In vitro activity: XMD17-109 potently inhibits ERK5 biochemically with an IC50 of 162 nM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 μM

Kinase Assay: Kinase activity was determined in an assay volume of 40 μL in kinase buffer (50 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 1 mM 2-mercaptoethanol) containing 200 ng of pure active ERK5 and the indicated amount of inhibitor. Reaction started by adding 10 mM magnesium acetate, and 50 μM [γ-32P]-ATP (500 cpm/pmol) and 250 μM PIMtide (ARKKRRHPSGPPTA) as substrates. Assays were carried out for 20 min at 30 °C, terminated by applying the reaction mixture onto p81 paper and the incorporated radioactivity measured as described previously.

Cell Assay: HeLa cells were serum starved overnight followed by treatment with inhibitors for 1 h. Cells were then stimulated with EGF (20 ng/mL) for 17 min and harvested in RIPA buffer (1× PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF and 1 mM sodium orthovanadate). Proteins from total cell lysates were resolved by 6% sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membrane, blocked in 5% nonfat milk, and blotted with anti-ERK5 antibody.

Eur J Med Chem. 2013;70:758-67.