RKI-1447

RKI-1447 is a cell-permeable pyridylthiazolyl-urea that acts as a potent, ATP site-targeting Rho Kinase inhibitor, displaying much reduced potency against PKA, PKN1/PRK1, p70S6K/RPS6kB1, AKT1, MRCKa/CDC42BPA (85.5%, 80.5%, 61.9%, 56.0%, and 50.4% inhibition, respectively, by 1 µM this compound) or 15 other kinases. Crystal structures of the this chemical/ROCK1 complex reveals that this inhibitor is a Type I kinase inhibitor that binds the ATP binding site through interactions with the hinge region and the DFG motif. It suppresses phosphorylation of the ROCK substrates MLC-2 and MYPT-1 in human cancer cells, but had no effect on the phosphorylation levels of the AKT, MEK, and S6 kinase at concentrations as high as 10 μM. This compound is also highly selective at inhibiting ROCK-mediated cytoskeleton re-organization (actin stress fiber formation) following LPA stimulation, but does not affect PAK-meditated lamellipodia and filopodia formation following PDGF and Bradykinin stimulation. It inhibits migration, invasion and anchorage-independent tumor growth of breast cancer cells. ^[1]

Cells are plated in a 96 well tissue culture plate (1200 cells per well) and incubated for 24 hours. After incubation the cells are treated with vehicle or increasing concentrations of RKI-1447 for 72 hours. After incubation, freshly prepared MTT (2mg/ml) is added to each well and incubated for 3 hours. After incubation the plates are read at 540 nm.

RKI-1447 is highly effective at inhibiting the outgrowth of mammary tumors in a transgenic mouse model. Tumors from mice treated with this compound increases in size with an average percent change in tumor volume of only 8.8%. Thus, it inhibited mammary tumor growth by 87%, and on average the mammary tumors from Compared with those tumors from mice treated with the vehicle control, this chemical treated mice are 7.7-fold smaller. This compound treatments does not result in mouse weight loss. ^[1]