Can the heptapeptide ASSIVSF of the ß2-adrenoceptor recognize ephedrine and pseudoephedrine epimers in a complex system?

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 ß2-AR functionalized silica gel (ß2-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.

Stepwise Frontal Analysis Coupled with Affinity Chromatography: A Fast and Reliable Method for Potential Ligand Isolation and Evaluation from Mahuang-Fuzi-Xixin Decoction.

Mahuang-Fuzi-Xixin Decoction (MFXD) is widely used in the treatment of asthma, however, the functional components in the decoction targeting beta2-adrenoceptor (ß2 -AR) remain unclear. Herein, we immobilized the haloalkane dehalogenase (Halo)-tagged ß2 -AR on the 6-chlorocaproic acid-modified microspheres. Using the affinity stationary phase, the interactions of four ligands with the receptor were analyzed by stepwise frontal analysis. The association constants were (4.75±0.28)×104  M-1 for salbutamol, (2.93±0.15)×104  M-1 for terbutaline, (1.23±0.03)×104  M-1 for methoxyphenamine, (5.67±0.38)×104  M-1 for clorprenaline at high-affinity binding site, and (2.73±0.05)×103  M-1 at low-affinity binding site. These association constants showed the same rank order as the radioligand binding assay, demonstrating that immobilized ß2 -AR had capacity to screen bioactive compounds binding to the receptor while stepwise frontal analysis could predict their binding affinities. Application of the immobilized receptor in analysis of MFXD by chromatographic method revealed that ephedrine, aconifine, karakoline, and chasmanine were the bioactive compounds targeting ß2 -AR. Among them, ephedrine and chasmanine exhibited association constants of (2.94±0.02)×104 M-1 and (4.60±0.15)×104  M-1 to the receptor by stepwise frontal analysis. Molecular docking analysis demonstrated that ephedrine, chasmanine, and the other two compounds interact with ß2 -AR through the same pocket involving the key amino acids such as Asn312, Asp113, Phe289, Trp286, Tyr316, and Val114. As such, we reasoned that the four compounds dominate the therapeutic effect of MFXD against asthma through ß2 -AR mediating pathway. This work shed light on the potential of immobilized ß2 -AR for drug discovery and provided a valuable methodology for rapid screening.

Identifying potential ligands specifically binding to beta1-adrenoceptor from Radix Aconiti Lateralis Praeparata extract by affinity chromatographic method.

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (ß1-AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of ß1-AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing ß1-AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged ß1-AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized ß1-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 ß1-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 ß1-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 ß1-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 ß1-AR is possible to provide a rapid method for screening potential ligands in herbal extract.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients' fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human's defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant's ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

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.

Revealing the Antiepileptic Effect of α-Asaronol on Pentylenetetrazole-Induced Seizure Rats Using NMR-Based Metabolomics.

α-Asaronol from Acorus tatarinowii (known as "Shichangpu" in Traditional Chinese medicine) has been proved to possess more efficient antiepileptic activity and lower toxicity than α-asarone (namely "Xixinnaojiaonang" as an antiepileptic drug in China) in our previous study. However, the molecular mechanism of α-asaronol against epilepsy needs to be known if to become a novel antiepileptic medicine. Nuclear magnetic resonance (NMR)-based metabolomics was applied to investigate the metabolic patterns of plasma and the brain tissue extract from pentylenetetrazole (PTZ)-induced seizure rats when treated with α-asaronol or α-asarone. The results showed that α-asaronol can regulate the metabolomic level of epileptic rats to normal to some extent, and four metabolic pathways were associated with the antiepileptic effect of α-asaronol, including alanine, aspartate, and glutamate metabolism; synthesis and degradation of ketone bodies; glutamine and glutamate metabolism; and glycine, serine, and threonine metabolism. It was concluded that α-asaronol plays a vital role in enhancing energy metabolism, regulating the balance of excitatory and inhibitory neurotransmitters, and inhibiting cell membrane damage to prevent the occurrence of epilepsy. These findings are of great significance in developing α-asaronol into a promising antiepileptic drug derived from Traditional Chinese medicine.

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.

Immobilized beta2-adrenergic receptor: A powerful chromatographic platform for drug discovery and evaluation of drug-like property for natural products.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized ß2- adrenergic recepotor (ß2-AR), an identified target of asthma, as the stationary phase in chromatographic column to screen compounds extracted from Stemonae Radix, Playtycodonis Radix, and Glycyrrhizae Radix et Rhizoma. To analyze binding properties of the extracted compounds to the immobilized receptors, we measured their retention behavior in the receptor chromatography and compared with six clinical asthma drugs. We identified tuberostemonine, platycodin D, and glycyrrhizic acid as the potential leads against asthma by our ß2-AR chromatography coupled with mass spectrum (MS). The association constants of the three compounds to ß2-AR were 2.85 × 10-5, 2.55 × 10-4, and 4.07 × 10-6 M with the dissociation rate constants of 6.91 ± 0.35, 11.88 ± 0.60, and 9.49 ± 0.64 min-1, respectively. Tuberostemonine, a pentacyclic Stemona alkaloids, presented the most optimum values of binding efficiency index (BEI) and surface efficiency index (SEI) as close to the diagonal of SEI-BEI optimization plane when it is compared with platycodin D, glycyrrhizic and the six clinical drugs. Our results suggest that tuberostemonine is a promising natural product to be developed for treating asthma because it exhibits better drug-like binding properties to ß2-AR than the clinical drugs. As such, we demonstrate a chromatographic strategy to identify bioactive natural products based on the ß2-AR immobilization, which can be widely adopted to screen natural products from mixture of herbal extracts.

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.

