Discovery of novel pyrrolidine-2,5-dione scaffold PICK1 PDZ inhibitors as anti-ischemic stroke agents.

Targeting PICK1 presents a promising therapeutic strategy for ischemic stroke. Through multi-level virtual screening, we identified a pyrrolidin-2-one-based hit 3a. Its optimized derivative, 6b, demonstrated potent PICK1 PDZ binding affinity (Ki = 27.73 μM) and robust neuroprotection in glutamate-induced HT22 cell and primary neuron models, improving cell survival. Mechanistically, 6b attenuated ROS production and significantly modulated apoptosis-related proteins, downregulating the levels of pro-apoptotic factors Cleaved-caspase-3 and Bax, while upregulating the anti-apoptotic protein Bcl-2. Critically, siRNA-mediated knockdown of PICK1 completely abolished the neuroprotective effects of 6b, confirming that its action is explicitly mediated through PICK1 inhibition. Furthermore, 6b exhibited high membrane permeability and, in a middle cerebral artery occlusion model, significantly reduced the cerebral infarct area by 32.51 %. Collectively, our findings underscore compound 6b as a highly promising, novel neuroprotective agent for ischemic stroke treatment.