Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System

Pei, Jian Zhong; Dishinger, John F.; Roman, David L.; Rungwanitcha, Chetwana; Neubig, Richard R.; Kennedy, Robert T.

doi:10.1021/ac800553g

Cited by 18 publications

(19 citation statements)

References 45 publications

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“…Use of novel microchip systems 55 or adoption to commercial microchip electrophoresis, e.g. CaliperLabChip EZ reader, will be required to make this a HTS tool suitable for screens of larger chemical collections.…”

Section: Resultsmentioning

confidence: 99%

Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

et al. 2013

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Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods.

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Section: Resultsmentioning

confidence: 99%

Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

et al. 2013

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“…15 In the pre-capillary format, the enzymatic reaction is done in a sample vial containing both an enzyme and a substrate, and a small portion of the reaction solution is periodically analyzed and the product is quantified by CE or micellar electrokinetic chromatography. Michaelis-Menten constants have been determined by the pre-capillary format for GTPase activity, 16 urease assay, 17 sirtuin assay, 18 hydrolysis of bovine -casein, 19 methionine sulfoxide reductase, 20 and N-demethylation of ketamine. 21 To investigate the reaction dynamics of an enzyme, a format of in-capillary reaction (EMMA) is more attractive.…”

Section: Introductionmentioning

confidence: 99%

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

2020

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A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During the CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related with the reaction rate, and it was used for the determination of a Michaelis-Menten constant through Lineweaver-Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.

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“…Higher parallelization may be possible. A 36 channel microfluidic device with gated injection has been demonstrated for a model enzyme inhibitor screen with 36 parallel assays completed in 30 s.[67] Parallelization on chip with fast optically-gated injection has been used to perform for a high throughput enzyme assay with 4 parallel separations and 30 s separation time. [21] The throughput of a droplet extraction ‘virtual walls’ device was improved by parallelization with three extraction channels on one device and was demonstrated for an enzyme assay achieving throughputs of 120 samples in 10 min.…”

Section: Improvements To Throughputmentioning

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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Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System

Cited by 18 publications

References 45 publications

Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

Advances in capillary electrophoresis and the implications for drug discovery

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