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

Our data indicate that furin cleaves at the S1/S2 site, whereas TMPRSS2 cleaves at the S2' 389 site. The effective processing of the S1/S2 site by furin was additionally confirmed by for furin cleavage at the S1/S2 site. Likewise, strong inhibition of SARS-CoV-2 replication by 397 knockdown of TMPRSS2 activity using T-ex5 PPMO or treatment of Calu-3 cells with 398 aprotinin, MI-432 and MI-1900, respectively, indicates that furin cannot compensate for the 399 lack of TMPRSS2 in S activation. This was further confirmed by using an analogous FRET 400 substrate derived from the S2' cleavage site of the SARS-CoV-2 S protein (Fig. S2) . Kinetic 401 measurements clearly revealed that this substrate cannot be cleaved by furin (Fig. S2) . Thus, 402