Development of immobilized beta1-adrenoceptor chromatography for rapid discovery of ligands specifically binding to the receptor from herbal extract.

The discovery of beta1-adrenoceptor (ß1-AR) ligands is viewed as an enormous demand for fighting ailments mediated by the receptor including cardiovascular diseases. Such pursuit is gravely challenged due to the lack of lead screening methods with high efficiency. This work developed a chromatographic method for pursuing ß1-AR ligand from the herbal extract by fusing epidermal growth factor receptor (EGFR) as a tag at its C-terminus to stably express the fusion receptor in E. coli, immobilizing the expressed EGFR-tagged ß1-AR onto ibrutinib-derivatized amino microspheres, and applying the immobilized receptor in the analysis of ligand-receptor interaction and herbal extract. Comprehensive characterizations like X-ray photoelectron spectroscopy and retention behaviors of canonical drugs demonstrated high specificity and good stability of the immobilized ß1-AR prepared through the covalent reaction between the EGFR and ibrutinib decorated on the microsphere surface. Frontal analysis of atenolol, metoprolol, and esmolol confirmed their bindings to ß1-AR with association constants of 1.07 × 104, 6.54 × 103, and 1.45 × 104 M-1. The thermodynamic analysis provided proof of electrostatic interaction, hydrogen bonds, and van der Waals force driving those interactions. Pulegone was recognized as a bioactive compound that specifically binding to ß1-AR from the extract of Ziziphora clinopodioides Lam by analyzing the retention peak through reverse-phase high performance liquid chromatography coupled with tandem mass spectrometry. These results, taken together, indicated that the current method is possible to provide an alternative for discovering ß1-AR ligands with high efficiency from complex matrices like herbal extract.

Screening of bioactive flavour compounds targeting muscarinic-3 acetylcholine receptor from Siraitia grosvenorii and evaluation of their synergistic anti-asthmatic activity.

Siraitia grosvenorii (Swingle) C. Jeffrey (SG) is widely used as a natural sweetener and traditional medicine for respiratory diseases. The anti-respiratory compounds in the plant and their mechanism remain elusive due to the lack of a high-throughput screening method. In this work, immobilization of the muscarinic-3 acetylcholine receptor (M3R) was used to establish an affinity chromatographic strategy for synchronously recognizing the flavour components in the SG extract binding to this receptor and evaluating their anti-asthmatic effect. The accuracy of the method was assessed by in vivo experiments. Mogroside V (Mog V) and 11-oxomogroside V (11-O MogV) were identified as functional flavour compounds binding to M3R. Their association constants were determined to be 3.32 × 104 and 2.40 × 104 M-1 by the injection amount-dependent method. The binding energies of the two compounds to M3R were calculated to be -80.52 and -48.20 kJ/mol by molecular dynamics simulation. The synergistic application of the two flavour compounds exhibited stronger anti-asthma activity than the original SG extract. These results indicated that immobilized M3R is a powerful alternative for the identification of flavour compounds in plants. Mog V and 11-O Mog V are the main functional flavour compounds contributing to SG's anti-asthma function. We reasoned that the two compounds have the potential to become functional food additives. This work has the possibility to contribute considerably to the pursuit of functional flavour compounds from natural plants in the field of functional food development.

Preparation and characterization of immobilized 5-HT1A receptor stationary phase for high throughput screening of the receptor-binding ligands from complex systems like Curcuma wenyujin Y. H. Chen et C. Ling extract.

