Discovery of bioactive compounds targeting endothelin A receptor and angiotensin II type 1 receptor in Gegen Qinlian decoction by co-immobilized receptor chromatography

Abstract

An increased interest in multi-target compounds screening from complex matrices has revolutionized drug development paradigms, as these compounds often exhibit superior therapeutic efficacy and reduced off-target liabilities compared to the single-target compound. However, conventional discovery methods have been constrained by their singular focus on single-target screening methodologies. To address this limitation, we introduce a co-immobilization strategy tailored for G protein-coupled receptors(GPCRs) implicated in cardiovascular pathologies-specifically the endothelin A receptor (ET_AR) and angiotensin II type 1 receptor(AT₁R)-to facilitate the identification of multi-target compound within traditional herbal formulations. This innovative approach involves the oriented co-immobilization of ET_AR and AT₁R onto silica gel surfaces via histidine-tag anchoring, ensuring precise spatial orientation and functional integrity. Rigorous chromatographic characterization using receptor-specific ligands validated the dual-receptor column’s functionality. Subsequent screening of Gegen Qinlian Decoction (GQD) identified puerarin as a novel multi-target compound capable of engaging both the two receptors. Zonal elution revealed that puerarin competes with native ligands for overlapping binding sites on ET_AR and AT₁R. Injection-amount-dependent method, demonstrated that puerarin binds ET_AR and AT₁R with association constants of 2.5 × 10⁵ M⁻¹ and 2.0 × 10⁵ M⁻¹, respectively. These findings validate our co-immobilization platform as a powerful tool for dissecting multi-target interactions in traditional Chinese medicines (TCMs), offering a transformative strategy to unlock the therapeutic potential of complex herbal mixtures.