Identification of a Potent and Selective Small-Molecule Inhibitor Targeting the nNOS-PDZ Domain that Exhibits Rapid In Vivo Antidepressant Efficacy.

The PDZ domain of neuronal nitric oxide synthase (nNOS-PDZ) plays a crucial role in regulating serotonin signaling in the forebrain and has emerged as a promising target for developing rapid-acting antidepressants. Here, we report the identification of N24, a potent and selective small-molecule inhibitor of nNOS-PDZ. N24 induces a substantial thermal shift (ΔTm) of 5.44 °C in a differential scanning fluorimetry (DSF) assay and displays an IC50 of 0.76 ± 0.07 μM in a fluorescence polarization (FP) assay. The cocrystal structure of the nNOS-PDZ-N24 complex reveals key binding interactions. In vivo, N24 produces rapid, dose-dependent antidepressant effects in mouse models of depression induced by chronic unpredictable mild stress (CUMS) and chronic corticosterone treatment, with no evidence of addictive potential or motility-related side effects. Together, these results establish N24 as a potent and selective nNOS-PDZ inhibitor, providing a promising lead compound for the development of antidepressants.