TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Dorothea Bestle; Miriam Ruth Heindl; Hannah Limburg; Thuy Van Lam van; Oliver Pilgram; Hong Moulton; David A. Stein; Kornelia Hardes; Markus Eickmann; Olga Dolnik; Cornelius Rohde; Stephan Becker; Hans-Dieter Klenk; Wolfgang Garten; Torsten Steinmetzer; Eva Böttcher-Friebertshäuser

. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.042085 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.042085 doi: bioRxiv preprint co-transfected with pCAGGS-S-Myc-6xHis and either empty vector or pCAGGS-TMPRSS2. Cells were then incubated in the absence or presence of aprotinin or furin inhibitor MI-1851 (50 µM each) for 48 h. Cell lysates were subjected to SDS-PAGE and Western blot analysis using antibodies against the C-terminal Myc-tag. Lanes are spliced together from one immunoblot from one experiment. 1-4) . T-ex5 treated cells were inoculated with SARS-CoV-2 as described above and incubated in the absence of PPMO for 72 h (lane 4). Total RNA was isolated and analysed by RT-PCR using primers designed to amplify 1228 nt of full-length TMPRSS2-mRNA. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.042085 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.042085 doi: bioRxiv preprint inhibitors of TMPRSS2 (aprotinin, MI-432, MI-1900), furin (MI-1851), and endosomal cathepsins (E64d), respectively, or DMSO (0.5 %), at the indicated concentrations. At 16, 24, 48, and 72 h p.i., supernatants were collected, and virus replication was determined by TCID 50 titration at indicated time points. Data are mean values ± SD from two to four independent experiments. B) Effect of inhibitor treatment on cell viability. Calu-3 cells were treated with the indicated protease inhibitor (50 µM) for 72 h. Untreated cells (w/o) and DMSO treated cells were used as controls. Cell viability of untreated cells was set as 100 %. Results are mean values ± SD (n=3). C) Antiviral activity of combinations of TMPRSS2 and furin inhibitors against SARS-CoV-2 in human airway epithelial cells. Calu-3 cells were inoculated with SARS-CoV-2 at a MOI of 0.001 as described above and then incubated in the presence of single protease inhibitors or inhibitor combinations at the indicated concentrations. Virus titers in supernatants were determined by TCID 50 at 16, 24, 48 and 72 h p.i.. Data are mean values ± SD of two to three independent experiments. D) Calu-3 cells were treated with PPMO for 24 h, then infected with SARS-CoV-2 as described above and incubated in the absence of PPMO (w/o, scramble and T-ex5) and with or without 10 µM of furin inhibitor treatment (MI-1851) for 72 h. At 16, 24, 48, and 72 h p.i., supernatants were collected, and viral titers were determined by TCID 50 at indicated time points. Data are mean values ± SD (n=2).