In vitro activity: PD 169316 is a novel potent, cell-permeable and specific/selective inhibitor of p38 MAPK kinase with IC50 value of 89 nM. PD169316 was also reported to show antiviral activity against Enterovirus71. PD169316 abrogates signaling initiated by both TGFbeta and Activin A, but not bone morphogenetic protein (BMP) 4. Inhibition of TGFbeta signaling is dose dependent and results in reduced Smad2 and Smad3 phosphorylation, nuclear translocation, and up-regulation of the TGFbeta target gene Smad7. Reduced TGFbeta signaling is not due to abrogation of p38 MAPK activity, since blocking p38 MAPK activity with a dominant negative form of p38 MAPK has no effect on TGFbeta/Smad signaling. The use of PD169316 at 5 MICROM or higher can block TGFbeta signaling activity and thus caution must be used when attributing cellular activities exclusively to p38 MAPK signaling when these inhibitors are used experimentally.

Kinase Assay: Sixteen hours after the removal of serum from Rat-1 fibroblasts or NGF from differentiated PC12 cells, the cells are incubated in the absence or presence of insulin (50 ng/mL) for 15 min at 37°C. After washing with 2 mL of ice-cold PBS, the cells are solubilized in 400 μL of ice-cold immunoprecipitation buffer containing 10 mM Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, and 0.2 mM phenylmethylsulfonyl fluoride. The cell lysates are centrifuged to remove insoluble material, and 200 μg of the supernatant protein (400 μL, total volume) are incubated with 1 μg of anti-p38 antibodies for 1 h at 4°C followed by incubation with 30 μL of Protein G Plus/Protein A-agarose for an additional hour. The immunocomplexes are pelleted and washed twice in immunoprecipitation buffer and then once in kinase ish buffer (50 mM β-glycerolphosphate, 1 mM EGTA, 20 mM MgCl2, 100 μM sodium orthovanadate). The protein kinase assay is initiated by the addition of 20 μL of 2× reaction buffer (50 mMβ-glycerolphosphate, 1 mM EGTA, 20 mM MgCl2, 100 μM sodium orthovanadate, 0.1 mg/mL ATF-2 (N-terminal half), 50 μg/mL IP20, a peptide inhibitor of c-AMP dependent protein kinase, 200 μM ATP, and 0.9 mCi/mL [32P]ATP) to 20 μL of immune complex. The reaction is allowed to proceed for 10 min at 30°C and then terminated by the addition of 2× LaemmLi sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis using 12% acrylamide gels. After electrophoresis, the gels are dried and subjected to phosphoimaging.

Cell Assay: PD169316 (10 μM) shows a significantly increased rate of proliferation in Nestin knockdown cells, and can rescue the effect of Nestin knockdown on cell viability in the absence of EGF.PD169316 significantly inhibits p38 MAP kinase activity with no significant change in ERK activity in PC12 cells. PD169316 (10 μM) blocks apoptosis induced by trophic factor withdrawal in differentiated PC12 cells.