RESUMO
A combinatorial method was devised and applied for the design and identification of substrate-analogue inhibitors of therapeutically relevant serine proteases, such as thrombin and factor Xa. We conceptualized imino acid derived diketomorpholines as generally applicable key intermediates prepared through solid-phase synthesis and prone to be cleaved with primary amines in a traceless fashion. The approach led to a compound library whose members were prepared under bioassay-compatible conditions and directly subjected to the in situ evaluation, allowing a fast prediction of hit compounds. Highly active inhibitors for serine proteases of the coagulation cascade have been identified. The most potent dual inhibitor, 16K, has a binding affinity of 23.9â¯nM to thrombin and 32.8â¯nM to factor Xa.
Assuntos
Fator Xa , Trombina , Fator Xa/química , Inibidores do Fator Xa , Serina Endopeptidases/metabolismo , Serina Proteases , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
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 cathepsinâ 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 500â m-1 s-1 , Ki =24.0â nm) and pyridyl ester 17 (kinac /Ki =29 100â m-1 s-1 , Ki =10.0â nm), promising drug candidates for further development have been discovered.