RKI-1447 is a potent inhibitor of the Rho-associated ROCK kinases with anti-invasive and antitumor activities in breast cancer.

The Rho-associated kinases ROCK1 and ROCK2 are critical for cancer cell migration and invasion, suggesting they may be useful therapeutic targets. In this study, we describe the discovery and development of RKI-1447, a potent small molecule inhibitor of ROCK1 and ROCK2. Crystal structures of the RKI-1447/ROCK1 complex revealed that RKI-1447 is a Type I kinase inhibitor that binds the ATP binding site through interactions with the hinge region and the DFG motif. RKI-1447 suppressed 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 μmol/L. RKI-1447 was 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, respectively. RKI-1447 inhibited migration, invasion and anchorage-independent tumor growth of breast cancer cells. In contrast, RKI-1313, a much weaker analog in vitro, had little effect on the phosphorylation levels of ROCK substrates, migration, invasion or anchorage-independent growth. Finally, RKI-1447 was highly effective at inhibiting the outgrowth of mammary tumors in a transgenic mouse model. In summary, our findings establish RKI-1447 as a potent and selective ROCK inhibitor with significant anti-invasive and antitumor activities and offer a preclinical proof-of-concept that justify further examination of RKI-1447 suitability as a potential clinical candidate.

Patel RA ，Forinash KD ，Pireddu R ，Sun Y ，Sun N ，Martin MP ，Schönbrunn E ，Lawrence NJ ，Sebti SM ... -  《-》

Identification of novel ROCK inhibitors with anti-migratory and anti-invasive activities.

Patel RA ，Liu Y ，Wang B ，Li R ，Sebti SM ... -  《-》

RKI-1447 suppresses colorectal carcinoma cell growth via disrupting cellular bioenergetics and mitochondrial dynamics.

Accumulated evidence suggested the importance of the Rho/Rho-kinase (ROCK) signaling pathway in cancer proliferation and invasion. However, its role in colorectal carcinoma (CRC) is not well understood. This study evaluated the effect of ROCK signaling pathway on CRC behavior on the basis of a novel Rho/ROCK inhibitor RKI-1447. Here, we found RKI-1447 could drastically suppress HCT-8 and HCT-116 cell growth and promoted apoptosis. Our in vitro data indicated suppressed cytoskeletal dynamics induced by RKI-1447 inhibition on mitochondrial respiration, which was evidenced by basal and maximal respiration rates, and ATP production. Simultaneously, cellular basal and maximal glycolytic rates, and glycolytic capacity were also reduced in response to RKI-1447. Moreover, RKI-1447 caused excessive reactive oxygen species generation and membrane depolarization as well as activated ER-stress. We also demonstrated CHOP is essential for RKI-1447 induced cell apoptosis. Finally, we proved inhibition of ROCK by RKI-1447 could effectively inhibit CRC growth in vivo. Taken together, this study demonstrated that inhibition of ROCK signaling pathway by RKI-1447 could suppress CRC via cytoskeleton associated mitochondrial dysfunction and cellular bioenergetics disruption. Our data suggest RKI-1447 may be an attractive antitumor drug candidate for the treatment of CRC.

Li L ，Chen Q ，Yu Y ，Chen H ，Lu M ，Huang Y ，Li P ，Chang H ... -  《-》

A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

Unbekandt M ，Croft DR ，Crighton D ，Mezna M ，McArthur D ，McConnell P ，Schüttelkopf AW ，Belshaw S ，Pannifer A ，Sime M ，Bower J ，Drysdale M ，Olson MF ... -  《Cell Communication and Signaling》

Discovery of novel pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors with anti-breast cancer migration and invasion activities.

Rho-associated coiled-coil kinase (ROCK) is involved in multiple cellular activities regulating the actin cytoskeleton, such as cell morphology, adhesion, and migration. The inhibition of ROCK is a feasible strategy to suppress breast cancer metastasis. Herein, based on Belumosudil, a series of pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors were designed and synthesized. Through systematic investigation of SARs, the piperazine analog 7u was identified with optimum ROCK2 inhibitory activity (IC50 = 36.8 nM) and excellent selectivity over the isoform protein ROCK1 (>250-fold). Intriguingly, upon treatment with 7u, the arrangement of the MDA-MB-231 cytoskeleton was affected accompanied by the alteration of morphology. Furthermore, cell scratch and transwell assays indicated that 7u inhibited MDA-MB-231 cell migration and invasion in a dose-dependent manner. Ultimately, the binding model of 7u with ROCK2 well accounted for the superior activities of 7u as a promising ROCK2 inhibitor with the potential application in breast cancer metastasis treatment.

Cao Z ，Wei X ，Xing S ，Zhang J ，Wang S ，Yue L ，Zhang J ，Jiang N ，Zhai X ... -  《-》