RESUMO
Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle.
Assuntos
Antivirais , Vírus da Dengue , Dengue , Proteínas não Estruturais Virais , Replicação Viral , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Humanos , Dengue/virologia , Dengue/tratamento farmacológico , Sorogrupo , RNA Helicases/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Ligação Proteica , Animais , Organelas/metabolismo , Organelas/efeitos dos fármacos , Proteases Virais , Aminofenóis , Proteínas de Membrana , Indóis , RNA Helicases DEAD-box , Nucleosídeo-Trifosfatase , ButiratosRESUMO
Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.