The Highly Selective Discovery of Allosteric Ligands from Herbal Extracts through Their Direct Interactions with the Immobilized Headless CaSR Truncation.

Allosteric modulators represent a novel paradigm to therapeutically target G-protein-coupled receptors (GPCRs), but the discovery of novel allosteric ligands from natural products remains challenging. Here, we developed a high-performance affinity chromatography method for screening allosteric ligands toward the human calcium-sensing receptor (CaSR) by immobilizing an extracellular domain-deleted CaSR truncation (ΔCaSR) onto silica gels as solid-phase materials for column packing. The immobilized ΔCaSR column demonstrated the greatest allosteric responsive feature when cinacalcet at 0.50 μM or NPS2143 at 0.25 μM was included in the mobile phase, suggesting that the binding affinity of Ca(2+) was increased 8% by cinacalcet and was decreased 77% by NPS2143. The column was applied to screen allosteric ligands from Epimedii Folium, which were identified as epimedin B, epimedin C, and icariin using HPLC-MS. The allosteric binding of the screened compounds was testified through competitive experiments, and their allosteric effects were verified by CaSR downstream signaling events, like the intracellular Ca(2+) levels and cAMP production. Our observations indicated that the three compounds exerted an allosteric effect similar to that of cinacalcet and might be potential allosteric ligands. The proposed approach features the immobilization of headless GPCR truncations, in which the transmembrane domain is exposed to interact directly with the ligands, realizing the highly selective discovery of allosteric ligands from complex herbal extracts.