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As the first and key step of traditional Chinese medicine (TCM)-guided drug development, lead discovery necessitates continuous exploration of new methodology for screening bioactive compounds from TCM. This work intends to establish a strategy for rapidly recognizing β2-adrenergic receptor (β2-AR) target compounds from the fruit of Siraitia grosvenorii (LHG). The method involved immobilization of β2-AR onto amino-microsphere to synthesize the receptor column, the combination of the column to high-performance liquid chromatography (HPLC) to screen bioactive compounds of LHG, the identification of the compounds by HPLC coupled with mass spectrometry (MS), and the evaluation of druggability through pharmacokinetic examination by HPLC–MS/MS. Mogroside V was screened and identified as the β2-AR-targeted bioactive compounds in LHG. This compound exhibited desired pharmacokinetic behavior including the time to reach peak plasma concentrations of 45 min, the relatively low elimination of 138.5 min, and the high bioavailability. These parameters indicated that mogroside V has a good druggability for the development of new drugs fighting β2-AR-mediated respiratory ailments like asthma. The combination of the methods in this work is probably becoming a powerful strategy for screening and early evaluating the bioactive compounds specifically binding to G-protein-coupled receptor target from complex matrices including TCM. [ABSTRACT FROM AUTHOR]

Owing to the promising clinical efficacy and relatively simple composition, Shuang-Huang-Lian prescription is widely prescribed for the treatment of acute upper respiratory tract infection and acute bronchitis in practice. This necessitates the understanding of the bioactive compounds of the prescription and their binding mechanism to β2-adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized β2-adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the β2-adrenoceptor column was identified as chlorogenic acid by using high-performance liquid chromatography coupled with ion trap mass spectrometry. Using an injection amount dependent method, chlorogenic acid proved the binding to β2-adrenoceptor through two kinds of sites. The numbers of the sites were (1.42 ± 0.03) × 10−8 and (9.06 ± 0.49) × 10−8 M. The association constants were (2.72 ± 0.01) × 105 and (2.80 ± 0.01) × 104 M−1, respectively. Molecular docking analysis of the interaction between chlorogenic acid and β2-adrenoceptor indicated that the binding mainly occurred on Ser169, Ser173, and Phe287 of β2-adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography. [ABSTRACT FROM AUTHOR]

β2-Adrenoceptor (β2-AR) was purified from the rabbit lung tissue by sepharose-salbutamol affinity chromatographic column. To prepare the β2-AR stationary phase, β2-AR was evenly immobilized on the surface of macro-pore silica with a mild chemical coupling method through covalent bond. The retention properties of β2-AR stationary phase were characterized by four ligands, salbutamol sulfate, noradrenaline bitartrate, adrenaline hydrochloride and propranolol hydrochloride, to establish the β2-AR affinity chromatography. Then, the method was used to screen the active compounds from the total extracts of Semen Armeniacae Amarum. The results showed that β2-AR on the surface of the stationary phase could keep its original bioactivity and selectivity. Amygdalin retained in the chromatographic column was proved to be the active compound of the total extracts of Semen Armeniacae Amarum. Compared with the existing chromatographic screening approaches, this method showed a good stability and high selectivity. The active compounds which could interact with β2-AR in traditional Chinese medicine (TCM) could be screened efficiently by this method, providing a new way to screen the active compounds in complicated samples such as TCM. [ABSTRACT FROM AUTHOR]

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 ( β 2 -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 β 2 -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 ( k d ) of salbutamol, terbutaline, methoxyphenamine, isoprenaline hydrochloride and ephedrine hydrochloride to β 2 -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 k d values for the five drugs bound to β 2 -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. [ABSTRACT FROM AUTHOR]

Beta2-adrenergic receptor (β 2 -AR) is believed as an attractive target for anti-asthmatic drugs. Its crystal structure and pharmacological activity have been clearly investigated. Yet the number of the approved anti-asthmatic drugs has declined in recent years. This work reports on the preparation of an immobilized β 2 -AR column through the specific trans-methylation reaction between SNAP tag and the benzyl-guanine derivative and application in anti-asthmatic compound screening from Raphani Semen. The characterization of the immobilized β 2 -AR was performed by scanning electron microscopy (SEM) and receptor-ligand interaction analysis by chromatographic methods. SEM analysis showed that the receptor has been successfully coated on the surface of PEGA amino microspheres. Binding constants of salbutamol and terbutaline calculated from frontal analysis within the temperature range of 10–30 ℃ confirmed the feasibility of the method in a thermodynamic viewpoint. Hydrogen bond was verified as the main driving force for drug-receptor interaction analysis. Sinapine was identified as the potential bioactive compound in Raphani Semen that specifically bind with β 2 -AR with a specific binding site of Ser 207. Taking together, the immobilized β 2 -AR column is promising in exploring drug-protein interaction analysis and anti-asthmatic drug screening. • SNAP was used for preparation of the immobilized β 2 -AR column. • Frontal analysis and perturbation peak method were used for the investigations. • Sinapine was screened in Raphani Semen targeting β 2 -AR. [ABSTRACT FROM AUTHOR]

