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.

N-methyl-D-aspartic acid receptor 2A functionalized stationary phase: A reliable method for pursuing potential ligands against Alzheimer's disease from natural products.

AIMS: N-methyl-D-aspartic acid (NMDA) receptors play subunit-specific role in central neuronal development. However, insights into the pharmacological modulation of NMDA receptors were mainly lack of subunit and synaptic selectivity. The purpose of the present study was to develop a novel strategy to rapidly recognize NMDA subunit 2A (NMDA-2A) ligands from natural products and provide subunit-selective drug candidates for Alzheimer's disease (AD). METHODS: The recombinant NMDA-2A containing a tag of epidermal growth factor receptor (EGFR) was expressed in Escherichia coli cells and immobilized on ibrutinib-modified microspheres based on the specific reaction between EGFR and its inhibitor ibrutinib. A novel affinity stationary phase was synthesized to screen NMDA-2A ligands from Gardenia jasminoides Ellis. RESULTS: The immobilized receptor column exhibited excellent receptor selectivity and ligand-binding activity. Crocetin was screened by using this method. In a cellular model of AD, the protein level of NMDA-2A was significantly decreased compared with the control group, while treatment with crocetin significantly increased NMDA-2A level in a concentration-dependent manner, confirming that crocetin could bind to NMDA-2A in vitro. CONCLUSION: In the present study, we developed a reliable method for the rapid identification of NMDA-2A ligands from natural products, which may be used as a platform for new drug discovery to generate high-quality drug candidates.

Enhancing Chromatographic Performance of Immobilized Angiotensin II Type 1 Receptor by Strain-Promoted Alkyne Azide Cycloaddition through Genetically Encoded Unnatural Amino Acid.

During integration to the solid surface, the effects of tags introduced for bioorthogonal reactions on protein activity have received far less investigation. This represents the major challenge of improving the performance of the immobilized protein-based assays. Herein, the relationship between the fusion tags and their reaction efficiency in mediating the assay performance was realized by determining the chromatographic performance using genetically encoded azide-alkyne cycloaddition, and Halo- and SNAP-tagged bioorthogonal reactions for synthesizing immobilized angiotensin II type 1 receptor (AT1R). We demonstrated that immobilization with the incorporation of unnatural amino acid in the receptor minimizes the peak tailings and broadenings of irbesartan, fimasartan, losartan, and tasosartan, while attachment via large tags (SNAP and Halo) leads to serious asymmetry peaks. Upon the first immobilization, the association constants of the four drugs to AT1R appeared to be 1 order of magnitude greater than the other two attachments. Such enhancement is likely reasoned by the improved association rate constants and the relatively identical dissociation rates due to the small tag. While demonstrating improved chromatographic performance, the immobilized AT1R prepared by the genetically encoded azide-alkyne reaction was applied in analyzing Uncaria Schreber nom. cons. extract, which identified hynchophylline as a specific ligand binding to the receptor. As immobilized proteins move toward diverse assays, our findings provide an unprecedented insight into the relation between fusion tags and their reaction efficiency in mediating the assay performance, which is thus dedicated to the creation of a protein-functionalized surface for precisely determining the drug-protein interaction and discovering the specific partner of the protein.

Covalent immobilization of beta2 adrenergic receptor through trans-methylation reaction by SNAP-tag and its application in anti-asthmatic compound screening from Raphani Semen.

Beta2-adrenergic receptor (ß2-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 ß2-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 ß2-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 â„ƒ 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 ß2-AR with a specific binding site of Ser 207. Taking together, the immobilized ß2-AR column is promising in exploring drug-protein interaction analysis and anti-asthmatic drug screening.

Development of an Allostery Responsive Chromatographic Method for Screening Potential Allosteric Modulator of Beta2-adrenoceptor from a Natural Product-Derived DNA-Encoded Chemical Library.

Allosteric ligands are promising drugs owing to their remote regulations of the orthosteric ligand signaling pathway. There are few allosteric ligands due to the lack of handy and efficacious method for the screening. Herein, we developed an affinity chromatographic method for allosteric ligand screening by immobilizing purified beta2 adrenoceptor (ß2-AR) onto macroporous silica gel by a two-point tethering method. The method relies on the occupation of the orthosteric site by an antagonist and the chelation of N-terminal His-tag of the receptor and Ni2+ coated on the gel. The immobilized ß2-AR demonstrated the greatest allosteric responsive feature when Cmpd-15 (0.25 µM) was included in the mobile phase. Under the same conditions, the association constants of three agonists (salbutamol, terbutaline, and tulobuterol) reduced to 47%, 19%, and 27% compared with the data without the inclusion of Cmpd-15 in the mobile phase. APF was screened as a potential allosteric modulator of ß2-AR by applying the immobilized receptor in a natural product-derived DNA-encoded chemical library (DEL). Relying on these results, we reasoned that the current method has potential in screening allosteric ligands of the receptor. We expect that it is applicable for the discovery of new allosteric binding sites of a target protein and screening allosteric modulators of the other receptors from complex samples.

Discovery of dual-target ligands binding to beta2-adrenoceptor and cysteinyl-leukotriene receptor for the potential treatment of asthma from natural products derived DNA-encoded library.

The design, synthesis, and discovery of dual-target compounds are considered as a promising strategy to develop new drugs with improved safety and efficacy compared with single-target drugs. This necessitates development of the methodologies that enable us to rapidly and accurately achieve the dual-target leads. Applying rosmarinic acid, 18ß-glycyrrhetinic acid, rhein, and ferulic acid as template building blocks, we introduced the self-assembling DNA encoded technique to build the library containing 1,000 compounds. These compounds were screened by receptor chromatography with immobilized beta2-adrenoceptor (ß2-AR) and cysteinyl-leukotriene receptor (CysLT), whereby we obtained a derivative of 18ß-glycyrrhetinic acid (XC267) that specifically binds to the two receptors. In vitro assessment demonstrated the desired binding affinity of 6.57 × 104 M-1 to ß2-AR, 2.82 × 104 M-1 to CysLT, and the dissociation rate constant of 7.52 s-1 to ß2-AR, 17.2 s-1 to CysLT. Pharmacological examination with ovalbumin-induced mice demonstrated that XC267 significantly reduced the levels of IL-4, IL-13, and IgE after oral administration of 10 mg/kg. By Western blot analysis, we observed an up-regulated expression of ß2-AR and a blocked level of CysLT with a dose-dependent manner in pulmonary bronchial. Our results suggest XC627 is a potential candidate to treat asthma by simultaneously regulating the signaling pathway of the two receptors.