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H 89 2HCl

Name: H 89 2HCl
Brand: Selleck Chemicals
SKU: S1582
Rating: 5 (180 reviews)

Catalog No.S1582 Purity:99.48%

H 89 2HCl is a potent PKA inhibitor with K_i of 48 nM in a cell-free assay, 10-fold selective for PKA than PKG,500-fold greater selectivity than PKC, MLCK, calmodulin kinase II and casein kinase I/II. H 89 2HCl induces autophagy.

H 89 2HCl Chemical Structure

CAS No. 130964-39-5

Purity & Quality Control

Batch: Purity: 99.48%

99.48

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Cell Data

Cell Lines	Assay Type	Concentration	Incubation Time	Formulation	Activity Description	PMID
CHO	Growth Inhibition Assay	10 μM	1 h		inhibits 5-HT induced cAMP production decrease	12569069
SK-N-MC	Function Assay	30 μM	24 h	DMSO	reduces the down-regulation of β1-AR by isoproterenol by 50%	11705454
Sf9	Function assay				Inhibition of His-tagged human MSK1 expressed in Sf9 cells, IC50 = 0.12 μM.	10998351
Sf9	Function assay				Inhibition of rat ROCK2 expressed in Sf9 cells, IC50 = 0.27 μM.	10998351
SF9	Function assay				Inhibition of human PKBalpha expressed in SF9 cells, IC50 = 2.6 μM.	10998351
OVCAR8	Cytotoxicity assay		72 hrs		Cytotoxicity against human OVCAR8 cells assessed as growth inhibition after 72 hrs by MTT assay, IC50 = 10.5 μM.	24508830
OVCAR8	Antiproliferative assay		72 hrs		Antiproliferative activity against human OVCAR8 cells after 72 hrs by MTT assay, IC50 = 10.5 μM.	24389511
HL60	Antiproliferative assay		72 hrs		Antiproliferative activity against human HL60 cells after 72 hrs by MTT assay, IC50 = 11.4 μM.	24389511
PC3M	Function assay				Inhibition of GSK3-beta in human PC3M cells by ELISA, IC50 = 15 μM.	18345609
U87MG	Growth inhibition assay				Growth inhibition of human U87MG cells by SRB assay, IC50 = 15 μM.	18345609
U87MG	Antiproliferative assay		96 hrs		Antiproliferative activity against human U87MG cells after 96 hrs by SRB assay, IC50 = 15 μM.	20151677
HCT116	Cytotoxicity assay		72 hrs		Cytotoxicity against human HCT116 cells assessed as growth inhibition after 72 hrs by MTT assay, IC50 = 15.7 μM.	24508830
HCT116	Antiproliferative assay		72 hrs		Antiproliferative activity against human HCT116 cells after 72 hrs by MTT assay, IC50 = 15.7 μM.	24389511
PC3M	Function assay		96 hrs		Inhibition of PC3M cells by SRB assay after 96 hrs, GI50 = 18 μM.	17451235
PC3M	Growth inhibition assay				Growth inhibition of human PC3M cells by SRB assay, IC50 = 18 μM.	18345609
LS174T	Function assay	20 uM	6 hrs		Inhibition of PGE2-stimulated TCF/LEF transactivation in human LS174T cells at 20 uM measured after 6 hrs by dual luciferase reporter gene assay	27736063
Click to View More Cell Line Experimental Data

Biological Activity

Description

Targets

PKA ^[1] (Cell-free assay)	S6K1 ^[2] (Cell-free assay)	PKG ^[1] (Cell-free assay)
48 nM(Ki)	80 nM	0.48 μM(Ki)