The incidence of depression has increased significantly during the COVID-19 pandemic. This disease is closely associated with serotonin 1A (5-HT1A) receptor and often treated by complex prescription containing Curcuma wenyujin Y. H. Chen et C. Ling. Therefore, we hypothesized that this herb contains bioactive compounds specially binding to the receptor. However, the rapid discovery of new ligands of 5-HT1A receptor is still challenging due to the lack of efficient screening methods. To address this problem, we developed and characterized a novel approach for the rapid screening of ligands by using immobilized 5-HT1A receptor as the chromatographic stationary phase. Briefly, haloalkane dehalogenase was fused at the C-terminal of 5-HT1A receptor, and the modified 5-HT1A receptor was immobilized on amino-microspheres by the reaction between haloalkane dehalogenase and 6-chlorohexanoic acid linker. Scanning electron microscope and X-ray photo-electron were used to characterize the morphology and element of the immobilized receptor. The binding of three specific ligands to 5-HT1A receptor was investigated by two different methods. Moreover, we examined the feasibility of 5-HT1A receptor colume in high throughput screening of new ligands from complex systems as exemplified by Curcuma wenyujin Y. H. Chen et C. Ling. Gweicurculactone, 2-hydroxy-1-(3,4-dihydroxybenzene)-7-(4'-hydroxybezene)-heptane and curcuminol F were identified as the ligands of 5-HT1A receptor with the binding energies of -7.06 kcal/mol, -7.77 kcal/mol and -5.26 kcal/mol, respectively. Collectively, these results indicated that the immobilized 5-HT1A receptor was capable of screening bioactive compound from complex system, providing an effective methodology for high throughput screening.

Site-selective covalently immobilized alpha 1A adrenergic receptor for thermodynamic and extra-thermodynamic study of four ligands binding to the receptor by chromatographic methods.

Immobilized G protein-coupled receptor is a versatile tool to study ligand-receptor interactions. In this work, we synthesized the immobilized alpha 1A adrenergic receptor (α1A-AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α1A-AR and macroporous silica gel coated with 6-chlorohexanoic acid. To investigate thermodynamic and extra-thermodynamic parameters for ligand binding, we utilized the covalently immobilized receptor as stationary phase to perform frontal analysis and injection-amount dependent analysis as well as compared with the random immobilization method. Terazosin gave the association constant of 1.48 × 105 M-1 to α1A-AR, indicating that the oriented immobilization of α1A-AR enhances the ligand-binding activity by one order of magnitude in comparison with the random immobilization method (7.9 × 104 M-1). The binding of phentolamine and tamsulosin to the receptor was accompanied by a large absolute heat capacity (ΔCp) of 1.28 ± 0.23 kJ mol-1, demonstrating that the binding enthalpy and entropy appear to compensate for one another. These results indicated that the covalent immobilization of the receptor onto solid support has a profound impact on the ligand-binding activity of the receptor and the determination of ligand-receptor binding parameters. The receptor immobilized through the site-selective method will act as a benchmark for chromatographic determination of binding parameters in ligand-receptor interactions and can be used as an effective approach for rapid analysis of drug-protein interactions with high accuracy.

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 (M3R) 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 M3R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M3R. The fusion M3R 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 M3R 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 M3R 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 M3R has high specificity, good stability, and the capacity to separate M3R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of M3R. The association constants of the five compounds to M3R were 4.44 × 104, 1.11 × 104, 7.20 × 104, 4.15 × 104, and 3.36 × 104 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.

Rapidly identifying bioactive compounds from Zhisou oral liquid by immobilized receptor-based high-performance affinity chromatography.

The identification of bioactive compounds in complex matrices remains a major challenge due to the lack of highly efficient and specific methods. This work developed an approach based on high-performance affinity chromatography to identify the potential antitussive compounds from Zhisou oral liquid . The main methods include the synthesis of immobilized beta2-adrenoceptor by a one-step method, the screening and identification of the potential bioactive compounds by the receptor column coupled with mass spectrometry, and the binding mechanism analysis of the compounds to the receptor by the in vivo experiment, injection amount dependent method and molecular simulation. We identified the potential bioactive compounds of Zhisou oral liquid as glycyrrhizic acid, platycodin D, tuberostemonine, and hesperidin. In vivo experiment showed that the combinational utilization of the four compounds was possible to present an equivalent antitussive effect to the formula. The docking results demonstrated that hydrogen bonds and Van der Waals forces were the main forces to drive the binding of the four compounds to beta2-adrenoceptor. We concluded that the four compounds are the effective components in Zhisou oral liquid. The proposed strategy is possible to provide an alternative for the development of highly efficient methods to pursue the bioactive compounds of complex matrices.