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.

Curcumin suppresses colorectal tumorigenesis via the Wnt/ß-catenin signaling pathway by downregulating Axin2.

Colorectal cancer (CRC) is the third most common cancer worldwide, with high incidence and mortality rates. Conventional therapies, including surgery, chemotherapy and radiation, are extensively used for the treatment of CRC. However, patients present with adverse effects, such as toxicity, hepatic injury and drug resistance. Thus, there is an urgent requirement to identify effective and safe therapy for CRC. Curcumin (CUR), a polyphenol substrate extracted from the rhizome of Curcuma longa, has been extensively studied for the treatment of CRC due to its high efficacy and fewer side effects. Previous studies have reported that several signaling pathways, such as NF-κB, Wnt/ß-catenin, are involved in the antitumor effects of CUR in vitro. However, the effect and mechanisms in vivo are not yet fully understood. The present study aimed to determine the molecular mechanism of colorectal cancer in vivo. Reverse transcription-quantitative PCR, western blot and immunohistochemistry analyses were performed to determine the underlying molecular mechanism of curcumin's anti-cancer effect in azoxymethane-dextran sodium sulfate induced colorectal cancer. The results of the present study demonstrated that CUR suppressed tumorigenesis in AOM-DSS induced CRC in mice, and anticancer effects were exerted by suppressing the expression of pro-inflammatory cytokines, and downregulating Axin2 in the Wnt/ß-catenin signaling pathway. Taken together, these results exhibit the potential in vivo mechanisms of the anticancer effects of CUR, and highlight Axin2 as a potential therapeutic target for CRC.

G protein-coupled receptor-in-paper, a versatile chromatographic platform to study receptor-drug interaction.

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 (ß2-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 ß2-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 ß2-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.

Reversible and site-specific immobilization of ß2-adrenergic receptor by aptamer-directed method for receptor-drug interaction analysis.

Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a ß2-adrenergic receptor (ß2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized ß2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized ß2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.

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.