Fig. 5: Mitoxantrone targets HS directly to inhibit SARS coronavirus entry.

a Mitoxantrone inhibits Spike-mediated entry of PP. ACE2-GFP HEK293 cells were transduced with SARS-Cov and SARS-CoV-2 PP for 48 h in the presence of Mitoxantrone as indicated. Error bars indicate SEM, n = 4. Cells treated without the virus were used to control drug cytotoxicity. b The subcellular distribution of Mitoxantrone. The scheme illustrates the experimental procedure. HEK293T cells were treated with 5 μM Mitoxantrone for 30 min at 37 °C before fractionation. The membrane pellet (P100) and the cytosol supernatant (S100) fractions were analyzed by immunoblotting and by spectrometry at the indicated wavelengths. Error bars indicate SEM, n = 3. c The membrane association of Mitoxantrone requires SLC35B2 (35B2). Cells of the indicated genotypes were treated with 5 μM Mitoxantrone for 30 min at 37 °C and then fractionated as in b. The absorbance of the P100 fractions was measured and the control sample at 680 nm was normalized to 1. Bottom panels show the cells stained with DAPI (blue) and GFP⁺ (green) to label DNA and HS (Green), respectively. Error bars indicate SEM, n = 3. d Mitoxantrone has a higher affinity for heparin and HS than chondroitin sulfate (CS). A₆₅₀ of Mitoxantrone (5 μM) mixed with the indicated oligosaccharides was determined. The decrease in absorbance after the addition of the oligosaccharides was plotted. Error bars indicate SEM, n = 2. e The chemical structures of Mitoxantrone (MTAN) and Banoxantrone (BANO). f Banoxantrone (BANO) has reduced antiviral activity. ACE2-GFP HEK293T cells were incubated with SARS-CoV-2 in the presence of the indicated drugs for 24 h before measuring the luciferase activity. Error bars indicate SEM, n = 4. g Mitoxantrone inhibits SARS-CoV-2 cell entry. ACE2-GFP HEK293T cells were pretreated with DMSO as a control or Mitoxantrone for 30 min before incubation with SARS-CoV-2 PP. Cells were fixed 3 h later and stained with anti-Spike antibodies (red) and DAPI (blue).