An orthoflavivirus inhibitor targeting multifunctional NS2A protein, a previously unidentified target.

Orthoflaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting orthoflaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlight the urgent need for a broad-spectrum antiviral agent targeting orthoflaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against orthoflaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A protein as a novel therapeutic target. Mechanistically, JNJ-1953 inhibits viral RNA synthesis, as demonstrated by robust reductions in intracellular viral RNA and infectious virus production. Additional experiments show that JNJ-1953 further impacts viral RNA packaging and interferes with the interaction between NS2A and prM, rendering the molecule a multimodal inhibitor.