4‐Dimethylaminophenol: A ‘Smaller’‐Molecule PPI Inhibitor Targeting the PSD95 GK Domain Against Ischemic Stroke

Protein-protein interactions (PPIs) represent challenging yet promising therapeutic targets. This study identifies 4-dimethylaminophenol, an ultralow molecular weight compound (137 Da) from a fragment-based library, as a novel PPI inhibitor targeting the Guanylate Kinase-like (GK) domain of postsynaptic density protein 95 (PSD95). The direct binding of 4-dimethylaminophenol with PSD95 GK was confirmed by X-ray crystallography. Crucially, we uncovered a previously unknown PDZ-independent interaction between the PSD95 GK domain and neuronal nitric oxide synthase (nNOS). 4-Dimethylaminophenol inhibited this PSD95/nNOS interaction, reducing nitric oxide (NO) overproduction and neuronal excitotoxicity, thus exerted neuroprotective effects in vitro and in vivo. Moreover, structure-activity relationship (SAR) study showed that the phenolic hydroxyl and dimethylamino groups were crucial to the activity, and the introduction of ortho-halogen substitution could enhance binding affinity. Leveraging its established clinical application as a cyanide antidote, low molecular weight, blood-brain barrier permeability (BBB) and proven neuroprotection, 4-dimethylaminophenol presents a promising neuroprotective lead for drug discovery targeting ischemic stroke. The study also highlights the potential of the smaller molecules as PPI inhibitors, offering new insights into drug development of small-molecule PPIs.