Screening of selective serotonin reuptake inhibitors from Cyperus rotundus using immobilized neurotransmitter transporter affinity chromatography for antidepressant discovery.

Despite advances in targeting neurotransmitter transporters, developing selective serotonin reuptake inhibitors (SSRIs) remains crucial for treating psychiatric disorders. Here, we developed an innovative affinity chromatography platform to discover novel SSRIs from Cyperus rotundus (CR), utilizing immobilized serotonin transporter (SERT) as the capture probe. Our approach employed genetically encoded unnatural amino acids (UAAs) and a "thiol-ene" click reaction to create a stable, functional SERT stationary phase. This immobilized SERT chromatography demonstrated superior specificity and longevity, enabling reliable ligand-receptor interaction analysis and inhibitor screening. Validation studies confirmed the platform's accuracy, with ligand affinity rankings matching conventional radioimmunoassay results. Through this method, we identified α-cyperone as a potent SERT inhibitor from CR, exhibiting high binding affinity (6.97 × 10⁴ M^-1) and favorable drug-like properties. In vivo studies revealed that α-cyperone alleviated depression-like behaviors in chronic unpredictable mild stress (CUMS) mice by modulating SERT function-inhibiting SERT reuptake and increasing synaptic 5-HT and NE concentrations. These findings position α-cyperone as a promising natural SSRI candidate. Importantly, this immobilization strategy is broadly applicable to various proteins, offering an efficient platform for high-throughput screening of targeted therapeutics from natural products.