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 IC(50) values <50.0 muM in the MK2-EGFP translocation assay, and a cross target query of the Lilly-RTP HTS database confirmed their inhibitory activity against in vitro kinase targets, including p38a. Compounds were confirmed structurally by LCMS analysis and profiled in cell-based imaging assays for MAPK signaling pathway selectivity. Three of the hit scaffolds identified in the MK2-EGFP translocation HCS run on the ArrayScan were selected for a p38a inhibitor hit-to-lead structure activity relationship (SAR) chemistry effort.
Trask
Oscar J
OJ
Sphinx RTP Laboratories, Eli Lilly and Company, Research Triangle Park, NC, USA.
Baker
Audrey
A
Williams
Rhonda Gates
RG
Nickischer
Debra
D
Kandasamy
Ramani
R
Laethem
Carmen
C
Johnston
Patricia A
PA
Johnston
Paul A
PA
D001681:Biological Assay / Q000379:methods; D002448:Cell Adhesion; D002467:Cell Nucleus / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000295:instrumentation* / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D014178:Translocation, Genetic; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry*; D000099190:MAP-Kinase-Activated Kinase 2
D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2