Abstract
Reversible phosphorylation of proteins, principally on serine, threonine, or tyrosine residues, is central to the regulation of most aspects of eukaryotic cell function. Dysregulation of protein kinases and protein phosphatases is linked to numerous human diseases. Consequently, many efforts have been made to target these enzymes with small molecules in order to develop new therapeutic agents. While protein kinase inhibitors have been successfully brought to the market, the development of specific protein phosphatase inhibitors is still in its infancy. The largest and most diverse protein phosphatase superfamily in humans is comprised by the protein tyrosine phosphatases, a group of over 100 enzymes. Here, we describe high-throughput screening methods to search for protein tyrosine phosphatase activity modulators. We illustrate the implementation of relatively simple phosphatase assays, using generic absorbance- or fluorescence-based substrates, in 384- or 1536-well microtiter plates. We discuss steps to optimize HTS assay quality and performance, and describe several PTP screening methods on the basis of previously performed successful HTS campaigns. Finally, we discuss how to confirm, follow up, and prioritize hit compounds, and point out a number of common pitfalls that are encountered in this process.