Rapid screening and identification of bioactive compounds specifically binding to beta 2-adrenoceptor from San-ao decoction using affinity magnetic fine particles coupled with high-performance liquid chromatography-mass spectrometry.

BACKGROUND: San-ao decoction (SAD) has been widely used in Chinese medicine against respiratory diseases, such as asthma and rhinallergosis. The bioactive compounds for such pharmacological action remain unknown. METHODS: We developed a methodology to isolate the bioactive compounds of SAD. The assay involved the immobilization of beta 2-adrenoceptor (ß 2-AR) onto magnetic fine particles, the capture of target compounds by the immobilized receptor, the identification of the receptor bound compounds by reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS: Vicenin, shaftoside, isoshaftoside, liquiritin apioside and isoliquiritin apioside were identified as ß 2-AR ligands in SAD extract. The binding of these compounds to ß 2-AR occurred on serine169, serine170 and phenylalanine256 of the receptor. CONCLUSIONS: The developed methodology has high stability and specificity for recognizing and isolating target compounds. It is an alternative method for rapidly screening bioactive compounds of immobilized receptor from Chinese prescriptions.

Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution.

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (ß2-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized ß2-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized ß2-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for ß2-AR ligand screening based on the loss of breakthrough time of an indicator drug caused by the inclusion of competitive drugs in the mobile phase. We concluded that the current strategy enables the reliable and accurate analysis of G protein-coupled receptor (GPCR)-drug interaction. The percentage change in the breakthrough time for drugs can provide useful information for estimating their binding affinity to the receptor. This approach builds a powerful platform for high-throughput ligand screening.

Tanshinol borneol ester on nanostructured lipid carriers has longer brain and systemic effector retention and better antioxidant activity in vivo.

BACKGROUND: Tanshinol borneol ester (DBZ) is a hybrid of danshensu (DSS) and borneol and has anti-ischemic activity in animals. However, its low water solubility and short half-life limit its clinical application. METHODS: We prepared polyethylene glycol (PEG)-modified and DBZ-loaded nanostructured lipid carriers (DBZ-PEG-NLC) and DBZ-NLC, and examined their physical characteristics, such as particle size, zeta potential, entrapment efficiency and drug loading. The in vitro stability and pharmacokinetics in rats as well as antioxidant activity of DBZ-PEG-NLC and DBZ-NLC in a C57BL/6 mouse model of ischemia/reperfusion-related brain injury were investigated. The levels of DBZ and its hydrolyzed DSS in rat plasma and brain microdialysates were determined by liquid chromatography-mass spectroscopy/mass spectroscopy analysis. RESULTS: We found that the mean particle size and entrapment efficacy of DBZ-PEG-NLC were similar to that of DBZ-NLC. The DBZ-PEG-NLC, like DBZ-NLC, released DBZ in a biphasic manner with initially burst release and then prolonged slow release in vitro. Intravenous injection of DBZ-PEG-NLC resulted in significantly higher levels and longer retention periods of DBZ and DSS in plasma and the brains than DBZ-NLC and DBZ in rats. Finally, treatment with DBZ-PEG-NLC achieved a better antioxidant activity than DBZ or DBZ-NLC in mouse model of ischemia/reperfusion by reducing the levels of brain malondialdehyde, but increasing the levels of brain superoxide dismutase and glutathione. CONCLUSION: DBZ-PEG-NLC is a preferable option to deliver DBZ for sustainable release of DSS and borneol in vivo, and may serve as a promising drug for effective therapy of ischemic cardiovascular and cerebrovascular diseases.

Binding kinetics of five drugs to beta2-adrenoceptor using peak profiling method and nonlinear chromatography.

Investigations of drug-protein interactions have advanced our knowledge of ways to design more rational drugs. In addition to extensive thermodynamic studies, ongoing works are needed to enhance the exploration of drug-protein binding kinetics. In this work, the beta2-adrenoceptor (ß2-AR) was immobilized on N, N'-carbonyldiimidazole activated amino polystyrene microspheres to prepare an affinity column (4.6 mm × 5.0 cm, 8 µm). The ß2-AR column was utilized to determine the binding kinetics of five drugs to the receptor. Introducing peak profiling method into this receptor chromatographic analysis, we determined the dissociation rate constants (kd) of salbutamol, terbutaline, methoxyphenamine, isoprenaline hydrochloride and ephedrine hydrochloride to ß2-AR to be 15 (±1), 22 (±1), 3.3 (±0.2), 2.3 (±0.2) and 2.1 (±0.1) s-1, respectively. The employment of nonlinear chromatography (NLC) in this case exhibited the same rank order of kd values for the five drugs bound to ß2-AR. We confirmed that both the peak profiling method and NLC were capable of routine measurement of receptor-drug binding kinetics. Compared with the peak profiling method, NLC was advantageous in the simultaneous assessment of the kinetic and apparent thermodynamic parameters. It will become a powerful method for high throughput drug-receptor interaction analysis.