Dichlorophenylpyridine-Based Molecules Inhibit Furin through anInduced-Fit Mechanism br

Inhibitors of the proprotein convertase furin mightserve as broad-spectrum antiviral therapeutics. High cellularpotency and antiviral activity against acute respiratory syndromecoronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we charac-terized the binding mechanism of this inhibitor class usingstructural, biophysical, and biochemical methods. We established aMALDI-TOF-MS-based furin activity assay, determined IC50values, and solved X-ray structures of (3,5-dichlorophenyl)-pyridine-derived compounds in complex with furin. The inhibitorsinduced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. Thesechanges formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into anewly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strongstructural stabilization in surface plasmon resonance and differential scanningfluorimetry experiments, respectively. The discoveredfurin conformation offers new opportunities for structure-based drug discovery.