Tie2 kinase inhibitor 1 (Tie2 kinase inhibitor), an optimized compound of SB-203580, is selective to Tie2 with IC50 of 0.25 μM, which is 200-fold more effective than p38.

Tie2:0.25 μM

In HEL cells, Tie2 kinase inhibitor 1（IC50=232 nM） displays medium cellular activities. Tie2 kinase inhibitor 1 shows a medium inhibitory activity against Tie2 tyrosine kinase 1. Tie2 kinase inhibitor 1 is more specific for Tie2 than p38 (IC50=50 μM), and is a >10-fold specificity than VEGFR2, VEGFR3, and PDGFR1β.

In a matrigel mouse model of angiogenesis, Tie2 kinase inhibitor 1 reduces angiogenesis. At doses of 25 and 50 mg/kg (i.p., b.i.d) for Tie2 kinase inhibitor 1, results on a reduction of 41% and 70% of angiogenesis, respectively. In a MOPC-315 plasmacytoma xenograft model, Tie2 kinase inhibitor treatment results in a modest dose-dependent delay in tumor growth.

Enzymatic HDAC activity assays: 40 μL enzyme buffer (15 mM Tris HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% v:v glycerol) containing HDAC1, 3, 6 or 8 activity, 29 μL enzyme buffer and 1 μL resminostat at different concentrations are added to a 96-well microtitre plate and the reaction started by the addition of 30μL substrate peptide Ac-NH-GGK(Ac)-AMC (HDAC1, 3 and 6 assays, final concentrations 6 μM for HDAC1, 10 μM for HDAC6 and 25 μM for HDAC3/DAD) or Ac-RHK(Ac)K(Ac)-AMC (HDAC8 assay, final concentration 50 μM). After incubation for 3 hours (HDAC1, HDAC6, HDAC8) or 2 hours (HDAC3) at 30°C, the reaction is terminated by the addition of 25 μL stop solution (50 mM Tris HCl pH 8, 100 mM NaCl, 0.5 mg/mL trypsin and 2 μM trichostatin A [TSA]). After incubation at room temperature for further 40 min, fluorescence is measured using a multilabel counter (extinction 355 nm, emission 460 nm) for quantification of AMC generated by tryptic cleavage of the deacetylated peptide. For the calculation of the IC50, the fluorescence in wells without test compound (1% DMSO, negative control) is set as 100% enzymatic activity and the fluorescence in wells with 2 μM TSA (positive control) is set at 0% enzymatic activity (background fluorescence substracted).

In MOPC-315 plasmacytoma xenograft model, Tie2 kinase inhibitor 1(≤50 mg), which is dissolved in 5% EtOH+5%cremophor+90% water, is injected intraperitoneally.