Development of cell-permeable and plasma-stable peptidomimetic inhibitors against PSD-95/nNOS interaction as potential anti-ischemic stroke agents

The N-methyl-d-aspartate receptor (NMDAR)-neuronal nitric oxide synthase (nNOS)-postsynaptic density-95 (PSD-95) ternary complex represents a promising therapeutic target for acute ischemic stroke treatment. Although the shortest peptide ETAV targeting the PDZ2 domain of PSD-95 shows therapeutic potential, its limit in vivo stability restricts its broad application. Therefore, we herein systematically optimized and developed a series of novel peptidomimetics based on ETAV. We identified the optimal candidate compound 32-2, which displays potent neuroprotective effects in both HT22 cells (cell viability: 58.31 % at 10 μM) and primary cortical neurons (cell viability: 63.50 % at 20 μM). Mechanistically, 32-2 significantly reduced glutamate-induced intracellular ROS levels by 80 % compared to the glutamate-induced group, and modulated apoptotic markers (Bcl-2, Bax, cleaved-caspase 3). Furthermore, 32-2 exhibited potent plasma stability and excellent cellular permeability in vitro model. In the tMCAO model, 32-2 significantly reduced cerebral infarct volume compared to model group, being similar to NA-1. These findings establish 32-2 as the first effective peptidomimetic inhibitor of PSD-95 PDZ2 in vivo, offering a promising lead for developing novel stroke therapeutics.