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Methods Mol Biol. 2009;565:107-26. doi: 10.1007/978-1-60327-258-2_5.

Enzyme assay design for high-throughput screening.

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  • 1Department of Pharmaceutical Sciences and BRITE, North Carolina Central University, Durham, NC, USA.

Abstract

Enzymes continue to be a major drug target class for the pharmaceutical industry with high-throughput screening the approach of choice for identifying initial active chemical compounds. The development of fluorescent- or absorbance-based readouts typically remains the formats of choice for enzyme screens and a wealth of experience from both industry and academia has led to a comprehensive set of standardized assay development and validation guidelines for enzyme assays. In this chapter, we generalize approaches to developing, validating, and troubleshooting assays that should be applicable in both industrial and academic settings. Real-life examples of various enzyme classes including kinases, proteases, transferases, and phosphatases are used to illustrate assay development approaches and solutions. Practical examples are given for how to deal with low-purity enzyme targets, compound interference, and identification of activators. Assay acceptance criteria and a number of assay notes on pitfalls to avoid should provide pointers on how to develop a suitable enzymatic assay applicable for HTS.

PMID:
19551359
[PubMed - indexed for MEDLINE]
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