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-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 nm) and pyridyl ester 17 (k(inac)/K-i=29 100 m(-1) s(-1), K-i=10.0 nm), promising drug candidates for further development have been discovered.