Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector

Li, Youxin; Liu, Danning; Bao, Jie

doi:10.1016/j.jchromb.2010.10.034

Cited by 22 publications

(10 citation statements)

References 26 publications

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“…Wang et al developed an integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized trypsin microreactor, and CZE for on‐line protein analysis . A CE–LIF procedure for studying the characterization of tyrosinekinase and drug screening was proposed by Liu et al . Electrophoretically mediated microanalysis (EMMA) and immobilized capillary enzyme reactors have also been use for enzyme assay and enzyme inhibitor screening.…”

Section: Introductionmentioning

confidence: 99%

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

Lin

Zhao

et al. 2013

J of Separation Science

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A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method.

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Section: Introductionmentioning

confidence: 99%

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

Lin

Zhao

et al. 2013

J of Separation Science

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“…An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

“…Assay sensitivity was increased by field-amplified sample injection techniques in CE methods for sirtuin enzymes [19,21] and flavin-containing monooxygenases [22]. Furthermore, the reversed electrode polarity stacking mode was employed in a MEKC method for the monitoring of purine nucleoside phosphorylase and adenosine deaminase activity [23].An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

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confidence: 99%

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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“…A CE–LIF assay was developed by Li et al. to study the enzymatic activity of TyrK in the absence or presence of inhibitors. A fluorescent polypeptide tagged with 5‐FAM (5‐carboxyfluorescein) was used as substrate.…”

Section: Precapillary Assaysmentioning

confidence: 99%

Advances in capillary electrophoresis for miniaturizing assays on kinase enzymes for drug discovery

Nehmé

Morin

2015

Electrophoresis

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CE has become a frequently used tool for miniaturizing enzyme assays due particularly to its well-recognized low sample consumption. CE-based enzyme assays cover all aspects of kinetic analysis including the evaluation of enzyme activity, substrate and modulator characterization, and identification. These assays are performed to conduct high-quality primary (hit finding) and secondary (confirmation by determining the half maximal inhibitory concentration, IC ) screening. Nowadays, the kinase family is among the most actively studied pharmaceutical targets in oncology, and in neurodegenerative and inflammation diseases. In this article, we review the fundamentals of the different approaches that may be employed for assaying kinase kinetics. Their advantages and limitations will also be discussed by covering the literature of CE-based assays for purified kinases as well as for kinases in living cells. The last section will be devoted to perspectives by showing some applications of multiplexed and of microchip devices for high-throughput screening kinase assays.

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Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector

Cited by 22 publications

References 26 publications

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in capillary electrophoresis for miniaturizing assays on kinase enzymes for drug discovery

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