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 is a member of the class of isoquinolines that is the sulfonamide obtained by formal condensation of the sulfo 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. It has a role as an EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitor. It is a member of isoquinolines, a sulfonamide, a member of bromobenzenes, an olefinic compound and a secondary amino compound. It is a conjugate base of a N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide(2+).

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A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including 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 were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.[1]

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The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.[2]

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H89 is marketed as a selective and potent inhibitor of protein kinase A (PKA). Since its discovery, it has been used extensively for evaluation of the role of PKA in the heart, osteoblasts, hepatocytes, smooth muscle cells, neuronal tissue, epithelial cells, etc. Despite the frequent use of H89, its mode of specific inhibition of PKA is still not completely understood. It has also been shown that H89 inhibits at least 8 other kinases, while having a relatively large number of PKA-independent effects which may seriously compromise interpretation of data. Thus, while recognizing its kinase inhibiting properties, it is advised that H89 should not be used as the single source of evidence of 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.)