Abstract: A highly selective assay was developed for screening compounds that bind to the porcine recombinant β2-adrenoceptor (β2-AR) with affinity chromatography coupled to quadrupole time-of-flight mass spectrometry (Q-TOF–MS). The methodology involved selective screening with immobilized β2-AR, a highly accurate identification via Q-TOF–MS, and a functional evaluation of the screened compounds with a sensitive myograph system. Ferulic acid, hydroxysafflor yellow A (HSYA), and naringin were confirmed to be the bioactive compounds in Huoxue capsule that specifically bound to the β2-AR. These compounds produced a concentration-dependent relaxation of arteries that were contracted by treatment with phenylephrine, and the relaxation caused by these compounds was attenuated in the presence of ICI 118551, a type of β2-AR antagonist. Our data indicate that the use of an immobilized receptor is potentially an alternative method for the rapid screening of bioactive compounds in a complex matrix because of its high specificity. β2-AR affinity chromatography was valuable in focusing attention on the further investigation of ferulic acid, HSYA, and naringin as β2-AR agonists. [Copyright &y& Elsevier]

[Display omitted] • Two immobilized GPCRs chromatography were prepared by a one-step method. • The immobilized GPCRs were applied to the screening of the DNA-encoded library. • The specific ligands were screened by the immobilized GPCRs chromatography. • The selected ligands have been evaluated in vitro and in vivo. The rapid growth of demands for drug discovery has necessitated the ongoing pursuit of new methods for specific ligands screening and identification. This work combined receptor-affinity chromatography (RAC) with high-throughput sequencing techniques to rapidly screen and identify the specific ligands. By this method, immobilized angiotensin II type I receptor (AT 1 R) and endothelin receptor A (ET A R) based on RAC were utilized for lead screening from a DNA-encoded library. The specific ligands of AT 1 R (ligand A 1 , A 2) and ET A R (ligand B 1 , B 2) were synthesized after decoding by high-throughput sequencing techniques. The dissociation rate constants (k d) of ligand A 1 , A 2 to AT 1 R and B 1 , B 2 to ET A R were 9.65 × 10−4, 31.1 × 10−4 and 0.66, 1.22 s−1 by peak profiling assay. The association constant (K A) to the receptors of four ligands was 5.4 × 106, 3.3 × 106 and 1.6 × 106, 2.2 × 105 by injection amount dependent method. The kinetic and thermodynamic parameters of the four specific ligands are similar to those of the positive drugs. This indicates that they are promising to drug candidates. The druggability of the four ligands through pharmacokinetic investigation by HPLC-MS/MS presented desired pharmacokinetic behavior including the fast absorption, the relatively slow elimination. These results, taking together, indicated that the RAC combined with high-throughput sequencing techniques can screen and identify the specific ligands according to various proteins, thus creating a general strategy for rapid discovery of promising drug candidates. [ABSTRACT FROM AUTHOR]

• Immobilized β 2 -adrenegic receptor (β 2 -AR) chromatography has been established by a trans-methylation reaction. • Bioactive compounds in Sanzi Yangqin Decoction targeting β 2 -AR were screened. • The pharmacodynamic effect of the screened bioactive compounds was evaluated by relaxation of the tracheal tissues in vitro. Screening bioactive compounds from traditional Chinese medicines plays pivotal role in preventing and curing diseases. Sanzi Yangqin Decoction (SYD) is a commonly used prescription for the treatment of cough, asthma and some other respiratory diseases for hundreds of years in practice. This reminds us that there may exist some bioactive compounds strongly binding with the recognized receptors mediating these diseases like β 2 -adrenegic receptor (β 2 -AR). Therefore, this work intends to screen bioactive compounds from SYD and revealed the binding mechanism by immobilized β 2 -AR chromatography and molecular docking. Taking advantages of a 3-high based enzymatic trans-methylation reaction (high speed, high specificity and high activity), the immobilization of β 2 -AR was successfully achieved. Representative chromatographic peaks of SYD on the immobilized β 2 -AR column was collected and recognized as rosmarinic acid and sinapine thiocyanate. Tension changes of the trachea ring showed that the two compounds were in a concentration-dependent manner when exerting their effects and the concentration ranges were 10−9-10−4 mol/L and 10−12−10−7 mol/L, respectively. Molecular docking revealed Ser203, Ser204, Ser207, Tyr316 and Asn312 were the main residues for the two compounds to bind with β 2 -AR. We concluded that the proposed method is becoming an alternative in rapid recognizing bioactive compounds from complex matrix. [ABSTRACT FROM AUTHOR]