PKA (Ki = 48 nM); S6K1 (IC50 = 80 nM); PKG (Ki = 0.48 μM)

Protein kinase A is competitively inhibited by H-89 when ATP is used. In PC12D cells, H-89 inhibited forskolin-induced protein phosphorylation in a dose-dependent manner without lowering intracellular cyclic AMP levels. In PC12D cells, H-89 strongly suppresses the neurite development caused by forskolin. In PC12D cell lysates, H-89 (30 μM) dramatically reduces cAMP-dependent histone IIb phosphorylation activity [1]. Rat cortical fiber reinitiation was markedly retarded by H-89 (1-2 μM), most likely as a result of its negative influence on T system potential. H-89 (10-100 μM) modifies the Ca32 sensitivity of the contractile apparatus in rat skin fibers and suppresses the net Ca2+ absorption of SR [2].

In PTZ-treated animals, H-89 (0.2 mg/100g, i.p.) markedly increased seizure latency and threshold. H-89 considerably raises epilepsy latency and epilepsy threshold and inhibits the epileptogenic action of bucladesin (300 nM) at doses of 0.05 and 0.2 mg/100 g, i.p. [3].

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Effect of H-89 pre-treatment on PTZ-induced seizure [3]
Effects of pretreatment with different doses of H-89 (0.05, 0.1 and 0.2 mg/100 g, i.p., 30 min) on PTZ (0.5% w/v i.v)-induced seizure are shown in Fig. 2A and B. The administration of H-89 at a dose of 0.2 mg/100 g significantly increased seizure latency and threshold compared to the control group (***P < 0.001). No significant differences were observed in seizure latency and threshold with two other doses of H-89 (0.05 and 0.1 mg/100 g) in comparison with control animals (Fig. 2A and B).

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Effects of pre-treatment with pentoxifylline and H-89 in combination on PTZ-induced seizure in mice [3]
All animals belonging to this combination group received PTX as the first component for 45 min and H-89 as the second one 30 min before PTZ infusion. Data obtained from groups that received PTX 50 mg/kg and H-89 0.2 mg/100 g, and PTX 100 mg/kg and H-89 0.2 mg/100 g, showed significant differences in seizure latency and threshold compared to controls (***P < 0.001) (Fig. 4A and B). The effect of H-89 (0.2 mg/100 g) on seizure threshold and latency was significantly attenuated by PTX (50 and 100 mg/kg) administration significantly (*P < 0.05) (Fig. 4A and B).

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.

Levels of intracellular cAMP are determined. After 48 hours of culture, PC12D cells are grown for 1 hour in test medium containing 30 μM H-89 before being exposed to brand-new medium containing 10 μM forskolin and 30 μM H-89. 0.5 ml of 6% trichloroacetic acid is added while cells are scraped off with a rubber policeman and sonicated. 2 ml of petroleum ether is added, the mixture is mixed, and the solution is centrifuged at 3000 rpm for 10 minutes to extract the trichloroacetic acid. The residue sample solution is used for analysis after aspiration of the top layer.

rat; mice
20 or 200 mg/kg (Rat); 0-5 mg/kg (Mice)
s.c. (Rat); i.p. (Mice)

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Pentoxifylline (25, 50, 100 mg/kg), bucladesine (50, 100, 300 nM/mouse) and H-89 (0.05, 0.1, 0.2 mg/100 g) were administered intraperitoneally (i.p.) 30 min before intravenous (i.v.) infusion of PTZ. In combination groups, the first and second components were injected 45 and 30 min before PTZ infusion. In all groups, the respective control animals received an appropriate volume of vehicle. For the i.v. infusion, the needle was inserted into the lateral tail vein, fixed to the tail vein by a narrow piece of adhesive tape, and the animal was allowed to move freely (Gholipour et al., 2008, 2009). PTZ solution was infused at a concentration rate of 1 ml/min.[3]

[1]. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem. 1990 Mar 25;265(9):5267-72.

[2]. Effects of the PKA inhibitor H-89 on excitation-contraction coupling in skinned and intact skeletal muscle fibres. J Muscle Res Cell Motil. 2001;22(3):277-86.

[3]. Effects of pentoxifylline and H-89 on epileptogenic activity of bucladesine in pentylenetetrazol-treated mice. Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.

