| Assay Method Information | |
| | p38 Inhibitory Potency Assay |
| Description: | Inhibition of MK2 and PRAK by Compound (P)-I and (M)-I, respectively, was investigated to understand selectivity of Compound (P)-I and/or Compound (M)-I in reducing inflammatory response. Compound (P)-I and (M)-I were evaluated in enzyme assays that compared inhibitor potency in blocking p38/MK2 versus p38/PRAK-induced phosphorylation of an HSP-27-derived peptide substrate. The ability of Compound (P)-I and (M)-I to inhibit activated phospho-p38α was evaluated using a p38α/MK2 and a p38α/PRAK cascade assay format. The kinase activity of p38α was determined by its ability to phosphorylate GST-MK2 or GST-PRAK. Activation of MK2 or PRAK by p38α was quantitated by measuring the phosphorylation of a fluorescently-labeled, MK2/PRAK specific peptide substrate, Hsp27 peptide. The phosphorylation of the Hsp27 peptide was quantified using IMAP technology (Molecular Devices, Sunnyvale CA). Kinase reactions were carried out in a 384-well plate (Greiner, 781280) in 20 mM HEPES pH 7.5, 10 mM MgCl2, 0.01% Triton X-100, 0.01% BSA, 1 mM DTT, and 2% DMSO. The concentration of inhibitor in the assays was varied between 0.02 nM to 30,000 nM, while the Hsp27 peptide substrate and MgATP were held constant at 1 μM and 10 μM, respectively. Activated p38α was added to a final concentration of 30 pM for reactions with non-phosphorylated 1 nM GST-MK2 in the cascade reaction. For the p38α/PRAK cascade, the concentration of non-activated GST-PRAK was held constant at 10 nM while p38α was added to a final concentration of 200 pM. Kinase reactions were incubated at room temperature and quenched after 120 minutes by the addition of IMAP Binding Solution. Under these conditions, approximately 20% of the substrate Hsp27 peptide was phosphorylated. Reactions were initiated by the addition of activated p38α except for pre-incubation experiments, where reactions were initiated by the addition of Hsp27 peptide and MgATP. Pre-incubation of p38α with inhibitor or p38α with non-activated GST-MK2 or non-activated GST-PRAK and inhibitor were performed at 2× final assay concentrations at room temperature 240 minutes prior to adding ATP and Hsp27 peptide to initiate catalysis. |
| Affinity data for this assay | |
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