title: A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.
abstract: The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany; Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Anton
authors.affiliation: Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany.
authors.forename: Silke M
authors.initials: SM
authors.lastname: Bauer
authors.affiliation: Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany.
authors.forename: Peter R W E F
authors.initials: PR
authors.lastname: Keck
authors.affiliation: Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Laufer
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany; Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C485184:enhanced green fluorescent protein
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