Fluorescence polarization binding assay to develop inhibitors of inactive p38alpha mitogen-activated protein kinase.

Development of inhibitors that target inactive kinase conformations is becoming a more attractive approach to kinase inhibitor research. The major advantage of this methodology is that targeting the inactive conformation reduces competition with high intracellular adenosine triphosphate (ATP) concentrations. p38alpha Mitogen-activated protein kinase (MAPK) signaling has been identified as the principal mediator of inflammation associated with a spectrum of disorders (e.g., arthritis, Alzheimer's disease, various malignancies). To allow identification and development of p38alpha MAPK inhibitors that preferentially bind to the inactive conformation, a novel fluorescence polarization-based binding assay is presented. The assay is homogeneous, requires low amounts of the kinase and fluoroprobe, and does not rely on radioactivity. It may, therefore, offer an inexpensive alternative to current p38alpha MAPK inhibitor screening methods. The validation of the system with known p38alpha MAPK inhibitors confirmed that the binding assay, rather than the conventional enzyme activity assay, correlates with cellular efficacy. Finally, we show that pyridinyl imidazoles that potently bind to the inactive p38alpha MAPK prevent activation of p38 MAPK in living cells, suggesting that pyridinyl imidazoles other than SB203580 are able to induce the DFG-out conformation that is incompatible with activation (where DFG is a single-letter amino acid code for the aspartate-phenylalanine-glycine sequence at the start of the activation loop).