Targeting the Main Protease of SARS‐CoV‐2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors

Abstract The main protease of SARS‐CoV‐2 (M pro ), the causative agent of COVID‐19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M pro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in‐depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k inac /K i =37 500 m −1 s −1 , K i =24.0 n m ) and pyridyl ester 17 (k inac /K i =29 100 m −1 s −1 , K i =10.0 n m ), promising drug candidates for further development have been discovered.