Kaur, B.; Abdo, A.; Cho, S.; Gabr, M.

NOS1AP is an adaptor for neuronal nitric oxide synthase that engages several partner proteins and is implicated in Alzheimer's disease. Efforts to drug NOS1AP have sought to disrupt these; we instead report pharmacological stabilization of one interaction. Affinity selection of a 1.4-billion-member DNA-encoded library identified a benzimidazole-5-carboxamide chemotype, optimized by fragment-directed SAR to NOSTRA with submicromolar NOS1AP binding affinity. Of six interactions, NOSTRA enhanced only SCRIB binding, with a cooperativity factor of 30, predominantly through a 14-fold slowing of dissociation; native mass spectrometry resolved a NOS1AP:NOSTRA:SCRIB ternary complex. In healthy human iPSC-derived cortical neurons, NOSTRA engaged the endogenous complex and activated Rac1 without cytotoxicity; in Alzheimer's patient-derived neurons, it lowered the A{beta}42/A{beta}40 ratio and p-tau217, reduced neurofilament light and improved network activity. With an in vitro ADME profile consistent with CNS exposure, NOSTRA is the first molecular glue for a NOS1AP complex, establishing interaction stabilization as a viable NOS1AP pharmacology.