In vitro
In vitro	H89 2HCl is a potent PKA (cAMP-dependent) protein kinase A inhibitor with K_i of 48 nM, exhibits 10-fold selectivity over PKG, exhibits >500-fold selectivity over PKC, MLCK, calmodulin kinase II and casein kinase I/II. Pretreatment of the cells with H-89 (30 μM) 1 h before the addition of forskolin markedly inhibits the forskolin-induced protein phosphorylation in a dose-dependent manner. ^[1] H89 also inhibits several other kinases with IC50 of 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b, respectively. ^[2]^[3] H89 also has activity at some cellular receptors and ion channels, including Kv1.3 K⁺ channels,β1AR and β2AR. ^[4]
Kinase Assay	PKA enzyme activity
Kinase Assay	cAMP-dependent protein kinase activity is assayed in a reaction mixture containing, in a final volume of 0.2 mL, 50 mM Tris-HC1 (pH 7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 μM cAMP or absence of cAMP, 3.3-20 μM [γ-³²P]ATP (4 × 10⁵ cpm), 0.5 μg of the enzyme, 100 μg of histone H2B, and each compound, as indicated.
Cell Research	Cell lines	PC12D
	Concentrations	~30 μM
	Incubation Time	1 h
	Method	Levels of intracellular cAMP are determined. After 48 h in culture, PC12D cells are cultured in test medium containing 30 μM H-89 for 1 h and then exposed to fresh medium that contained both 10 μM forskolin and 30 μM H-89. Cells are scraped off with a rubber policeman and sonicated in the presence of 0.5 ml of 6% trichloroacetic acid. To extract trichloroacetic acid, 2 ml of petroleum ether is added, the preparation mixed and centrifuged at 3000 rpm for 10 min. After aspiration of the upper layer, the residue sample solution is used for determination.
Experimental Result Images	Methods	Biomarkers	Images	PMID
	Western blot	p-eNOS / T-eNOS / p-PKA p-GPIbβ / GPIbβ / p-BAD p-S6K1 / p-rpS6 / p-GSK3β		31035633
	Immunofluorescence	p-eNOS / eNOS iNOS / NFκB p65 myopodin		30513637

In Vivo
In vivo	H89 causes distinct modifications of protein phosphorylation, with the most robust changes in phosphorylation are fructose-1,6-biphosphatase, heterogeneous nuclear ribonucleoprotein (hnRNP), NSFL1 cofactor p47, all which have potentially regulatory connections to cAMP/PKA. ^[5]
Animal Research	Animal Models	rat; mice
	Dosages	20 or 200 mg/kg (Rat); 0-5 mg/kg (Mice)
	Administration	s.c. (Rat); i.p. (Mice)

References

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Chemical Information & Solubility

Molecular Weight	519.28	Formula	C₂₀H₂₀BrN₃O₂S.2HCl
CAS No.	130964-39-5	SDF	Download H 89 2HCl SDF
Smiles	C1=CC2=C(C=CN=C2)C(=C1)S(=O)(=O)NCCNCC=CC3=CC=C(C=C3)Br.Cl.Cl
Storage (From the date of receipt)	3 years-20°Cpowder	Storage of Stock Solutions Aliquot the stock solutions to avoid repeated freeze-thaw cycles	1 year-80°Cin solvent
Storage (From the date of receipt)	3 years-20°Cpowder		1 month-20°Cin solvent

In vitro
Batch:

DMSO : 100 mg/mL ( (192.57 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : 10 mg/mL

Ethanol : Insoluble

Molecular Weight Calculator

In vivo Batch: Add solvents to the product individually and in order.				In vivo Formulation Calculator
Clear solution	saline	10.000mg/ml (19.26mM)	Taking the 1 mL working solution as an example, add 10 mg of this product to 1 ml of physiological saline (0.9% NaCL solution), mix evenly to make it clear, The mixed solution should be used immediately for optimal results.
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.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	5.000mg/ml (9.63mM)	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to make it clear; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Clear solution	saline	4.000mg/ml (7.70mM)	Taking the 1 mL working solution as an example, add 4 mg of this product to 1 ml of physiological saline (0.9% NaCL solution), mix evenly to make it clear, The mixed solution should be used immediately for optimal results.
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.
Clear solution	saline	6.000mg/ml (11.55mM)	Taking the 1 mL working solution as an example, add 6 mg of this product to 1 ml of physiological saline (0.9% NaCL solution), mix evenly to make it clear, The mixed solution should be used immediately for optimal results.
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.
Clear solution	saline	6.000mg/ml (11.55mM)	Taking the 1 mL working solution as an example, add 6 mg of this product to 1 ml of physiological saline (0.9% NaCL solution), mix evenly to make it clear, The mixed solution should be used immediately for optimal results.
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.
Clear solution	5%DMSO 1 Corn oil Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	5.000mg/ml (9.63mM)	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.

Preparing Stock Solutions

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
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Dilution Calculator Molecular Weight Calculator

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

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Calculation results:

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.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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