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.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Farmacorresistência Viral/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/toxicidade , Linhagem Celular Tumoral , Chlorocebus aethiops , Dengue , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Descoberta de Drogas , Farmacorresistência Viral/efeitos dos fármacos , Hepacivirus/genética , Humanos , Mutação , RNA Viral/genética , Replicon/efeitos dos fármacos , Análise de Sequência de RNA , Bibliotecas de Moléculas Pequenas , Células VeroRESUMO
Cell-based high-throughput screening campaigns are widely used to identify novel antiviral compounds, for example, against human immunodeficiency virus type 1 (HIV-1). Typically, these assays enable identification of compounds that potentially target any viral or cellular factor involved in the viral replication cycle. Unraveling the mechanism of action of these active compounds is an important step to facilitate further drug development. Time-of-addition (TOA) assays are an elegant tool to achieve this goal by comparing the TOA profile of novel compounds with those of well-studied reference compounds. Downscaling to a 384-well format and automation significantly increase the capacity of the TOA assay, enabling compound handling around the clock. Mechanical liquid dispensing with optimized time points for compound addition ensures robustness (Z'>0.8) and maximal resolution in profiling novel antiviral compounds. The presented methodology has been optimized for routine use and allows for fully automated high-throughput screening to support the process in search for novel inhibitors of HIV-1.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Fármacos Anti-HIV/administração & dosagem , Automação , Bioensaio , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Apresentação de Dados , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Indicadores e Reagentes , Padrões de Referência , Reprodutibilidade dos Testes , Replicação Viral/efeitos dos fármacosRESUMO
Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.