Rapid screening of bioactive compound in Sanzi Yangqin Decoction and investigating of binding mechanism by immobilized ß2-adrenogic receptor chromatography coupled with molecular docking.

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 ß2-adrenegic receptor (ß2-AR). Therefore, this work intends to screen bioactive compounds from SYD and revealed the binding mechanism by immobilized ß2-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 ß2-AR was successfully achieved. Representative chromatographic peaks of SYD on the immobilized ß2-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 ß2-AR. We concluded that the proposed method is becoming an alternative in rapid recognizing bioactive compounds from complex matrix.

Screening of bioactive components from traditional Chinese medicine by immobilized ß2 adrenergic receptor coupled with high performance liquid chromatography/mass spectrometry.

Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where ß2 adrenergic receptor (ß2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6 × 50 mm, 7 µm) to form ß2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four ß2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between ß2-AR and tetrahydroberberine (9.04 × 104/M) as well as fumarine (4.30 × 104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized ß2-AR with corresponding concentrations of 6.67 × 10-4 M and 5.88 × 10-4 M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.

Screening Bioactive Compounds of Siraitia grosvenorii by Immobilized ß2-Adrenergic Receptor Chromatography and Druggability Evaluation.

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 ß2-adrenergic receptor (ß2-AR) target compounds from the fruit of Siraitia grosvenorii (LHG). The method involved immobilization of ß2-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 ß2-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 ß2-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.

Site-Specific Immobilization of ß2-AR Using O6-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta2-adrenergic receptor (ß2-AR), the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of ß2-AR on the resin. The immobilized ß2-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to ß2-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting ß2-AR in Fructus Perillae. We concluded that O6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.

Screening bioactive compounds with multi-targets from Rhodiola crenulata by a single column containing co-immobilized beta2-adrenergic receptor and voltage dependent anion channel isoform 1.

The pursuit of drugs having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. This work synthesized a chromatographic stationary phase containing co-immobilized beta2-adrenergic receptor (ß2-AR) and voltage dependent anion channel isoform 1 (VDAC-1) to achieve such purpose. Specific ligands of the two receptors (e.g. salbutamol, methoxyphenamine, ATP and NADH) were utilized to characterize the specificity and bioactivity of the column. Validated application of the stationary phase was performed by screening multi-target compounds of Rhodiola crenulata using high performance affinity chromatography coupled with ESI-Q-TOF-MS. By zonal elution, we identified salidroside as a bioactive compound simultaneously binding to ß2-AR and VDAC-1. The compound exhibited the binding sites of 1.0 × 10-7 and 4.0 × 10-7 M on the ß2-AR and VDAC-1. On these sites, the association constants were calculated to be 3.3 × 104 and 1.0 × 104 M-1. Molecular docking indicated that the binding of salidroside to the two receptors occurred on Ser169 and Phe255of ß2-AR, and the channel wall of VDAC-1. Taking together, we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.

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.

Bioactive compounds of Shuang-Huang-Lian prescription and an insight into its binding mechanism by ß2 -adrenoceptor chromatography coupled with site-directed molecular docking.

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 ß2 -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized ß2 -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the ß2 -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 ß2 -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 ß2 -adrenoceptor indicated that the binding mainly occurred on Ser169 , Ser173 , and Phe287 of ß2 -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography.

Confirming therapeutic target of protopine using immobilized ß2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized ß2 -adrenoceptor (ß2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized ß2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to ß2 -AR by the 2 methods were (1.00 ± 0.06) × 105 M-1 and (1.52 ± 0.14) × 104 M-1 . The numbers of binding sites were (1.23 ± 0.07) × 10-7 M and (9.09 ± 0.06) × 10-7 M, respectively. These results indicated that ß2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.

Screening bioactive compounds from Ligusticum chuanxiong by high density immobilized human umbilical vein endothelial cells.

High throughput screening methodologies play a very important role in screening bioactive compounds from complex media. In this work, a new strategy for attaching cells onto amino microspheres using human umbilical vein endothelial cells (HUVECs) as a probe was developed. The immobilization depended on the specific affinity between integrin on the cells and the RGD peptide, which was coated on poly[oligo (ethylene glycol) methacrylate] by atom transfer radical polymerization. Validated application of the stationary phase was performed in the analysis of Ligusticum chuanxiong extraction by high performance affinity chromatography-mass spectrometry. Three compounds were screened as the bioactive compounds of Ligusticum chuanxiong. Two of them were identified as 3-butyl-hexahydroisobenzofuran-1(3H)-one and tetramethylpyrazine (TMP), whereas the other one remains indistinct. The association constant of vascular endothelial growth factor (VEGF) and TMP binding to VEGF receptor (VEGFR) on HUVECs were calculated to be (1.04 ± 0.08) × 10(11) M(-1) and (9.84 ± 1.11) × 10(8) M(-1) by zonal elution. Molecular docking showed that one hydrogen bond was formed between N atom of TMP and 3-N atom of imidazole group in histidine(223) of VEGFR. Both zonal elution and molecular docking indicated that TMP and VEGF bind to the same site of VEGFR on HUVECs. It is possible to become a promising tool for high throughput screening of the bioactive compounds binding to HUVECs through broad application of the stationary phase.

Target-directed screening of the bioactive compounds specifically binding to ß2-adrenoceptor in Semen brassicae by high-performance affinity chromatography.

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized ß2-adrenoceptor (ß2-AR) and target-directed molecular docking. The methods involved the attachment of ß2-AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high-performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to ß2-AR was determined to be 1.36 × 10(5) M(-1) with a value of 1.27 × 10(-6) M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and ß2-AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug-receptor interaction.