RKI-1447 Is a Potent Inhibitor of the Rho-Associated ROCK Kinases with Anti-Invasive and Antitumor Activities in Breast Cancer

Abstract

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 μM. 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. Lastly 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 anti-tumor activities and offer a preclinical proof-of-concept that justify further examination of RKI-1447 suitability as a potential clinical candidate.

Ocular Hypotension, Actin Stress Fiber Disruption and Phagocytosis Increase by RKI-1447, a Rho-Kinase Inhibitor

“…In the current study, we utilized a porcine anterior segment perfusion model that mimicks the TM changes and IOP phenotype of human PG to examine the hypotensive effect of RKI-1447, an oncology drug used in breat cancer 4 . Pigment dispersion caused a significant IOP elevation at the 48-hour time interval, while RKI-1447 partially reversed this effect throughout the study.…”

“…The only drugs that increase the conventional outflow that are readily available at the time of this writing are cholinergic agonists who have miosis and ciliary spasm as significant side effects 2 although NO donors are on the horizon that relax the TM to increase outflow 3 . In the present study we examine the hypotensive effect of RKI-1447, which was originally introduced as an anti-tumor agent in breast cancer treatment 4 .…”

“…Several trials that employed functionally and structurally similar compounds were discontinued due to the low therapeutic efficiency and high incidence of adverse events , RKI-1447 inhibits the type I kinase by binding to the ATP binding site through interaction with the Asp-Phe-Gly motif and hinge region (Figure 1) 4,20 . In vitro, it selectively inhibits the ROCK1/2-mediated actin stress fiber formation at the low level of IC-50 (14.5nM and 6.2nM for ROCK1 and ROCK2, respectively 21 ) following lysophosphatidic acid stimulation.…”

“…In vitro, it selectively inhibits the ROCK1/2-mediated actin stress fiber formation at the low level of IC-50 (14.5nM and 6.2nM for ROCK1 and ROCK2, respectively 21 ) following lysophosphatidic acid stimulation. Unlike other ROCK inhibitors, RKI-1447 does not exhibit cross-reactivity with AKT, MEK, PKA, and PKG signaling, even at a high concentration of up to 10 μM 4,21 , reducing the risk of side effects. Recently, our group developed an ex vivo pigmentary glaucoma model that exhibited a IOP elevation, a typical TM stress fiber formation, and a reduction in TM phagocytosis 22,23 .…”

Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels

“…eIF5A Regulates RhoA and ROCK2 Protein Expression in PDAC Cells-Although Rho/ROCK signaling has been linked to cell motility and metastasis in various cancers including PDAC, little is known about how Rho/ROCK protein levels are regulated in tumor cells, especially in regard to translation (37)(38)(39)(40)(41). Therefore, we decided to focus on RhoA and ROCK2, established components of the Rho/ROCK pathway whose protein levels were reduced upon eIF5A knockdown (Fig.…”