Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation.

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.

Discovery of JNJ-1802, a First-in-Class Pan-Serotype Dengue Virus NS4B Inhibitor.

Dengue is a global public health threat, with about half of the world's population at risk of contracting this mosquito-borne viral disease. Climate change, urbanization, and global travel accelerate the spread of dengue virus (DENV) to new areas, including southern parts of Europe and the US. Currently, no dengue-specific small-molecule antiviral for prophylaxis or treatment is available. Here, we report the discovery of JNJ-1802 as a potent, pan-serotype DENV inhibitor (EC50's ranging from 0.057 to 11 nM against the four DENV serotypes). The observed oral bioavailability of JNJ-1802 across preclinical species, its low clearance in human hepatocytes, the absence of major in vitro pharmacology safety alerts, and a dose-proportional increase in efficacy against DENV-2 infection in mice were all supportive of its selection as a development candidate against dengue. JNJ-1802 is being progressed in clinical studies for the prevention or treatment of dengue.

Multiplexed multicolor antiviral assay amenable for high-throughput research.

To curb viral epidemics and pandemics, antiviral drugs are needed with activity against entire genera or families of viruses. Here, we develop a cell-based multiplex antiviral assay for high-throughput screening against multiple viruses at once, as demonstrated by using three distantly related orthoflaviviruses: dengue, Japanese encephalitis and yellow fever virus. Each virus is tagged with a distinct fluorescent protein, enabling individual monitoring in cell culture through high-content imaging. Specific antisera and small-molecule inhibitors are employed to validate that multiplexing approach yields comparable inhibition profiles to single-virus infection assays. To facilitate downstream analysis, a kernel is developed to deconvolute and reduce the multidimensional quantitative data to three cartesian coordinates. The methodology is applicable to viruses from different families as exemplified by co-infections with chikungunya, parainfluenza and Bunyamwera viruses. The multiplex approach is expected to facilitate the discovery of broader-spectrum antivirals, as shown in a pilot screen of approximately 1200 drug-like small-molecules.