We report an online ligand screening method that targets human glucose transporter 1 (hGlut1) under approximately physiological conditions, named nonimmobilized biomaterial capillary electrophoresis (NIBCE), and we investigated the interactions between drugs/candidate compounds and HEK293 cells, hGlut1-overexpressing HEK293 cells, non-small-cell lung cancer A549 cells, A549 tumor tissue, and normal lung tissue by simulating the interactions between drugs and moving target cells or the space-occupying tumor. NIBCE omits the trouble of isolating and purifying target receptors from cell membrane while maintaining their native conformation and binding activity. The biomaterials were intercepted by porous frits in capillary columns and cannot flow through the detection window, thereby solving the problem of interference detection, and they can be renewed any time flexibly, thus effectively maintaining their surface bioactivity. Furthermore, the binding kinetic parameters (K, ka, kd, and k′) were calculated by nonlinear chromatography (NLC) theory, and competitive binding experiments, ligand docking studies, and antitumor activity assays in vitro and in vivo were performed to verify the feasibility of NIBCE.

Alzheimer's disease (AD) is the main cause of dementia, but precise diagnosis and treatment are not sufficient so far. The purpose of this study is to develop biomarkers and therapeutic targets for diagnosis and better understanding of AD. As a result, lysophosphatidylcholine and intermediates of sphingolipid metabolism including sphinganine-1-phosphate, sphingosine-1-phosphate, sphingomyelin, and sphingosine in plasma were annotated as potential biomarkers by using UPLC-Q-TOF-MS and UHPLC-Q-Exactive-MS. Besides, glutathione S-transferases (GSTs) including GstA3, Gstm1, Gstm5, Gstm3, Gstk1 and Gstp1 were significantly enhanced in AD hippocampus by using label free nano-LC-MS/MS. Thus, pathogenesis of AD was involved with increasing of choline, decreasing of ACh, enhancement of GSTs and increasing of glutamate which led to oxidative stress and excitotoxity. Effects of donepezil and a natural medicine were evaluated through metabolomics and proteomics. In summary, proteomic and metabolomic analysis on constructed AD rat model were performed through rapid, sensitive and high resolution LC-MS methods to reveal candidate biomarkers. The data suggested that GSTs have great value as therapeutic targets. This study provided valuable information for the diagnosis mechanism and drug discovery of AD.Download high-res image (242KB)Download full-size image

•A new screening method of HIV-1 inhibitor from natural extracts without isolation.•The CE-MS method of seeking active ingredients in licorice extract is developed.•We identified the structure of main ingredients from licorice active mixture.•The Kb of compounds isolated from licorice extract with R15K are calculated by ACE.•Glycyrrhizin could be HIV-1 entry inhibitor through binding to gp120-V3 loop.The binding of envelope protein gp120 to glycosphingolipids is very important during the human immunodeficiency virus entering into the host cell. This step occurs in the V3 loop region in particularly. The conserved core sequence of V3 loop in gp120 was named R15K. Anti-HIV drug targeting to R15K would avoid the drug-resistance caused by HIV-1 genetic diversity. Here, for the first time, affinity capillary electrophoresis (ACE) and capillary electrophoresis-mass spectrometry (CE-MS) were used for establishing a simple, rapid and effective method of screening the licorice extract for biological activity (anti-HIV), which avoided the complicated isolation and purification process. R15K, 3′-sialyllactose (the positive control), and d-galactose (the negative control) were used for the development and validation of ACE method. After the interaction between licorice extract and R15K was confirmed by ACE, the relative active ingredients were isolated by SPE and their structures were determined by CE-ESI-MS online. In this research, two mixtures from licorice extract were found to be active. Furthermore, glycyrrhizin and licorice saponin G2 were verified as the main ingredients that significantly interacted with R15K via CE-MS and LC–MS. The results of quantitative assays showed that the active mixture contained glycyrrhizin of 74.23% and licorice saponin G2 of 9.52%. Calculated by Scatchard analysis method, glycyrrhizin/R15K complex had the highest binding constant (1.69 ± 0.08) × 107 L/mol among 27 compounds isolated from licorice extract. The anti-HIV activity of glycyrrhizin was further confirmed by bioactive experiment of cellular level. This strategy might provide a high throughput screening and identifying platform for seeking HIV-1 inhibitors in natural products.Here, the interaction between licorice extracts and R15K was investigated by ACE for the first time. CE-ESI-MS was performed to identify the structures of the active ingredients.

A rapid, simple, and sensitive on-line solid-phase extraction HPLC–DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05–50.0 μg/ml for dapsone and omeprazole, 0.1–50.0 μg/ml for caffeine and 0.2–50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R2) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8–109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.Highlights► On-line solid-phase extraction HPLC–DAD method was applied to determine five specific probe substrates in vivo. ► The method was applied to evaluate the effects of TM208 on rat five CYP450 isoforms. ► TM208 had the potential to induce the metabolic activities of CYP2E1 and CYP3A4 in rats. ► TM208 might not significantly affect CYP1A2, CYP2D6 and CYP2C19-mediated metabolism in rats.

ML40 is the equivalent peptide derived from the N terminal of CCC4 (CC chemokine receptor 4), which plays a pivotal role in allergic inflammation. A new capillary electrophoresis method was developed to study the interactions between ML40 and its potential ligands in which ML40 was immobilized on the inner wall of capillary as the stationary phase based on the covalent linking technique. The interaction between S009, a known CCR4 antagonist, and the immobilized ML40 was studied to validate the bioactivity of ML40. The electropherogram of S009 showed that the peak height was reduced and the peak width was broadened in the ML40 immobilized capillary. Otherwise, 25 computer-aided design and drafting compounds were screened out using this method. Four compounds’ peak widths were broadened and their peak heights were reduced, as with S009. Meanwhile, nonlinear chromatography was used to calculate the constants for the ligand–receptor complex formation. Furthermore, the tertiary amine compounds belonging to the chiral tertiary amines of the type NRR′R″, which are optically inactive resulting from rapid pyramide inversion, were chiral separated by our protein immobilization method for the first time. In general, the methodology presented would be applicable to study compound–ML40 interactions as a reliable and robust screening method for CCR4 antagonist discovery.

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-(2-(4-chloro-phenyl)-5-{[(naphthalen-1-ylmethyl)-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-N-(3-morpholin-4-yl-propyl)-acetamide (S009) and the N-terminal extracellular tail (ML40) of CCR4 has been validated to be high affinity by capillary zone electrophoresis (CZE). The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-11, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases.