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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. [ABSTRACT FROM AUTHOR]

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10-10 M and (1.39 ± 0.12) × 109 M-1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 104/ m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 n m. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug-protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Highlights: [•] We constructed a novel model for revealing drug–protein interactions. [•] Validated application of the model is performed two proteins as probes. [•] The model is drug-saving and rapid comparing with batch affinity assays. [Copyright &y& Elsevier]

A new oriented method using a diazonium salt reaction was developed for linking β2-adrenoceptor (β2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10−4 M. Thermodynamic studies showed that the binding of the two compounds to β2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β2-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs. [ABSTRACT FROM AUTHOR]

Self-competitive displacement is used to examine changes in the association constant and the binding site for berberine binding to immobilized β2-adrenoceptor (β2-AR) at different temperatures and using a varying mobile phase. The compound was confirmed to have a single kind of binding site to β2-AR under all the tested conditions. At 298.15 K, the association constant and the binding site are (2.12 ± 0.02) × 104 M−1and (1.56 ± 0.02) × 102 M. Thermodynamic investigation indicates that the binding of berberine to β2-AR has a positive change in energy due to enthalpy and negative response to entropy changes. Accordingly, this interaction is thought to be an endothermal process with entropy increase. The binding of berberine to the receptor was found to be more sensitive to ionic strength than pH. The placing of 1-propanol up to 5.0% in the mobile phases generates an 18% decrease in retention for berberine. These results indicate that the immobilized receptor would probably be an alternative for exploring the binding mechanism of ligand and receptor. [ABSTRACT FROM AUTHOR]

Abstract: A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β 2-adrenoceptor (β 2-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β 2-AR affinity column. The retention compounds eluted from the β 2-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β 2-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β 2-AR. Their association constants were (2.28±0.11)×104/M and (3.00±0.10)×104/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β 2-AR, and the corresponding association constants were (2.20±0.09)×10−4/M and (6.78±0.001)×105/M. [Copyright &y& Elsevier]

The study of protein-drug interaction plays a crucial role in understanding drug mechanisms, identifying new drug targets and biomarkers, and facilitating drug development and disease treatment. In recent years, significant progress has been made in various protein-drug interaction research methods due to the rapid development and in-depth application of mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and other technologies. The progress has enhanced the sensitivity, precision, accuracy, and applicability of analytical methods, enabling the establishment of drug-protein interaction networks. This review discusses various emerging research methods, such as native mass spectrometry, infrared spectroscopy, nuclear magnetic resonance and spectrum, biosensor technologies employing surface enhanced Raman, electrochemistry, and magneto resistive signals, as well as affinity magnetic levitation and affinity chromatography. The article also delves into the principles, applications, advantages, and limitations of these technologies. • Up-to-date overview of emerging methods for protein-drug interactions. • Overview of the principles and applications of interaction methods based on different detection technologies. • Examination of advantages and disadvantages of each interaction method. • Future development direction for protein-drug interaction research. [ABSTRACT FROM AUTHOR]

Immobilized proteins hold promise as the basic units that have enabled a broad range of analytical applications within chemical measurement science. As yet, the co-immobilization of diverse proteins at precise ratio and whether they give rise to improved analytical performance remain challengeable. Herein, we utilized a circularly permuted HaloTag (cpHaloTag) to achieve the co-immobilization of two proteins at precise ratio, which was applied in developing a chromatographic method with improved specificity for pursuing dual-target compounds. The methodology involved the fusion 3A and 2C at N- and C-terminuses of cpHaloTag, the immobilization of the fusion protein onto silica gel through bioorthogonal reaction, the morphological and functional characterization, the application in finding dual-target compounds. Expression of the fusion protein in E. coli system showed a yield of milligram level with the presence of 3A and 2C domains. Immobilization of the protein was achieved in 10 min with a reaction efficiency more than 88.5 %. Immobilized 3A-cpHalo-2C exhibited higher specificity and better retentions of canonical compounds of the two enzymes in comparison with the column containing immobilized 3A or 2C alone. In real sample application, screening analysis found that hyperoside, cymaroside, and baicalin were dual-target compounds in concert with 3A and 2C in Shuanghuanglian extract. Taking 3A and 2C as probe, we proposed a simple method for direct co-immobilization of diverse proteins from cell lysates and demonstrated an affinity chromatographic-based dual-target compound screening platform. The implications of these methodology are possible to insight the de novo design of multi-target surface for fabricating new bioanalytical methods with improved performance. [Display omitted] • Proposed a strategy for co-immobilizing of 3A and 2C proteins at precise ratio. • Co-immobilized fusion protein by cpHaloTag improved chromatographic specificity. • Highly selective screening of dual-target compounds in natural products. [ABSTRACT FROM AUTHOR]

