A FRET Probe for Cell-Based Imaging of Ganglioside-Processing Enzyme Activity and High-Throughput Screening

2015 ◽  
Vol 54 (18) ◽  
pp. 5389-5393 ◽  
Author(s):  
Guang-Yu Yang ◽  
Caishun Li ◽  
Michael Fischer ◽  
Christopher W. Cairo ◽  
Yan Feng ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Processing Enzyme

A FRET Probe for Cell-Based Imaging of Ganglioside-Processing Enzyme Activity and High-Throughput Screening

2015 ◽  
Vol 127 (18) ◽  
pp. 5479-5483 ◽  
Author(s):  
Guang-Yu Yang ◽  
Caishun Li ◽  
Michael Fischer ◽  
Christopher W. Cairo ◽  
Yan Feng ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Processing Enzyme

scholarly journals Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

2009 ◽  
Vol 14 (6) ◽  
pp. 668-678 ◽  
Author(s):  
Sumitra M. Sukumaran ◽  
Benjamin Potsaid ◽  
Moo-Yeal Lee ◽  
Douglas S. Clark ◽  
Jonathan S. Dordick
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
High Throughput ◽  
High Throughput Screening ◽  
Imaging System ◽  
Early Stage ◽  
Wide Field ◽  
Cytochrome P450 Enzyme ◽  
Assay Sensitivity ◽  
Screening Platform

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 × 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z′ factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. ( Journal of Biomolecular Screening 2009:668-678)

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR).Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS.Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals High-Throughput Scintillation Proximity Assay for Stearoyl-CoA Desaturase-1

2011 ◽  
Vol 16 (5) ◽  
pp. 506-517 ◽  
Author(s):  
Paul Tawa ◽  
Jean-Pierre Falgueyret ◽  
Sebastien Guiral ◽  
Elise Isabel ◽  
David A. Powell ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Enzyme Activity Assay ◽  
Scintillation Proximity Assay ◽  
Binding Characteristics ◽  
Obesity And Diabetes ◽  
High Throughput Screening Assay ◽  
Stearoyl Coa Desaturase

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([3H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z′ factor (0.8). A screening collection of 1.6 million compounds was screened at 11 µM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [3H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.

scholarly journals Development of a Simple High-Throughput Screening Protocol Based on Biosynthetic Activity of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2006 ◽  
Vol 11 (8) ◽  
pp. 1035-1042 ◽  
Author(s):  
Upasana Singh ◽  
Dhiman Sarkar
Keyword(s):  
Enzyme Activity ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Quantitative Estimation ◽  
Signal To Noise ◽  
Chemical Library ◽  
Validation Data ◽  
Screening Protocol

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The Km values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH4Cl, 3.25 mM for MgCl2, and 2.5 mM for adenosine triphosphate. The Km value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25° C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z′ factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 μg/ml concentration. The IC50 values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 μM and 30 μM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z′ and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.

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