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

The S1/S2 cleavage site of the novel emerged SARS-CoV-2 has been shown to possess a 145 minimal furin consensus motif of the sequence R-R-A-R↓ with an alanine instead of a basic 146 substrates possess a nonbasic residue in P2 position, such as Pseudomonas aeruginosa 148 exotoxin A or Shiga toxin (Rockwell et al., 2002; Garten, 2018) . To test, whether the S1/S2 149 sequence of SARS-CoV-2 S protein is efficiently cleaved by furin, a small series of 150 Fluorescence Resonance Energy Transfer (FRET) substrates was synthesized ( Fig. 2A ). All 151 compounds possess a 3-nitrotyrosine amide as P4ˈ residue and a 2-amino-benzoyl 152 fluorophore in P7 position. The analogous sequences of the S proteins from MERS-CoV, 153 SARS-CoV, and avian infectious bronchitis virus (IBV) strain Beaudette were prepared as 154 reference substrates. Moreover, two FRET substrates of the SARS-CoV-2 S1/S2 cleavage 155 site with P2 AK and AR mutations were synthesized, to evaluate whether they could 156 constitute even more efficient cleavage sites for furin than the wild-type. The FRET 2_M1, which contains an optimized furin recognition site by virtue of an AK mutation in P2 164 position, was cleaved with similar efficiency compared to the wild-type sequence. However, 165 substitution of AR in the P2 position strongly enhanced cleavage by furin. As expected, the 166 analogous reference sequence of IBV was also processed by furin very efficiently. The data 167