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

The main protease of SARS-CoV-2 (M ), 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 . 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 and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k /K =37 500 m  s , K =24.0 nm) and pyridyl ester 17 (k /K =29 100 m  s , K =10.0 nm), promising drug candidates for further development have been discovered.