Assay Development and Case History of a 32K‐Biased Library High‐Content MK2‐EGFP Translocation Screen to Identify p38 Mitogen‐Activated Protein Kinase Inhibitors on the ArrayScan 3.1 Imaging Platform

This chapter describes the conversion and assay development of a 96‐well MK2‐EGFP translocation assay into a higher density 384‐well format high‐content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well‐substantiated hypothesis that mitogen‐activated protein kinase‐activating protein kinase‐2 (MK2) is a substrate of p38 MAPK kinase and that p38‐induced phosphorylation of MK‐2 induces a nucleus‐to‐cytoplasm translocation. This chapter also presents a case history of the performance of the MK2‐EGFP translocation assay, run as a “high‐content” screen of a 32K kinase‐biased library to identify p38 inhibitors. The assay performed very well and a number of putative p38 inhibitor hits were identified. Through the use of multiparameter data provided by the nuclear translocation algorithm and by checking images, a number of compounds were identified that were potential artifacts due to interference with the imaging format. These included fluorescent compounds, or compounds that dramatically reduced cell numbers due to cytotoxicity or by disrupting cell adherence. A total of 145 compounds produced IC50 values