RESUMO
A recombinant form of yellow fever virus (YFV) NS3 protease, linked via a nonapeptide to the minimal NS2B co-factor sequence (CF40-gly-NS3pro190), was expressed in Escherichia coli and shown to be catalytically active. It efficiently cleaved the fluorogenic tetrapeptide substrate Bz-norleucine-lysine-arginine-arginine-AMC, which was previously optimized for dengue virus NS2B/3 protease. A series of small peptidic inhibitors based on this substrate sequence readily inhibited its enzymic activity. To understand the structure-activity relationship of the inhibitors, they were docked into a homology model of the YFV NS2B/NS3 protease structure. The results revealed that the P1 and P2 positions are most important for inhibitor binding, whilst the P3 and P4 positions have much less effect. These findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.
Assuntos
Inibidores Enzimáticos/farmacologia , Oligopeptídeos/metabolismo , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Vírus da Febre Amarela/enzimologia , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação/fisiologia , Cinética , Dados de Sequência Molecular , Oligopeptídeos/química , RNA Helicases/química , RNA Helicases/efeitos dos fármacos , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.
