RKI-1447 1342278-01-6

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Molecular Weight:

326.37 RKI-1447 is a Potent Inhibitor of ROCK1 and ROCK2, with IC50 of 14.5 nM and 6.2 nM, respectively, has anti-invasive and antitumor activities.


Biological Activity


RKI-1447 is a cell-permeable pyridylthiazolyl-urea that acts as a
potent, ATP site-targeting Rho Kinase inhibitor, displaying much reduced
potency against PKA, PKN1/PRK1, p70S6K/RPS6kB1, AKT1, MRCKa/CDC42BPA
(85.5%, 80.5%, 61.9%, 56.0%, and 50.4% inhibition, respectively, by 1 µM
RKI-1447) or 15 other kinases. Crystal structures of the RKI-1447/ROCK1
complex reveals 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 suppresses 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 is 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. RKI-1447 inhibits migration, invasion and
anchorage-independent tumor growth of breast cancer cells.


RKI-1447 is highly effective at inhibiting the outgrowth of mammary
tumors in a transgenic mouse model. Tumors from mice treated with the
RKI-1447 increases in size with an average percent change in tumor
volume of only 8.8%. Thus, RKI-1447 inhibited mammary tumor growth by
87%, and on average the mammary tumors from Compared with those tumors
from mice treated with the vehicle control, RKI-1447 treated mice are
7.7-fold smaller. RKI-1447 treatments does not result in mouse weight
loss.


Protocol(Only for Reference)


Kinase Assay: [2]

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.

Cell Assay: [1]

Cell lines	MDA-MB-231
Concentrations	~600 μM
Incubation Time	72 h
Method	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 (2mg/ml) is added to each well and incubated for 3 hours. After incubation the plates are read at 540 nm.

Animal Study: [1]

Animal Models	MMTV/neu transgenic mice [FVB/N-Tg (MMTVneu) 202 Mul/J]
Formulation	20%-2-hydroxypropyl-betacyclodextrin(HPCD)
Dosages	200 mpk/day
Administration	i.p.
Solubility	15% Captisol, 30 mg/mL
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Baboon	Dog	Monkey	Rabbit	Guinea pig	Rat	Hamster	Mouse
Weight (kg)	12	10	3	1.8	0.4	0.15	0.08	0.02
Body Surface Area (m2)	0.6	0.5	0.24	0.15	0.05	0.025	0.02	0.007
Km factor	20	20	12	12	8	6	5	3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×	mouse Km(3)	= 11.2 mg/kg
	rat Km(6)

Chemical Information

Molecular Weight (MW)	326.37
Formula	C16H14N4O2S
CAS No.	1342278-01-6

Storage	3 years -20℃Powder
	6 months-80℃in solvent (DMSO, water, etc.)
Synonyms

Solubility (25°C) *	In vitro	DMSO	65 mg/mL (199.16 mM)
		Water	<1 mg/mL (
		Ethanol	<1 mg/mL (
	In vivo	15% Captisol	30 mg/mL
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Name	Urea, N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]-

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