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.
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Acknowledgments
The authors would like to thank Dr. Li-An Yeh, the Director of the BRITE at NCCU, for her continued support. We want to thank Mark Hughes and Ginger Smith from the automation group at BRITE for their excellent technical support, and Dr. Tiffany Graves for kindly providing data. We are grateful to them and all our colleagues in the Biomanufacturing Research Institute & Technology Enterprise (BRITE) at North Carolina Central University; including Dr. Gordon Ibeanu, Dr. Weifan Zheng, Dr. Al Williams, and Dr. Jonathan Sexton for their intellectual support. Finally, we would like to thank the Golden LEAF Foundation and the BIOIMPACT Initiative of the State of North Carolina through the Biomanufacturing Research Institute & Technology Enterprise (BRITE) Center at North Carolina Central University for financial support.
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Williams, K.P., Scott, J.E. (2009). Enzyme Assay Design for High-Throughput Screening. In: Janzen, W., Bernasconi, P. (eds) High Throughput Screening. Methods in Molecular Biology, vol 565. Humana Press. https://doi.org/10.1007/978-1-60327-258-2_5
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DOI: https://doi.org/10.1007/978-1-60327-258-2_5
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