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

Nehmé, Reine; Morin, Pascal

doi:10.1002/elps.201500239

Cited by 19 publications

(8 citation statements)

References 75 publications

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“…These studies include determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors or substrates. Latest developments and applications in this area are presented in two recent reviews .…”

Section: Applicationsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Kašička

2017

Electrophoresis

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The review brings a comprehensive overview of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) to analysis, microscale isolation, purification, and physicochemical and biochemical characterization of peptides in the years 2015, 2016, and ca. up to the middle of 2017. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression and EOF control, and detection) are described. New developments in particular CE and CEC methods are presented and several types of their applications to peptide analysis are reported: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC methods to provide important physicochemical characteristics of peptides are demonstrated.

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Section: Applicationsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Kašička

2017

Electrophoresis

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“…FIA-HRMS methodology feasibility was also validated by comparing to CE-UV [42]. As stated previously, FIA-HRMS methodology could bring imprecisions coming from no compound separation, ionisation fluctuation and in-source fragmentation.…”

Section: Capillary Electrophoresis (Ce-uv)mentioning

confidence: 92%

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry

Ferey

Silva

Colas

et al. 2019

Analytica Chimica Acta

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Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

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“…One such alternative is CE. The rapid development of CE for the purposes of pharmaceutical analysis, including its use in drug discovery, has been recently recognized . In general CE benefits from its simplicity, minimal sample consumption, short analysis time, and its ability to cover a broad range of applications .…”

Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β‐secretase as the Alzheimer's disease drug target*

et al. 2019

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Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β‐secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β‐secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.

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

Cited by 19 publications

References 75 publications

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β‐secretase as the Alzheimer's disease drug target*

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