Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis

Wang, Hairong; Xu, Wenfang; Cao, Jiyue; Wang, Weihong

doi:10.1002/elps.201400283

Cited by 10 publications

(4 citation statements)

References 27 publications

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“…Online enzyme assay integrates together enzyme incubation, reaction, and product detection in a single capillary within one run. Comparing off‐line enzyme assay, it thus greatly simplified enzyme assay process, and has developed two mainly operation modes, at inlet and electrophoretically mediated microanalysis (EMMA) .…”

Section: Online Screening Of Enzyme Inhibitorsmentioning

confidence: 99%

Advances in screening enzyme inhibitors by capillary electrophoresis

Wang

Yang

2019

Electrophoresis

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Capillary electrophoresis (CE) has attracted lots of attention due to its simplicity, low sample consumption, low solvent volume, high resolution, and high speed. Based on these advantages, it has been widely used in enzyme inhibitor screening. There are two main operation modes on enzyme inhibitor screening: off‐line (precapillary enzyme assays) in which process CE was used as an analytical tool; online (in‐capillary enzyme assays) which combined the sample injection, mix, reaction, separation, and detection within a single run. Additionally, diverse of new materials were introduced to immobilize enzyme, which has been coupled with CE for the study of enzyme activity and its inhibitor screening. This review gives an overview of the developments and applications for the CE‐based enzyme inhibitor screening.

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Section: Online Screening Of Enzyme Inhibitorsmentioning

confidence: 99%

Advances in screening enzyme inhibitors by capillary electrophoresis

Wang

Yang

2019

Electrophoresis

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“…On the other hand, the on-line approach initiates enzymatic reaction through mixing in-capillary. The on-line mixing of enzyme, substrate, and inhibitor has been accomplished through electrophoretically mediated microanalysis [58,59,62,63,66,73], transverse diffusion of laminar flow profiles [21,31,41,74,75], and pressure mediated microanalysis [45]. On-line analysis with the use of immobilized enzyme for inhibitor studies have also been developed and high throughput analysis was achieved [18,22,23,56,68,76–78].…”

Section: Evaluating Enzyme Inhibitors Using Capillary Electrophoresismentioning

confidence: 99%

Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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“…Lower observed inhibition has been attributed to the decreased incubation time typical in EMMA. [86] EMMA was also demonstrated for a two substrate enzyme, glycerol kinase, with four reactant plugs: incubation buffer, enzyme and two distinct substrate plugs, mixed in-capillary. [88] Small-scale screening of Chinese herbs and other crude products against enzymatic targets by EMMA with UV detection demonstrated the utility of CE in screening of complex test compound mixtures, where potential optical interference is reduced compared to other optical platforms.…”

Section: Targetsmentioning

confidence: 99%

Advances in capillary electrophoresis and the implications for drug discovery

Ouimet

D'Amico

Kennedy

2016

Expert Opinion on Drug Discovery

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Introduction Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery. Areas Covered This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described. Expert opinion Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.

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Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis

Cited by 10 publications

References 27 publications

Advances in screening enzyme inhibitors by capillary electrophoresis

Advances in screening enzyme inhibitors by capillary electrophoresis

Advances in enzyme substrate analysis with capillary electrophoresis

Advances in capillary electrophoresis and the implications for drug discovery

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