Development of a NanoBiT based High throughput screening assay for discovery of NOS1-NOS1AP interaction inhibitors

Abstract The interaction between neuronal nitric oxide synthase (NOS1) and its adaptor protein NOS1AP (CAPON) plays a critical role in various neurological processes and has been implicated in cardiovascular and neuropsychiatric disorders. Disruption of this protein-protein interaction represents a potential therapeutic strategy, yet identifying small molecule inhibitors has been challenging. Here, we present the development and validation of a NanoBiT-based luminescence complementation assay optimized for high-throughput screening (HTS) of NOS1-NOS1AP interaction inhibitors. We engineered NOS1 and NOS1AP fusion proteins with HiBiT and LgBiT complementary subunits, respectively, and established stable CHO-K1 cell lines for robust signal generation. The assay demonstrated excellent performance characteristics with a signal-to-background ratio exceeding 240-fold, and was validated using TAT-GESV, a known peptide inhibitor that showed time- and dose-dependent inhibition. We successfully screened a diverse library of 10,240 compounds and identified 19 validated hits with IC₅₀ values ranging from 2.54 to greater than 30 μM, with the majority exhibiting IC₅₀ values below 30 μM. The top three compounds exhibited potent inhibitory activity with IC₅₀ values of less than 5 μM. This NanoBiT-based assay provides a reliable and efficient platform for discovering novel NOS1-NOS1AP interaction inhibitors and can be adapted for other protein-protein interaction studies.