RKI-1447 is a potent small molecule inhibitor of ROCK1 and ROCK2 with IC₅₀ values of 14.5 nM and 6.2 nM, respectively.

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 suppresses phosphorylation of the ROCK substrates mLC-2 and MYPT-1 in human cancer cells, but has no effect on the phosphorylation levels of the AKT, MEK and S6 kinase at concentrations as high as 10 μM. RKI-1447 is also highly selective at inhibiting ROCK-mediated cytoskeleton re-organization. RKI-1447 inhibits migration, invasion and anchorage-independent tumor growth of breast cancer cells^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

RKI-1447 is highly effective at inhibiting the outgrowth of mammary tumors in a transgenic mouse model. RKI-1447 inhibits mammary tumor growth by 87% and on average the mammary tumors from RKI-1447 treated mice are 7.7 fold smaller compared to those tumors from mice treated with the vehicle control^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

326.37

C₁₆H₁₄N₄O₂S

1342278-01-6

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : ≥ 50 mg/mL (153.20 mM)

* “≥” means soluble, but saturation unknown.

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (7.66 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (7.66 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (7.66 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (7.66 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Patel RA, et al. RKI-1447 is a potent inhibitor of the Rho-associated ROCK kinases with anti-invasive and antitumor activities in breast cancer. Cancer Res. 2012 Oct 1;72(19):5025-34.

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. The ratio of the signals at 445 nm and 520 nm is measured. IC₅₀ values are determined using fitted curves with GraphPad Prism 5 software^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

NIH 3T3 cells are plated at 8000 cells/well in 8-chamber slides in serum free media for 24 hours, and treated with vehicle, 1 μM RKI-1447 or 1 μM RKI-1313 for 1 hour. The cells are then stimulated with complete media, 10 μM LPA, 200 ng/mL bradykinin or 30 ng/mL PDGF for 30 mins. After stimulation, the cells are fixed in 4% paraformaldehyde and stained with Texas-Red Phalloidin. Mounting medium containing DAPI is then added and at least 100 cells per well are observed^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice: MMTV/neu transgenic mice are treated i.p. daily for 14 days with either Vehicle [20%-2-hydroxypropyl-beta-cyclodextrin (HPCD)] or 200 mpk RKI-1447 dissolved in freshly prepared HPCD. The percent change in volume is calculated on the basis of the tumor volume on the last day of treatment (Vf) relative to that on the day of initiation of treatment (V0). The average percent change in tumor volume is then calculated for each treatment group^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Patel RA, et al. RKI-1447 is a potent inhibitor of the Rho-associated ROCK kinases with anti-invasive and antitumor activities in breast cancer. Cancer Res. 2012 Oct 1;72(19):5025-34.