310.1

Open access
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Figures and data

Nsp10-16 overview and active site architecture
Structural representation of nsp10-16 with nsp10 as blue and nsp16 as beige surface (based on PDB entry: 7JIB). A) The active site is highlighted with a black frame and shows the substrates SAM and Cap0-analog as stick models with carbon in gray, oxygen in red, sulfur in yellow, nitrogen in blue, and phosphate in orange. B) Schematic representation of the active site with ligands as stick models as in A). The relative positions of the gate loops and the sub division of the Cap0 and SAM site is shown as described in the text. The extended cavity at the SAM site extends from N7 at the adenosyl cavity.

Chemical structures of SAM-site binders.
The compounds on top represent adenosine (green labels) and on the bottom tubercidin derivatives (blue labels). Structures of SARS-CoV-2 nsp10-16 with SAM, SAH, Sinefungin, WZ16 and SS148 have been described previously. The protein complex structures of the remaining compounds are reported in this study. Chemical differences of the derivatives compared to tubercidin are highlighted with green/blue circles or ellipsoids.

Overview of interactions of ligands with nsp10-16.
A) Overlay of ligands (stick representation) bound to SAM binding site of nsp16 (cartoon representation). B) SAM site architecture adapted from figure 1 B). C) - M) interaction of different ligands with nsp16. The top of each panel shows the ligand poses in the crystal including possible interactions as yellow dotted lines. On the bottom, possible interactions as 2D-plots only including relevant residues without the protein backbone are shown. Plots were created and edited with PoseView and PoseEdit (https://proteins.plus, Schöning-Stierand et al. 2022; Diedrich et al. 2023). Complex structures with partial occupancy by SAM are marked with an asterisk. For clarity, SAM is not shown even if present in these crystal structure.

Overview of interactions of ligands with nsp10-16.
A) Overlay of ligands (stick representation) bound to SAM binding site of nsp16 (cartoon representation). B) SAM site architecture adapted from figure 1 B). C) - M) interaction of different ligands with nsp16. The top of each panel shows the ligand poses in the crystal including possible interactions as yellow dotted lines. On the bottom, possible interactions as 2D-plots only including relevant residues without the protein backbone are shown. Plots were created and edited with PoseView and PoseEdit (https://proteins.plus, Schöning-Stierand et al. 2022; Diedrich et al. 2023). Complex structures with partial occupancy by SAM are marked with an asterisk. For clarity, SAM is not shown even if present in these crystal structure.

Cap binding and gate loop motions.
A) Gate loop conformations for nsp16 with bound SAM (blue ribbon) in overlay with the SAM and Cap0-analog bound structure (green ribbon). SAM and Cap0-site positions are indicated by circles. B) Overlay of nsp10-16 structures with bound toyocamycin and Cap0-analog (yellow ribbon and sticks) with the structure resulting from soaking sangivamycin and Cap0-analog (pink ribbon and sticks). In the latter structure, SAM and SAH are present aside from sangivamycin and a fraction of the Cap0- analog was converted into Cap1. The orientation is identical to A). C) Interactions of Cap0-analog and toyocamycin (TO1) with nsp16. D) Interactions of SAH/sangivamycin (SGV) and Cap0/Cap1-analog with nsp16.

Effect of selected compounds on SARS-CoV-2 replication in Vero E6 cells.
The viral titers (half-solid circles) and cell viability (empty circles) were determined by immunofocus assays and the CCK-8 method, respectively. Individual data points represent means ± SD from three independent replicates in one experiment. Values below the methodological detection limit are shaded in gray. The dose-response curves were generated by fitting the data to the sigmoidal four parameter logistic function with 1/Y² weighting using GraphPad Prism version 9.5.1 for Mac OS X (GraphPad Software, San Diego, California USA, www.graphpad.com). The maximum values were fixed to 100 %, and the minimum values were constrained to be equal to or greater than the methodological detection limit. Remdesivir was used as positive control.

Cytotoxicity, in vitro antiviral activity, and selectivity of selected compounds against SARS-CoV-2.
CC₅₀- half-maximal cytotoxic concentration; EC₅₀- half-maximal effective concentration; SI- selectivity index. Cell viability and viral titers were determined by the CCK-8 method and immunofocus assays, respectively. Compounds that reduced infectious particles by at least a hundred-fold in combination with SI greater than five are considered antivirally active and are marked with an asterisk. Except for the SI, values were calculated from three independent replicates in one experiment by fitting the data to the sigmoidal four parameter logistic nonlinear regression function.

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