RESUMEN
West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Furina/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Virus del Nilo Occidental/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Virus del Dengue/enzimología , Virus del Dengue/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Furina/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/crecimiento & desarrolloRESUMEN
A series of cyclic active-site-directed inhibitors of the NS2B-NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue-4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d-lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α-amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with Ki values <5â nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin-like serine proteases and furin-like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure-activity relationships were observed for these enzymes, thus suggesting their potential application as pan-flaviviral protease inhibitors.
Asunto(s)
Compuestos Macrocíclicos/farmacología , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virus del Dengue/enzimología , Relación Dosis-Respuesta a Droga , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Virus Zika/enzimologíaRESUMEN
Zika virus NS2B-NS3 protease plays an essential role in viral replication by processing the viral polyprotein into individual proteins. The viral protease is therefore considered as an ideal antiviral drug target. To facilitate the development of protease inhibitors, we report three high-resolution co-crystal structures of bZiPro with peptidomimetic inhibitors composed of a P1-P4 segment and different P1' residues. Compounds 1 and 2 possess small P1' groups that are split off by bZiPro, which could be detected by mass spectrometry. On the other hand, the more potent compound 3 contains a bulky P1' benzylamide structure that is resistant to cleavage by bZiPro, demonstrating that presence of an uncleavable C-terminal cap contributes to a slightly improved inhibitory potency. The N-terminal phenylacetyl residue occupies a position above the P1 side chain and therefore stabilizes a horseshoe-like backbone conformation of the bound inhibitors. The P4 moieties show unique intra- and intermolecular interactions. Our work reports the detailed binding mode interactions of substrate-analogue inhibitors within the S4-S1' pockets and explains the preference of bZiPro for basic P1-P3 residues. These new structures of protease-inhibitor complexes will guide the design of more effective NS2B-NS3 protease inhibitors with improved potency and bioavailability.