High-Content Screening Analysis of the p38 Pathway: Profiling of Structurally Related p38 α Kinase Inhibitors Using Cell-Based Assays

The complexity of the p38 mitogen-activated protein kinase (MAPK) signaling pathway presents challenges to understanding the efficacy of p38 inhibitors. Biochemical recombinant kinase assays and tumor necrosis factor α (TNFα) secretion assays are typically used to evaluate p38α inhibitors, but they do not provide insight into proximal intracellular events. Stimulation of the pathway evokes a cascade of phosphorylation events, accompanied by movement of molecules to different cellular compartments. Herein, we describe the profiling and potency comparison of a large set of p38α inhibitors with a pyrimidinone, imidazopyrimidine, or triazolopyrimidine core against biochemical recombinant p38α kinase activity, lipopolysaccharide (LPS)-mediated TNFα secretion by THP-1 cells, and a set of cellular imaging assays in SW1353 chondrocytes and baby hamster kidney cells. These pathway assays included p38 phosphorylation, MAPK-activated protein kinase 2 translocation, and heat shock protein (HSP) 27 phosphorylation. We established that HSP27 phosphorylation correlates well with LPS-induced TNFα secretion, validating our cellular imaging assays. We also found that the choice of cells and inducer can profoundly affect cellular potency results. High-content analysis may reveal signaling details, enriching our understanding of the mechanism of action of p38α inhibitors.