Abstract
The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts (˜ 20,000 wells per day) to ultra-high throughput environments (˜ 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.
Keywords: Malachite green, high throughput screening, pyrophosphatase, assay techniques, phosphate
Combinatorial Chemistry & High Throughput Screening
Title: A Universal, Fully Automated High Throughput Screening Assay for Pyrophosphate and Phosphate Release from Enzymatic Reactions
Volume: 13 Issue: 1
Author(s): Scott D. Pegan, Yang Tian, Valerie Sershon and Andrew D. Mesecar
Affiliation:
Keywords: Malachite green, high throughput screening, pyrophosphatase, assay techniques, phosphate
Abstract: The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts (˜ 20,000 wells per day) to ultra-high throughput environments (˜ 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.
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Pegan D. Scott, Tian Yang, Sershon Valerie and Mesecar D. Andrew, A Universal, Fully Automated High Throughput Screening Assay for Pyrophosphate and Phosphate Release from Enzymatic Reactions, Combinatorial Chemistry & High Throughput Screening 2010; 13 (1) . https://dx.doi.org/10.2174/138620710790218203
DOI https://dx.doi.org/10.2174/138620710790218203 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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