用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

Establishment of a High Expression of α1A Adrenergic Receptor Cell Membrane Chromatography-RPLC Method for Screening Target Components from Radix Caulophylli

We describe here an analytical method of HEK293 α_1A AR cell membrane chromatography (HEK293 α_1A AR/CMC) combined with reverse phase liquid chromatography (RPLC) for recognition, separation and identification of target components from Traditional Chinese Medicines (TCMs) Radix Caulophylli. The HEK 293 α_1A cells with high expressing α_1A adrenergic receptors were used to prepare the stationary phase in the CMC model. Retention fractions on the α_1A AR–CMC model were collected using an automated fraction collection and injection module (FC/I). And each fraction was analyzed by RPLC under optimized conditions. 5-Methylurapidil (5-MU) and tamsulosin hydrochloride were used as standard compounds to investigate the suitability and reliability of the HEK 293 α_1A AR–CMC–RPLC method prior to screening target component from Radix Caulophylli total alkaloid. The results indicated that caulophine was the target component acting on the α_1A AR. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.     

A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A₄₃₁ cells) are an important target for drug discovery. We have developed a new A₄₃₁/cell membrane chromatography (A₄₃₁/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A₄₃₁ cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A₄₃₁/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A₄₃₁ cell growth in vitro. This new A₄₃₁/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

EGFR/cell membrane chromatography-online-high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Radix Angelicae Pubescentis

The intracellular kinase domains of the epidermal growth factor receptor (EGFR) in some tumor cells are significant targets for drug discovery. We have developed a new EGFR cell membrane chromatography (EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis. In this study, the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase (CMSP) in the EGFR/CMC model. The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online. As a result, osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. This new EGFR/CMConline-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs, for instance, Radix Angelicae Pubescentis. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae

A high‐expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high‐performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high‐expression epidermal growth factor receptor cell in a dose‐dependent manner and even achieved a better inhibition effect than gifitinib in the low‐dosage range.     

Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system

Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well‐developed biological chromatographic technique. In this study, we have developed combined SMMC‐7721/CMC and HepG2/CMC with high‐performance liquid chromatography and time‐of‐flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R² = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti‐inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two‐dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose‐dependent manner in the range of dose from 0.10 to 102.4 μM. This two‐dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.     

Screening anti‐tumor compounds from Ligusticum wallichii using cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry

Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry was developed. Tyrosine 367 Cysteine‐HEK293 cells were used as the cell membrane stationary phase. The specificity and reproducibility of the cell membrane chromatography was evaluated using 1‐tert‐butyl‐3‐{2‐[4‐(diethylamino)butylamino]‐6‐(3,5‐dimethoxyphenyl)pyrido[2,3‐d]pyrimidin‐7‐yl}urea, nimodipine and dexamethasone acetate. Then, anti‐tumor components acting on Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 were screened and identified from extracts of Ligusticum wallichii. Components from the extract were retained on the cell membrane chromatographic column. The retained fraction was directly eluted into high‐performance liquid chromatography with mass spectrometry system for separation and identification. Finally, Levistolide A was identified as an active component from Ligusticum wallichii extracts. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide‐formazan colorimetric assay revealed that Levistolide A inhibits proliferation of overexpressing the mutated receptor cells with dose‐dependent manner. Phosphorylation of fibroblast growth factor receptor 4 was also decrease under Levistolide A treatment. Flex dock simulation verified that Levistolide A could bind with the tyrosine kinase domain of fibroblast growth factor receptor 4. Therefore, Levistolide A screened by the cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry can arrest cell growth. In conclusion, the two‐dimensional high‐performance liquid chromatography method can screen and identify potential anti‐tumor ingredients that specifically act on the tyrosine kinase domain of the mutated fibroblast growth factor receptor 4.     

Enhanced stability designs of cell membrane chromatography for screening drug leads

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.     

A vascular smooth muscle/cell membrane chromatography–offline-gas chromatography/mass spectrometry method for recognition,separation and identification of active components from traditional Chinese medicines

We describe an analytical method of vascular smooth muscle cell membrane chromatography (VSM/CMC) combined with gas chromatography/mass spectrometry (GC/MS) for recognition, separation and identification of active components from traditional Chinese medicines (TCMs). VSM cells by means of primary culture with rat thoracic aortas were used for preparation of the stationary phase in the CMC model. Retention components by the VSM–CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the VSM/CMC–offline-GC/MS method using nifedipine and nitrendipine as standard compounds, this method was applied in screening active components from the extracts of TCMs such as Radix Angelicae Dahuricae (RAD), Rhizomza Seu Radix Notopterygii (RSRN), Radix Glehniae (RG) and Fructus Cnidii (FC). Retention components from the extracts in the VSM–CMC model were imperatorin and osthole identified by the GC/MS method. In vitro pharmacological trials indicated that imperatorin and osthole could concentration dependently relax the rat thoracic artery pre-contracted by KCl (P < 0.05). The maximum relaxation effects (R_max) were 63 ± 5% and 40 ± 6% for imperatorin and osthole, respectively. The VSM/CMC–offline-GC/MS method is an effective screening system that can rapidly detect and enrich target components from a complex sample and then accurately identify them.     

