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

In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at 36 two different sites by host cell proteases, which therefore represent potential drug targets. In 37 the present study we investigated which host cell proteases activate the SARS-CoV-2 S 38 protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the 39 proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 40 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of 41 SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 42 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two 43 synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor 44 aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic 45 furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more 46 potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine 47 protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus 48