Die Hauptprotease von SARS‐CoV‐2 als Zielstruktur: Von der Etablierung eines Hochdurchsatz‐Screenings zum Design maßgeschneiderter Inhibitoren

Die Hauptprotease von SARS‐CoV‐2 (Mpro), dem Auslöser von COVID‐19, ist ein wichtiges Arzneistoff‐Target. Ein neues fluorogenes Substrat, das kinetisch mit einem intern gequenchten fluoreszierenden Peptid verglichen wurde, erwies sich als ideal geeignet für ein Hochdurchsatz‐Screening mit rekombinant exprimierter Mpro. Zwei Klassen von Protease‐Inhibitoren, Azanitrile und Pyridylester, wurden identifiziert, optimiert und biochemisch charakterisiert. Maßgeschneiderte Peptide mit einer reaktiven Azanitril‐Kopfgruppe zeigten eine duale Inhibition von Mpro und Cathepsin L, einer Protease, welche die virale Zellinvasion befördert. Zur Optimierung der Pyridylindolester wurde ein Positions‐Scanning durchgeführt. Unser fokussierter Ansatz zur Entwicklung von Mpro‐Inhibitoren erwies sich dem virtuellen Screening als überlegen. Mit den beiden irreversiblen Inhibitoren Azanitril 8 (kinac/Ki=37 500 m−1 s−1, Ki=24.0 nm) und Pyridylester 17 (kinac/Ki=29 100 m−1 s−1, Ki=10.0 nm) wurden vielversprechende Kandidaten für die zukünftige Arzneistoffentwicklung entdeckt.

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 (Mpro), 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 Mpro. 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 Mpro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac/Ki = 37,500 M-1s-1, Ki = 24.0 nM) and pyridyl ester 17 (kinac/Ki = 29,100 M-1s-1, Ki = 10.0 nM), promising drug candidates for further development have been discovered.

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 (Mpro ), 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 Mpro . 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 Mpro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac /Ki =37 500 m-1 s-1 , Ki =24.0 nm) and pyridyl ester 17 (kinac /Ki =29 100 m-1 s-1 , Ki =10.0 nm), promising drug candidates for further development have been discovered.