N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide belongs to the isoquinoline class of compounds. It is a sulfonamide formed by the condensation of the sulfonic acid group of isoquinoline-5-sulfonic acid with the primary amino group of N(1)-[3-(4-bromophenyl)prop-2-en-1-yl]ethane-1,2-diamine. It is a protein kinase A inhibitor, belonging to EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitors. It belongs to the isoquinoline class, sulfonamide class, bromobenzene class, olefin class, and secondary amino class. It is the conjugate base of N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide (2+). The newly synthesized isoquinoline sulfonamide H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide) was shown to have potent and selective inhibitory activity against cyclic adenosine monophosphate-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 ± 0.008 μM. H-89 exhibited weaker inhibitory activity against other kinases, with Ki values of 0.48 ± 0.13 μM, 31.7 ± 15.9 μM, 38.3 ± 6.0 μM, 136.7 ± 17.0 μM, 28.3 ± 17.5 μM, and 29.7 ± 8.1 μM for cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II, respectively. Kinetic analysis showed that H-89 inhibits protein kinase A in a competitive manner with ATP. To investigate the role of protein kinase A in neurite growth of PC12 cells, we treated PC12 cells with H-89, nerve growth factor (NGF), fosskine, or dibutyryl cAMP, respectively. H-89 pretreatment dose-dependently inhibited fosskine-induced protein phosphorylation without reducing intracellular cAMP levels in PC12D cells; however, NGF-induced protein phosphorylation was not inhibited. H-89 also significantly inhibited fosskine-induced neurite growth in PC12D cells. This inhibitory effect was also observed when H-89 was added before dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 μM) significantly inhibited cAMP-dependent histone IIb phosphorylation activity in cell lysates, but had no effect on the phosphorylation activities of other proteins, such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and α-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit NGF-induced neurite growth in PC12D cells. Therefore, fosskelin and dibutyryl cAMP-induced neurite growth is clearly mediated by protein kinase A, while NGF-induced neurite growth is mediated by a protein kinase A-independent pathway. [1]

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We examined the specificity of 28 commercially available compounds that are reported to be relatively selective inhibitors of specific serine/threonine-specific protein kinases and tested against a wide range of protein kinases. The results showed that compounds KT 5720, Rottlerin, and quercetin inhibited multiple protein kinases, sometimes with inhibitory potency far exceeding their presumed targets. Therefore, conclusions drawn from cell experiments based on these compounds may contain errors. Ro 318220 and its related bisindolemaleimide compounds, as well as H89, HA1077, and Y 27632, were more selective inhibitors, but they still inhibited two or more protein kinases with similar potency. The study found that LY 294002 exhibited inhibitory potency against casein kinase-2 comparable to its inhibitory potency against phosphatidylinositol 3-kinase. The most selective compounds included KN62, PD 98059, U0126, PD 184352, rapamycin, vortmannin, SB 203580, and SB 202190. Similar to PD 98059, U0126 and PD 184352 block the mitogen-activated protein kinase (MAPK) cascade in cell experiments by inhibiting the activation of mitogen-activated protein kinase (MAPK) rather than directly inhibiting the activity of MKK1. Apart from rapamycin and PD 184352, even the most selective inhibitors affect at least one other protein kinase. Our results suggest that the specificity of protein kinase inhibitors cannot be assessed by studying only the effects of the inhibitors on kinases closely related to the primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based experiments. [2]

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H89 is a selective and potent inhibitor of protein kinase A (PKA). Since its discovery, it has been widely used to evaluate the effects of PKA in tissues such as the heart, osteoblasts, hepatocytes, smooth muscle cells, nerve tissue, and epithelial cells. Despite the widespread use of H89, the mechanism by which it specifically inhibits PKA remains not fully elucidated. The study also showed that H89 inhibits at least eight other kinases and has a considerable number of PKA-independent effects, which may seriously affect the interpretation of the data. Therefore, despite its kinase-inhibiting properties, H89 should not be used as the sole source of evidence for PKA involvement. H-89 should be used in conjunction with other PKA inhibitors, such as Rp-cAMPS or PKA analogs. [3]

C20H20BRN3O2S

446.363

445.045

C, 53.82; H, 4.52; Br, 17.90; N, 9.41; O, 7.17; S, 7.18

127243-85-0

H-89 dihydrochloride;130964-39-5; 127243-85-0 ; 1000995-75-4; 1049740-55-7 (2HCl hydrate)

449241

Typically exists as off-white to light brown solids at room temperature

1.4±0.1 g/cm3

639.7±65.0 °C at 760 mmHg

195-200°C

340.7±34.3 °C

0.0±1.9 mmHg at 25°C

1.653

3.03

2

5

8

27

570

0

BrC1C([H])=C([H])C(=C([H])C=1[H])/C(/[H])=C(\[H])/C([H])([H])N([H])C([H])([H])C([H])([H])N([H])S(C1=C([H])C([H])=C([H])C2C([H])=NC([H])=C([H])C1=2)(=O)=O

N-[2-[[3-(4-Bromophenyl)-2-propen-1-yl]amino]ethyl]-5-Isoquinolinesulfonamide

ZKZXNDJNWUTGDK-NSCUHMNNSA-N

H-89 free base H-89 free base H 89 H89.

h-89; 127243-85-0; H 89; N-(2-(4-Bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; H-89 DIHYDROCHLORIDE; N-[2-[[(E)-3-(4-bromophenyl)prop-2-enyl]amino]ethyl]isoquinoline-5-sulfonamide; Protein kinase inhibitor H-89; N-[2-(P-BROMOCINNAMYLAMINO)ETHYL]-5-ISOQUINOLINE SULFONAMIDE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~224.03 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)