Figure 1. In silico screening identified NSP16 inhibitors with potent anti‐SARS‐CoV‐2 activity.

• A
  Schematic representation of the target‐based antiviral drug discovery pipeline employed herein. A total of 4,991 chemical compounds from DrugBank were docked to the SAM‐binding pocket in the crystal structure of SARS‐CoV‐2 NSP16 (PDB ID: 6W4H), obtaining 14 commercially available compounds with high docking score that were used, along a control, in an in vitro antiviral assay. UMAP dimensionality reduction according to MACCS structural keys. Plots depict structural diversity of the shortlisted compounds alongside the compounds used in the in silico screen (contour lines), overlaid on the top of density distribution of ~800,000 bioactive small molecules (Duran‐Frigola et al, 2020).
• B
  Docking score from the in silico screen, depicted for all (black) and shortlisted (beige, table) compounds (full list provided in Dataset EV1).
• C
  Results of in vitro antiviral assay for compounds according to (B). A549‐ACE2 cells were pretreated with indicated compounds at 1 μM concentration for 3 h prior to infection with SARS‐CoV‐2 at MOI 0.01. Twenty‐four hours post‐infection, expression of SARS‐CoV‐2 transcript encoding envelope protein (E) was quantified by RT–qPCR as a measure of SARS‐CoV‐2 replication and is shown as a percent of vehicle‐treated control. NA, not assayed.
• D
  The docking poses of SAM (top) or tubercidin (bottom) in the SAM‐binding pocket of SARS‐CoV‐2 NSP10/16 (PDB 6W4H).
• E
  Disintegrations per minute (DPM 3H) originating from in vitro‐transcribed cap0 RNA methylated by the NSP10/16 complex (left) or Vaccinia virus VP39 (right) with optional addition of 10 mM tubercidin. SAM[³H] was provided as a substrate. Error bars correspond to mean ± SD of three reaction replicates; statistics were calculated using Student's two‐sided t‐test between indicated conditions.
• F
  A549‐ACE2 or control A549‐Venus cells were pretreated with tubercidin or vehicle (DMSO) 3 h prior to infection with SARS‐CoV‐2 at MOI 0.1. After 24 h, the abundance of SARS‐CoV‐2 nucleoprotein (N), ACE2, Venus, and β‐actin (ACTB, loading control) was visualized using Western blotting. The presented data are representative of three independent repeats.
• G
  A549‐ACE2 cells were pretreated with tubercidin or vehicle (DMSO) 3 h prior to infection with SARS‐CoV (MOI 0.01) or SARS‐CoV‐2 (MOI 0.1). After 24 h, protein content of the cells was isolated and subjected to LC–MS/MS‐based proteomics analysis. Label‐free quantification (LFQ)‐based abundance of detected viral proteins is depicted.
• H
  A549‐ACE2 cells were pretreated with tubercidin or vehicle (DMSO) 3 h prior to infection with SARS‐CoV‐2 at MOI 0.01. At 1 h post‐infection, medium change was performed. At the indicated days post‐infection, infectious viral progeny was quantified in the supernatants from three independently infected wells by plaque assay on Vero cells. ND, not detected. The measurements are representative of two independent repeats.
• I
  A549‐ACE2 cells were pretreated with tubercidin or vehicle (DMSO) 3 h prior to infection with indicated strains of SARS‐CoV‐2 at MOI 0.01. At 24 h post‐infection, relative expression of SARS‐CoV‐2 E was quantified by RT–qPCR. Error bars correspond to mean ± SD of n = 3 independently infected wells, and the measurements are representative of two independent repeats; statistics were calculated using Student's two‐sided t‐test between indicated conditions.

Source data are available online for this figure.