• The specific DNA aptamer of PPARγ was obtained by an on-column SELEX method. • PPARγ was immobilized by a bivalent affinity binding-inspired method. • Enhanced conformation selectivity and ligand-binding activity was found for PPARγ. Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M−1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M−1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M−1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors. [ABSTRACT FROM AUTHOR]

• Heptapeptide of β 2 -AR recognizes ephedrine and pseudoephedrine epimers in solution. • Immobilized peptide separates the epimers in herbal extract and blood samples. • An enhanced hydrogen bonding was found in heptapeptide-pseudoephedrine complex. Epimer separation is crucial in the field of analytical chemistry, separation science, and the pharmaceutical industry. No reported methods could separate simultaneously epimers or even isomers and remove other unwanted, co-existing, interfering substances from complex systems like herbal extracts. Herein, we prepared a heptapeptide-modified stationary phase for the separation of 1R,2S -(-)-ephedrine [(-)-Ephe] and 1S,2S -(+)-pseudoephedrine [(+)-Pse] epimers from Ephedra sinica Stapf extract and blood samples. The heptapeptide stationary phase was comprehensively characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The separation efficiency of the heptapeptide column was compared with an affinity column packed with full-length β 2 -AR functionalized silica gel (β 2 -AR column). The binding affinity of the heptapeptide with (+)-Pse was 3-fold greater than that with (-)-Ephe. Their binding mechanisms were extensively characterized by chromatographic analysis, ultraviolet spectra, circular dichroism analysis, isothermal titration calorimetry, and molecule docking. An enhanced hydrogen bonding was clearly observed in the heptapeptide-(+)-Pse complex. Such results demonstrated that the heptapeptide can recognize (+)-Pse and (-)-Ephe epimers in a complex system. This work, we believe, was the first report to simultaneously separate epimers and remove non-specific interfering substances from complex samples. The method was potentially applicable to more challenging sample separation, such as chiral separation from complex systems. [ABSTRACT FROM AUTHOR]

• Proposed a label-free strategy for immobilizing GPCR using genetically encoded nnAAs. • Immobilized 5-HT 1A R O-ALTyr improved chromatographic performance. • High selective screening for 5-HT 1A R ligands in natural products. • Confirmed magnoflorine as a potential 5-HT 1A R agonist. G protein-coupled receptors (GPCRs) are one of the most prominent targets for drug discovery. Immobilizing GPCRs has proven to be an effective strategy for expanding the utility of GPCRs into nonbiological contexts. However, traditional strategies of immobilizing GPCRs have been severely challenged due to the loss of receptor function. Here, we reported a novel and general approach to realize the label-free and site-selective immobilization of 5-hydroxytryptamine 1A receptor (5-HT 1A R) and the application in developing a chromatographic method with improved specificity for pursuing 5-HT 1A R ligands from natural products. This method involved the use of a clickable non-natural amino acid, O-allyl-L-tyrosine (O-ALTyr) to immobilize the receptor onto thiol-functionalized silica gels through a 'thiol-ene' click chemistry, which allowed us to avoid the purification step and directly immobilize 5-HT 1A R on silica gels. The immobilized receptor was characterized using immunofluorescence assay, and receptor-ligand interaction analysis was conducted through frontal analysis. To test the feasibility of the immobilized 5-HT 1A R O-ALTyr in complex matrices, bioactive compounds in Ziziphi Spinosae Semen (ZSS) were screened and their interaction with the receptor was assessed using zonal elution. Our findings indicated that immobilizing the receptor through nnAAs effectively minimizes the chromatographic peak tailing and broadening of specific ligands, leading to a significant improvement in chromatographic performance. The association constants of the three ligands to 5-HT 1A R were approximately one order of magnitude greater than those of Halo-tag attachment. These results demonstrated that the immobilized 5-HT 1A R exhibits high specificity and the ability to recognize receptor ligands from complex matrices. This allowed us to identify magnoflorine (Mag) as a potential ligand of 5-HT 1A R from ZSS extract. In vivo assay also proved that Mag presented a promising anxiolytic effect by promoting the expression of 5-HT 1A R in mice brain. The above findings pointed to the fact that the immobilized 5-HT 1A R affinity chromatographic strategy relying on the site-specific encoded non-natural amino acid is a powerful alternative for precisely determining the drug-protein interaction and discovering the specific ligand of GPCRs from complex matrixes. [ABSTRACT FROM AUTHOR]

