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 (MR) 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 MR ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of MR. The fusion MR 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 MR 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 MR 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 MR has high specificity, good stability, and the capacity to separate MR ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of MR. The association constants of the five compounds to MR were 4.44 × 10, 1.11 × 10, 7.20 × 10, 4.15 × 10, and 3.36 × 10 M. 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.