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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-HT1AR) and the application in developing a chromatographic method with improved specificity for pursuing 5-HT1AR 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-HT1AR 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-HT1ARO-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-HT1AR were approximately one order of magnitude greater than those of Halo-tag attachment. These results demonstrated that the immobilized 5-HT1AR 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-HT1AR from ZSS extract. In vivo assay also proved that Mag presented a promising anxiolytic effect by promoting the expression of 5-HT1AR in mice brain. The above findings pointed to the fact that the immobilized 5-HT1AR 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.

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The platforms based on immobilization of transmembrane proteins have become an effective way to study drug-protein interaction and identify new leads for drug discovery. Herein, we exploited the protein superglue (i.e. SpyTag-SpyCatcher chemistry) for site-specific, oriented, and in-situ one-step beta2-adrenoceptor (ß2-AR) immobilization. SpyCatcher was used as a fusion tag at the C-terminal of ß2-AR and the macroporous silica gels were functionalized with the SpyTag peptide. Immobilization was realized by immersing the gels into the E.coli cell lysate containing ß2-AR-SpyCatcher. Characterization of the functionalized gels was performed by X-ray photoelectron spectroscopy and fluorescence microscopy. Adsorption energy distribution calculation, injection amount dependent analysis (IADA) and nonlinear chromatographic were used for receptor-ligand interaction analysis. The affinity rank order of four ligands to the receptor was tulobuterol> chlorprenaline> salbutamol> terbutaline, which showed highly consistent with data from the radioligand binding assay and the ß2-AR column prepared by HaloTag technology. Magnolol and honokiol were screened from Cortex Magnoliae Officinalis and proved to promote the expression of the receptor in human airway smooth muscle cells. Our work unraveled the great potential to generate good bioactivity of the immobilized ß2-AR through Spy toolbox. This technology can be extended to the immobilization of other functional proteins, providing a better alternative in the field of bioanalysis, biosensing, and separation science.

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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.

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Thrombin (THR) inhibitors play an important role in the treatment of thrombotic diseases. This study established a THR-based bio-specific extraction coupled with affinity chromatography and ultra-high performance liquid chromatography-high resolution mass spectroscopy (UPLC-HR-MS) analysis method to screen and identify THR ligands in Leech. After evaluating the reliability of the screening method using positive control drug (hirudin), it was successfully used to screen the potential active constituents in leech. And a comprehensive analysis of the peptides in leech elution was performed by UPLC-HR-MS, a total of 34 peptides were identified. At the same time, anti-THR activity was explored and inferred by searching databases and published literature. As a result, six peptides were discovered to be potential active compounds in leech. Further, the six peptides were synthesized and in vitro enzymatic activity assay was performed. Finally, SYELPDGQVITIGNER was screened as an anti-THR peptide with an IC50 value of 255.75 µM and it was discovered for the first time from Whitmania pigra Whitman and Hirudo nipponica Whitman. The molecular docking study showed that THR inhibitory activity of the polypeptide was mainly attributed to the hydrogen bond interactions, van der Waals forces and electrostatic interactions interaction between polypeptide and THR. These results suggest that the polypeptide is a potential natural THR inhibitor that can be used as anticoagulant.

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Immobilization of biomaterials developed rapidly due to the great promise in improving their stability, activity and even selectivity. In this review, the immobilization strategies of biomaterials, including physical adsorption, encapsulation, covalent attachment, cross-linking and affinity linkage, were briefly introduced. Then, the major emphasis was focused on the reported various types of immobilized biomaterials, including proteins, enzymes, cell membrane and artificial membrane, living cells, carbohydrates and bacteria, used in the herbal analysis for bioactive compound screening, drug-target interaction evaluation and chiral separation. In addition, a series of carrier materials applied in biomaterials immobilization, such as magnetic nanoparticles, metal-organic frameworks, silica capillary column, cellulose filter paper, cell membrane chromatography, immobilized artificial membrane chromatography and hollow fiber, were also discussed. Perspectives on further applications of immobilized biomaterials in herbal analysis were finally presented.

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Thrombin (THR) plays a significant role in thromboembolic diseases, direct THR inhibitors are a class of important clinical anticoagulant drugs. This study established a THR in-solution based biospecific extraction combined with ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry analysis (TUA) method to screen and identify ligands for THR in Rhizoma Chuanxiong. After evaluating the reliability of the present TUA method using positive (argatroban) and negative (adenosine, tirofiban, ticagrelor) control drugs, this method was successfully applied to detect eight potential active compounds in Rhizoma Chuanxiong. Two new THR-targeted compounds isochlorogenic acid C and senkyunolide I with high THR inhibitory activity (IC50 206.48 and 197.23µM, respectively) were identified by liquid chromatography/mass spectrometry and enzyme inhibitory activity test finally. They were reported with direct THR inhibition activity for the first time and their ligand-THR interactions were explored by in silico molecular docking research. In addition, based on the TUA screening result, four compounds gained similar structure with the two hit compounds were also investigated as promising candidates targeting THR with high binding energy (>5.0kcal/mol). These results may prove that the proposed method could effectively screen THR inhibitors in complex mixtures.

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