RKI-1447 is a potent inhibitor of ROCK1 and ROCK2. It has anti-invasive and antitumor activities.

ROCK1:14.5 nM, ROCK2:6.2 nM

RKI-1447抑制87％乳腺肿瘤的生长,并且和对照小鼠相比,乳腺肿瘤平均体积减小7.7倍.RKI-1447在转基因小鼠模型中,有效抑制乳腺肿瘤的生长.小鼠肿瘤用RKI-1447处理时,肿瘤体积增加的平均百分比仅为8.8％.

RKI-1447抑制人癌细胞中ROCK底物MLC-2和MYPT-1的磷酸化,但对浓度高达10 μM的AKT,MEK和S6激酶的磷酸化水平没有影响。RKI-1447抑制乳腺癌细胞的迁移,侵袭和锚定非依赖性肿瘤的生长。RKI-1447/ROCK1复合物的晶体结构显示RKI-1447是I型激酶抑制剂,其通过与铰链区和DFG基序的相互作用结合ATP结合位点。

Z-Lyte FRET kinase assay: Kinase inhibition is measured using the Invitrogen Z-Lyte? FRET kinase assay with Ser/Thr 13 peptide substrate based on the myosin light chain sequence KKRPQRRYSNVF. Compounds are tested on three separate days with 8 point dilutions performed in duplicate to determine average IC50 values. The assay conditions are optimized to 15 μL of kinase reaction volume with 5 ng of enzyme in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35. The reaction is incubated for 1 h at room temperature in the presence of 1.5 μM of peptide substrate with 12.5 μM of ATP (for ROCK1) or 2 μM of substrate with 50 μM of ATP (for ROCK2). The reaction is then stopped and the ratio of phosphorylated to unphosphorylated peptides is determined by selective cleavage of only the unphosphorylated peptide as described by the manufacturer. This is followed by excitation of coumarin at 400 nm resulting in emission at 445 nm and energy transfer to fluorescein and final emission at 520 nm. The substrate contains both coumarin and fluorescein and only uncleaved phosphorylated substrate will undergo FRET. The ratio of the signals at 445 nm and 520 nm is measured using a Wallac EnVision Plate Reader, model 2102 plate-reader.

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 (2 mg/ml) is added to each well and incubated for 3 hours. After incubation the plates are read at 540 nm. (Only for Reference)