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H 89 2HCl

H 89 2HCl is a potent PKA inhibitor with Ki 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

H 89 2HCl Chemical Structure

CAS No. 130964-39-5

Purity & Quality Control

H 89 2HCl Related Products

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
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Biological Activity

Description H 89 2HCl is a potent PKA inhibitor with Ki 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.
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 Ki 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
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)

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)

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


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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.

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