Development and characterization of a selective chromatographic approach to the rapid discovery of ligands binding to muscarinic-3 acetylcholine receptor

The pursuit of new ligands binding to muscarinic-3 acetylcholine receptor (M 3 R) is viewed as challenging due to the lack of screening methods with high efficiency. To address such challenges, this work developed and characterized an approach to the rapid discovery of M 3 R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M 3 R. The fusion M 3 R was immobilized on 6-chlorocaproic acid-activated ammino-microspheres by the specific covalent reaction between the Halo-tag and the linker. Comprehensive characterizations of the immobilized M 3 R were performed by scanning electron microscope, X-ray photoelectron spectroscopy, and the investigation on the binding of three specific ligands to the receptor. The feasibility of the immobilized M 3 R in complex matrices was tested by screening the bioactive compounds in Zhisou oral liquid, assessing the interaction between the screened compounds and the receptor using zonal elution, and evaluating the in vivo activity of the targeted compounds. The results evidenced that the immobilized M 3 R has high specificity, good stability, and the capacity to separate M 3 R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of M 3 R. The association constants of the five compounds to M 3 R were 4.44 × 10 4 , 1.11 × 10 4 , 7.20 × 10 4 , 4.15 × 10 4 , and 3.36 × 10 4 M −1 . The synergistic application of the five compounds exhibited an equivalent expectorant activity to the original formula. We reasoned that the current method is possible to provide a highly efficient strategy for the discovery of receptor ligands.