Engineered Subcellular Transmembrane Protein Biomimetic Platform for In Situ Drug Discovery

ABSTRACT Subcellular membrane environments pose major challenges to target‐oriented drug discovery due to the structural complexity and dynamic conformational transitions of transmembrane proteins. Conventional affinity chromatography techniques are limited by prolonged preparation period, lack of binding‐site resolution, and interference from endogenous membrane proteins. This study introduces i n situ synthesized t ransmembrane‐protein a ffinity chromatography (iSTAC), a biomimetic platform integrating cell‐free protein synthesis, engineered silica‐phase modification, and tethered lipid bilayers to reconstitute functional multipass transmembrane receptors under native‐like conditions. iSTAC enables the rapid column preparation within 5 h (a reduction in time of 33.6‐fold), while preserving structural integrity and conformational dynamics. This facilitates high‐specificity drug screening and binding‐site analysis. We used the 5 hydroxytryptamine receptor 1A (5‐HT 1A ) as a model to identify two natural agonists, crocin I and crocin II, from herbal extracts and validated their anti‐insomnia and neuroprotective effects in vivo. Mutational iSTAC analysis precisely mapped their binding interactions at the residues N386, T196, and S199, demonstrating that both compounds activate 5‐HT 1A via canonical signaling pathways.