Recognition and identification of active components from Radix Bupleuri using human neuroblastoma SH‐SY5Y cells

The aim of the study was to screen active components of Radix Bupleuri (a traditional Chinese herb) and discover novel anti‐schizophrenic candidate drugs using human neuroblastoma SH‐SY5Y cells. SH‐SY5Y cells were used for preparation of the stationary phase in the cell membrane chromatography model. Retention components by the SH‐SY5Y/CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the SH‐SY5Y/CMC method using amisulpride and haloperidol as standard compounds, this method was applied to screening active components from the extracts of Radix Bupleuri. Retention components of SH‐SY5Y/CMC model were saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C and saikosaponin D, which were identified by the GC/MS method. In vitro pharmacological trials‐MTT, saikosaponin B1, saikosaponin B2 and saikosaponin C could protect SY5Y cells. The protective effects of saikosaponin B1 and saikosaponin C were concentration dependent. Saikosaponin A and saikosaponin D inhibited cell viability at concentrations >30 µg/mL (p < 0.05). Via SH‐SY5Y/CMC method and SH‐SY5Y MTT trial, we rapidly detected target components from Radix Bupleuri, accurately identified them and determined their different effects on SH‐SY5Y cells. Saikosaponin B1, saikosaponin B2 and saikosaponin C may be anti‐schizophrenic candidate drugs. Copyright © 2015 John Wiley & Sons, Ltd.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Prostate Cell Membrane Chromatography�CLC/MS Method for Screening ��1A-Adrenoceptor Antagonists from Lotus Plumule

The ??_1A-adrenoceptor (??_1A-AR) plays an important role in drug discovery and development. An online analytical method coupling prostate cell membrane chromatography (CMC) with high-performance liquid chromatography/mass spectrometry (LC/MS) was established to screen ??_1A-AR antagonists in traditional Chinese medicines (TCMs). The prostate cell membrane stationary phase (CMSP) was prepared by immobilizing the prostate cell membrane onto the surface of the silica carrier. The surface and chromatographic characteristics of the prostate CMSP were studied using tamsulosin as a model molecule. The retained fractions from the prostate CMC were analyzed by transferring them to an LC/MS system through a 10-port switch valve. The active component, which could act on the prostate cell membrane and receptor on it (such as ??_1A-AR), was determined using a displacement experiment. The results indicated that liensinine, isoliensine, and neferine from Lotus Plumule exhibited similar retention characteristics to the control drug tamsulosin when utilizing the prostate CMC model. This new prostate CMC?CLC/MS method is applicable for screening ??_1A-AR antagonists from TCMs such as Lotus Plumule and could be employed as a drug discovery tool for natural medicinal herbs.     

High‐expression β1 adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines

β‐Adrenergic receptors are important targets for drug discovery. We have developed a new β₁‐adrenergic receptor cell membrane chromatography (β₁AR‐CMC) with offline ultra‐performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary‐S cells with high β₁AR expression levels were established and used to prepare a cell membrane stationary phase in a β₁AR‐CMC model. The retention fractions were separated and identified by the UPLC–MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β₁AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β₁AR antagonist. It will be a useful method for drug discovery as a leading compound resource.     

Determination of Pharmaceuticals by Dynamic Cell Membrane Chromatography Coupled with High-Performance Liquid Chromatography

As a biological affinity chromatographic method, cell membrane chromatography (CMC) using a silica stationary phase covered with specific cell membrane has been used in screening active components. The innovation of this work is that the bioactive cell membrane and the chromatographic packing are mixed and absorbed for the first time to form the pre-column. The pre-column was placed in front of a C₁₈ column to create dynamic CMC online high-performance liquid chromatography (HPLC) system. The retention behavior and dynamic changes of pharmaceuticals were studied for this system. The results indicate that the retention time of the drug was increased and the symmetry factor reached the analytical level after the addition of the dynamic cell membrane pre-column. Therefore, the dynamic CMC coupled with HPLC system may be a potentially rapid and efficient drug analysis approach for the interaction of drug molecule and receptor on red blood cell membranes.     

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC–ESI–IT–TOF–MS

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two‐dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two‐dimensional HPLC–IT–TOF–MS. Molecular docking and 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose‐dependent manner in the range of 5–50 μmol/L. In conclusion, the two‐dimensional EGFR/CMC–HPLC–IT–TOF–MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.     

Preparation and Application of Modified VEGFR-2 Cell Membrane Chromatographic Separation System

The vascular endothelial growth factor receptor-2 (VEGFR-2) in some tumor cells is a significant target for drug discovery. In this work, a modified model of VEGFR-2 cell membrane stationary phase (CMSP) was prepared by immobilizing U251 cell membrane onto the surface of chitosan-silica (CTS-SiO₂) hybrid carrier. The surface and chromatographic characteristics of VEGFR-2 CMSP were studied. We have developed modified VEGFR-2 cell membrane chromatography for screening drugs and sunitinib malate was used as a positive control. The interaction between the new compounds and membrane receptor was determined by the capacity factors (kʹ). The in vitro cytotoxicity of 10 new compounds on U251 cell viability was determined by MTT test separately to verify the potential pharmacological activity. The modified VEGFR-2 cell membrane chromatographic system demonstrated fast and effective characteristics for screening leading compounds.

     

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF alpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.