Cell experiment:

HeLa cells are maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin. Before use HeLa cells are serum starved for 16 h in DMEM supplemented with 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin. HeLa cells are then incubated with ERK5-IN-1 at the indicated concentrations for 1 h prior to stimulation with 0.5mol/Lsorbitol for 30 min. Cells are lysed in Triton lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.27mol/Lsucrose, 1 μM microcystin-LR, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol) and 20 μg of protein loaded per well. Samples are run on 8% polyacrylamide gels using standard methods. Proteins are transferred onto nitrocellulose membranes and specific proteins detected by immunoblotting.

XMD17-109 is a new inhibitor of ERK5, IC50 value in HeLa cell is 0.09 ± 0.03 μM, and in vitro, Enzymatic IC50 value is 0.162 ± 0.006 μM.

XMD17-109 is capable of inhibiting the ERK5 autophosphorylation in cells.[1]

Through intravenous injection and oral delivery of XMD17-109 in mice, the pharmacokinetic properties of this compound are as bellows: the T1/2 (half time) is 8.2 h, the plasma clearance is 8.64 mL/min/Kg (data of intravenous injection), the AUC (area under the curve) of oral delivery is 15745 h*ng/mL and the oral bioavailability is 90%.

Reference:
1.  Deng, X., et al., Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones. European Journal of Medicinal Chemistry, 2013. 70: p. 758-767.