American Association for Cancer Research
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Supplementary Figure 3 from RKI-1447 Is a Potent Inhibitor of the Rho-Associated ROCK Kinases with Anti-Invasive and Antitumor Activities in Breast Cancer

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journal contribution
posted on 2023-03-30, 21:34 authored by Ronil A. Patel, Kara D. Forinash, Roberta Pireddu, Ying Sun, Nan Sun, Mathew P. Martin, Ernst Schönbrunn, Nicholas J. Lawrence, Saïd M. Sebti

PDF file, 86K, RKI-1447 and RKI-1313 have little effect on cell cycle progression and apoptosis. (Panel A) MDA-MB-231 breast cancer cells were treated with either vehicle, RKI-1447 (10 μM) or RKI-1313 (10 μM) for 48 hours, fixed with 70% methanol overnight and stained with Propidium Iodide/Triton X-100. Cell cycle analysis was performed on BD Bioscience FACS Scan system as described below. (Panel B) MDA-MB-231 breast cancer cells were treated with either vehicle, RKI-1447 or RKI-1313 for 24 hours and processed for western immunoblotting with the indicated antibodies as described under Materials and Methods. Cell Cycle Analysis Method: MDA-MB-231 cells were plated in a 60mm dish and treated the next day with either vehicle, RKI- 1447 (10 �M) or RKI- 1313 (10 �M) for 48 hours. After incubation the cells were trypsinized and washed with cold PBS. The cells were resuspended in 0.5ml cold PBS and were fixed by adding 70% ethanol drop wise with gentle vortexing to obtain a single-cell suspension. Cells were left in the fixative overnight at 4{degree sign}C. The next day the cells were centrifuged and resuspended in 5ml PBS and washed. The cells were then incubated at room temperature for 1 hour in 1ml Propidium Iodide/Triton X-100 staining solution with RNase. After incubation, cell cycle analysis was performed on BD Bioscience FACS Scan system. The data was acquired and analyzed using BD CellQuest Pro software and ModFit, respectively.



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. Cancer Res; 72(19); 5025–34. ©2012 AACR.