• A new equation for drug-protein interaction analysis was derivatized. • Co-injection of two drugs was realized for measurement of drug-protein binding. • Two-fold higher throughput than typical chromatographic assays was confirmed. Quantifying drug-protein interactions has a pivotal role in both early phase drug development and clinical processes. Diverse affinity chromatographic methods like nonlinear chromatography can realize such quantification, however, their throughputs are challenged due to the loading of a single ligand during each run. This work derivatized a new equation for simultaneously determining the bindings of two ligands to a protein relying on assumption that the retention factors of the ligands are dependent on their injection amounts. Experimental validation of the derivatization was performed on an immobilized endothelin A receptor (ET A R) column taking ambrisentan, bosentan, and macitentan as injecting solutes. All three ligands presented a decrease in retention times along with increasing moles of injection when they were singly injected into the column. Likewise, negative relationships between the retentions and the injection amounts were observed when co-injection of ambrisentan/bosentan or bosentan/macitentan was performed, thus confirming the assumption of the derivatization. The association constants of ambrisentan, bosentan, and macitentan binding to ET A R were (1.42 ± 0.78)×104, (1.81 ± 0.22)×104, and (1.71 ± 0.41)×104 L/mol when each of them was singly loaded on the column. Such data displayed insignificant changes in four weeks thereby providing a proof of good stability of the column during the period. Co-injections of the two ligand pairs resulted in the association constants of (2.97 ± 0.13)×104 for ambrisentan, (2.51 ± 0.87)×104 for bosentan, and (2.88 ± 0.34)×104 L/mol for macitentan. These results were in good agreement with the calculation when each of the ligands was injected alone into the column and demonstrated little differences from the data by nonlinear chromatography. Owning to the simultaneous analysis of two ligands, the throughput of the proposed method was twofold higher than the typical assays including frontal analysis, zonal elution, and nonlinear chromatography. It is possible to become an alternative for rapid analysis of drug-protein interaction. [ABSTRACT FROM AUTHOR]

• M 3 R was immobilized on the microspheres through a biological orthogonal reaction. • We screened anti-asthmatic flavour compounds from Siraitia grosvenorii by immobilized M 3 R. • In vitro and in vivo tests were applied to prove the effects of the two flavour compounds. • Two flavour compounds show a better anti-asthmatic effect than original extract in the same dose. 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 (M 3 R) 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 M 3 R. 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 M 3 R 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 M 3 R 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. [ABSTRACT FROM AUTHOR]

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (β 1 -AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of β 1 -AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing β 1 -AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged β 1 -AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized β 1 -AR in the analysis of ligand-receptor interaction. The morphology was characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). The chromatographic specificity of the immobilized receptor column was evaluated by determining the association constants of atenolol, esmolol and metoprolol using stepwise frontal analysis plus injection amount-dependent method. The potential ligands binding to β 1 -AR was screened by collecting the peak with retention time longer than the void time, and identified the collection by reverse phase liquid chromatography coupled with tandem mass spectrometry. The association constants of the three drugs to β 1 -AR were (3.33 ± 0.29)× 106 M−1, (2.33 ± 0.23)× 106 M−1 and (2.06 ± 0.03)× 106 M−1, indicating a desired specificity of the immobilized receptor for recognizing its ligands. Molecular docking showed that van der Waals, hydrogen bonds, and hydrophobic interactions were the principal interaction forces for the receptor-drug complexes. Benzoylmesaconine was screened as the potential ligand of β 1 -AR in Radix Aconiti Lateralis Praeparata extract. The association constant of the ligand was (1.06 ± 0.02)× 105 M−1, hinting structural modification may be required before clinical application. The immobilized β 1 -AR is possible to provide a rapid method for screening potential ligands in herbal extract. • Preparation of β 1 -AR stationary phase by the bioorthogonal click reaction. • Revealing the binding mechanism between drugs and β 1 -AR by chromatographic methods. • Screening the bioactive compounds targeting β 1 -AR from herbal medicine. [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]

