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

ABSTRACT 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.