Targeting Nipah virus replication: A fluorescence polarization assay for high-throughput modulators screening against...

Nipah virus (NiV) is a biosafety level 4 pathogen for which no licensed antiviral or vaccine is currently available. Given its pandemic potential and the serious threat it poses to human health, there is an urgent need for robust assays enabling the screening of chemical libraries in search of antiviral candidates. Here, we report the development of a high-throughput competitive fluorescence polarization assay designed to screen compound libraries and identify modulators of the NiV N_TAIL-P_XD interaction. Using an initial minimal setup, we successfully titrated and displaced this interaction with a nonlabeled peptide serving as a reference competitor. To enhance both assay window and dynamic range, we engineered a thioredoxin-XD fusion construct. We then assessed the assay's tolerance to dimethyl sulfoxide and found that the interaction remained stable at concentrations as high as 10%. Benchmarking of the final assay conditions using a screening-scale Z'-factor calculation yielded an excellent mean value of 0.8. Finally, we applied this optimized assay to screen a protein-protein interaction-focused chemical library of more than 10,000 compounds, resulting in a primary hit rate of 0.81%, encompassing both stabilizers and inhibitors. This assay expands the drug-discovery toolkit available against NiV and holds promise for adaptation to other Henipaviruses and, more broadly, to paramyxoviruses.