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RepSox (E-616452) TGF-beta/Smad inhibitor

Name: RepSox (E-616452)
Brand: Selleck Chemicals
SKU: S7223
Rating: 5 (82 reviews)

Cat.No.S7223

RepSox (E-616452, SJN 2511, ALK5 Inhibitor II) is a potent and selective inhibitor of TGFβR-1/ALK5 with an IC50 of 23 nM and 4 nM for ATP binding to ALK5 and ALK5 autophosphorylation in cell-free assays, respectively.

Chemical Structure

Molecular Weight: 287.32

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Quality Control

Batch: Purity: 99.92%

99.92

Related Targets	PKC ROCK Bcr-Abl
Other TGF-beta/Smad Inhibitors	SB431542 Vactosertib (TEW-7197) LDN-193189 A-83-01 LDN-193189 Dihydrochloride Galunisertib (LY2157299) SRI-011381 (C381) LY2109761 SIS3 Hydrochloride SB-525334

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Concentration	Incubation Time	Activity Description	PMID
baculovirus/Sf9 cells	Function assay			Inhibition of recombinant Activin A receptor type II-like kinase (ALK5) expressed in baculovirus/Sf9 cells, IC50=4 nM	15317461
HepG2 cells	Function assay			Stimulation of Transforming growth factor beta receptor I kinase in HepG2 cells, IC50=10 nM	15317461
Sf9	Function assay			Inhibition of human ALK5 expressed in Sf9 insect cells using casein as substrate in presence of [gamma-33P]ATP, IC50 = 0.181 μM.	28135685
Sf9	Function assay			Inhibition of mouse ABL expressed in Sf9 insect cells using GGEAIYAAPFKK as substrate in presence of [gamma-33P]ATP, IC50 = 21 μM.	28135685
HeLa	Function assay	1 uM	30 mins	Inhibition of ALK5 in serum starved human HeLa cells assessed as reduction in TGF-beta-induced SMAD2/3 nuclear transaction at 1 uM pre-incubated for 30 mins before TGF-beta stimulation for 30 mins by immunofluorescence analysis	28135685
Click to View More Cell Line Experimental Data

Chemical Information, Storage & Stability

Molecular Weight	287.32	Formula	C₁₇H₁₃N₅	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	446859-33-2	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent
Synonyms	SJN 2511,ALK5 Inhibitor II			Smiles	CC1=NC(=CC=C1)C2=C(C=NN2)C3=NC4=C(C=C3)N=CC=C4

Solubility

In vitro
Batch:

DMSO : 57 mg/mL (198.38 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
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Dilution Calculator Molecular Weight Calculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

In vivo Formulation Calculator (Clear solution)

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Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
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Mechanism of Action

Targets/IC₅₀/Ki	TGFβR1(ALK5) (Cell-free assay) 4 nM
In vitro	RepSox (E-616452) inhibits ATP binding to ALK5 and ALK5 autophosphorylation with IC50 of 23 nM and 4 nM, respectively. It also inhibits TGF-β induced cellular PAI-1 luciferase activity with IC50 of 18 nM. This compound is able to successfully replace Sox2 in reprogramming by inhibiting transforming growth factor-β (Tgf-β) signalling, which in turn induces Nanog expression. Effect of RepSox inducing reprogramming does not require chromatin remodelling. It is found to be efficient at generating iPSCs.
Kinase Assay	ALK5 fluorescence polarisation binding assay
Kinase Assay	Compound binding to ALK5 is tested on purified recombinant GST-ALK5 (residues 198-503). Displacement of rhodamine green fluorescently labelled ATP competitive inhibitor by different concentrations of test compounds is used to calculate a binding pIC50. GST-ALK5 is added to a buffer containing 62.5 mM Hepes, pH 7.5, 1 mM DTT, 12.5 mM MgCl₂, 1.25 mM CHAPS, and 1 nM rhodamine green-labelled ligand so that the final ALK5 concentration is 10 nM based on active site titration of the enzyme. Forty microlitres of the enzyme/ligand reagent is added to 384 well assay plates containing 1 μL of different concentrations of RepSox (E-616452). The plates are read immediately on a fluorescence reader with excitation, emission, and dichroic filters of 485, 530, and 505 nm, respectively. The fluorescence polarisation for each well is calculated by the Acquest and is then imported into curve fitting software for construction of concentration-response curves.
In vivo	When injected into blastocysts, RepSox (E-616452) is able to contribute to forming chimeric embryos in vivo. A one-day treatment with this compound is sufficient to replace transgenic Sox2.
References	[1] https://pubmed.ncbi.nlm.nih.gov/15317461/ [2] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335195/ [3] https://pubmed.ncbi.nlm.nih.gov/36207615/ [4] https://pubmed.ncbi.nlm.nih.gov/32066676/

Applications

Methods	Biomarkers	Images	PMID
Western blot	α-SMA / Calponin / Histone H3		30365043
Immunofluorescence	Smad2/3 / cytokeratin		28729719

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