Zonal elution and nonlinear chromatography are two mainstream models for the determination of drug-protein interaction in affinity chromatography. This work intended to compare the results by zonal elution with that by nonlinear chromatography when it comes to the analysis of the interaction between seven drugs and immobilised β 2 -adrenoceptor (β 2 -AR). The results of the zonal elution showed that clorprenaline, clenbuterol, methoxyphenamine, salbutamol, terbutaline, tulobuterol and bambuterol have only one type of binding site on immobilised β 2 -AR, while nonlinear chromatography confirmed the existence of at least two types of binding sites between β 2 -AR and clorprenaline, clenbuterol and bambuterol. On these sites, both zonal elution and nonlinear chromatography presented the same rank order for the association constants of the seven drugs. Compared with the data from zonal elution, the association constants calculated using nonlinear chromatography gave a good linear response to the corresponding values by radio-ligand binding assay. The sampling efficiencies of nonlinear chromatography were clearly higher than zonal elution. Nonlinear chromatography will probably become a powerful alternative for the high throughput determination of drug-protein interaction. [ABSTRACT FROM AUTHOR]

• Preparaion of a new stationary phase by immobilizing the beta1-adrenoceptor (β 1 -AR) onto ibrutinib-coated microspheres. • Exploration of the binding mechanism between a ligand and the β 1 -AR by the thermodynamic analysis. • Development of a chromatographic method for pursuing β 1 -AR ligand from complex matrices. The discovery of beta1-adrenoceptor (β 1 -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 β 1 -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 β 1 -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 β 1 -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 β 1 -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 β 1 -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 β 1 -AR ligands with high efficiency from complex matrices like herbal extract. [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]

• We immobilize Halo-tagged α 1A -AR by a site-specific method • A strategy was developed for GPCR-ligand interaction analysis • Ligand binding thermodynamic and extra-thermodynamic were investigated • Molecular mechanisms were studied by enthalpy and entropy compensation 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 (α 1A -AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α 1A -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 α 1A -AR, indicating that the oriented immobilization of α 1A -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 (Δ C p) 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. [ABSTRACT FROM AUTHOR]

• Developed and characterized a new stationary phase by attaching M 3 R onto the amino microsphere through the specific covalent reaction. • The use of M 3 R chromatography in exploring the binding of ligands to the receptor. • First application of the immobilized M 3 R chromatographic method to systematically recognize and separate new ligands of the receptor from complex matrices. • Western blot analysis validated the specificity and accuracy of the immobilized receptor when it is utilized to screen bioactive compounds binding to the 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 × 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. [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]

• We construct a GPCR-in-paper chromatographic platform. • β2-AR was immobilized onto the surface of paper discs by a one-step method. • FTIR, fluorescence analysis and XPS was used to characterize β2-AR on paper. • The platform was evaluated by receptor-drug interaction analysis. High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (β 2 -AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that β 2 -AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to β 2 -AR were calculated to be 2.02 × 104 M−1, 1.15 × 104 M−1, 1.75 × 104 M−1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications. [ABSTRACT FROM AUTHOR]

• Immobilized AT 1 R chromatography was prepared by a one-step method. • Thermodynamic and kinetic parameters of four ARBs have been measured by injection amount-dependent method and peak profiling method. • The hit identification of puerarin and rosmarinic acid has been evaluated by the immobilized AT 1 R chromatography. The enormous growth in drug discovery paradigm has necessitated continuous exploration of new methods for drug-protein interaction analysis. To enhance the role of these methodologies in designing rational drugs, this work extended an immobilized angiotensin II type I receptor (AT 1 R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT 1 R and expressed the fusion receptor in E. coli. The expressed receptor was covalently immobilized onto 8.0 μm microspheres by mixing the cell lysate with 6-chlorocaproic acid-modified amino polystyrene microspheres. The immobilized AT 1 R was utilized for thermodynamic and kinetic interaction analysis between the receptor and four specific ligands. Following confirmation of these interactions by molecular docking, we identified puerarin and rosmarinic acid by determining their binding to the receptor. Azilsartan, candesartan, valsartan and olmesartan displayed two kinds of binding sites to AT 1 R by injection amount-dependent method. By molecular docking, we recognize the driving forces of the interaction as electrostatic interaction, hydrogen bonds and van der Waals force. The dissociation rate constants (k d) of azilsartan, candesartan, valsartan and olmesartan to AT 1 R were 0.01138 ± 0.003, 0.05142 ± 0.003, 0.07547 ± 0.004 and 0.01310 ± 0.005 min−1 by peak profiling assay. Comparing with these parameters, puerarin and rosmarinic acid presented lower affinity (K A : 0.12 × 104 and 1.5 × 104/M) and slower kinetics (k d : 0.6864 ± 0.03 and 0.3005 ± 0.01 min−1) to the receptor. These results, taking together, indicated that the immobilized AT 1 R has the capacity to probe antihypertensive compounds. [ABSTRACT FROM AUTHOR]