Identification, validation, and characterization of approved and investigational drugs interfering with the SARS‐CoV‐2 endoribonuclease Nsp15

FIGURE 1. Schematic overview of the workflow followed in this study.

FIGURE 2. Characterization of Nsp15 enzymatic parameters. (a) Determination of K m. 1 μM Nsp15 was incubated with increasing substrate concentration and signal was detected every minute for a maximum of 5 min after substrate addition. (b) Titration of Nsp15. The enzyme was incubated in increasing concentrations, with increasing concentrations of the substrate or buffer (0 nM substrate). (c) Tolerance of Nsp15 enzymatic activity to increasing DMSO concentration in the assay, using 0–5 v/v % DMSO in assay. (d) Effect of EDTA addition and absence of Mn2+ in assay buffer on Nsp15 enzymatic activity. (e) Inhibition of Nsp15 using Congo red, calculated IC50 7.5 μM. Each data point represents n = 3, error bars ±SD.

FIGURE 3. Primary screen (PS) and hit confirmation (HC) of the repurposing collection against SARS‐CoV‐2 Nsp15. (a) Frequency distribution of Z′ values calculated for screened assay plates. (b) Frequency distribution of calculated Nsp15 inhibition including the negative control/DMSO control and the positive control/100% inhibition (Congo red at 50 μM).

FIGURE 4. Structure of the found UNC compounds and their dose response curve series. Nsp15 was preincubated for 30 min with increasing concentrations of UNC‐compounds in the presence of 1 mM DTT or 1 mM l‐cysteine. Each data point represents N = 3, error bars ±SD.

FIGURE 5. (a) The Nsp15 hexamer in two orientations (rotated 90 degrees). Each Nsp15 subunit has a unique color. For one subunit, circled in dashed line, the three domains of the protein are shown (Nsp15 N‐terminal domain in raspberry, the middle domain in cyan and the EndoU in slate blue). (b) Domain organization and domain boundaries of Nsp15. The catalytic triad is in ball and stick. (c) DAMMIN fit (solid line) and the hexamer model (produced from PDB ID 6wlc, in green) overlaid to the ab‐initio model (gray beads) shows that Nsp15 is mostly hexameric in solution as well.

FIGURE 6. (a) Localization of two identified inhibitors YM‐155 (pink) and TAS‐103 (green) in the catalytic EndoU domain of Nsp15 (for domain color coding see Figure 5). (b) Calculated mFo‐DFc omit map for YM‐155 and TAS‐103 (contoured at a level of 3.0σ for YM‐155 and 2.5σ for TAS‐103, shown as orange mesh). (c) Electrostatic potentials for the catalytic domains of the YM‐155 and TAS‐103 structures.

FIGURE 7. (a) Close up on the interactions of YM‐155 with key residues of the EndoU. (b) The positioning of YM‐155 (in magenta) substantially narrows the uridine binding pocket.

FIGURE 8. Molecular docking on Nsp15 and the found inhibitors. Identified binding patches in the Nsp15 subunit (a) and in the Nsp15 hexamer (b).