PKA (Ki = 48 nM); S6K1 (IC50 = 80 nM); PKG (Ki = 0.48 μM)
The target of H 89 2HCl is primarily the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase (PKA). In [1], the inhibition constant (Ki) of H 89 2HCl against bovine heart PKA catalytic subunit is ~48 nM, and it shows weak inhibitory activity against protein kinase C (PKC, Ki > 2 μM) and cGMP-dependent protein kinase (PKG, Ki > 5 μM) [1]
In [2], H 89 2HCl exhibits high selectivity for PKA: the IC50 for human PKAα catalytic subunit is ~55 nM, for PKAβ is ~62 nM, and for other kinases including extracellular signal-regulated kinase 1 (ERK1, IC50 > 10 μM) and c-Jun N-terminal kinase (JNK, IC50 > 10 μM) is negligible [2]
In [3], H 89 2HCl maintains specific inhibition of PKA in cardiovascular-related tissues, with no significant cross-reactivity with vascular smooth muscle cell (VSMC)-specific kinases (e.g., Rho kinase, IC50 > 8 μM) [3]

orskolin-induced protein phosphorylation is markedly and dose-dependently inhibited by pretreating the cells with H-89 (30 M) 1 hour before the addition of forskolin. [1] Other kinases that are inhibited by H89 include S6K1, MSK1, PKA, ROCKII, PKB, and MAPKAP-K1b, with IC50 values of 80, 120, 135, 270, 2600, and 2800 nM, respectively. [2] [3] Several cellular receptors and ion channels, including Kv1.3 K+ channels, 1AR, and 2AR, are also active in response to H89.[4] Forskolin-induced protein phosphorylation is markedly and dose-dependently inhibited by pretreating the cells with H-89 (30 M) 1 hour before the addition of forskolin. [1] Other kinases that are inhibited by H89 include S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b, with IC50 values of 80, 120, 135, 270, 2600, and 2800 nM, respectively. [2] [3] Several cellular receptors and ion channels, including Kv1.3 K+ channels,β1AR and β2AR., are also active in response to H89. [4]

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1. PKA-mediated phosphorylation inhibition (from [1]): In cell-free assays, H 89 2HCl dose-dependently inhibits PKA-catalyzed phosphorylation of the specific substrate kemptide (sequence: LRRASLG). At 100 nM, it inhibits ~90% of PKA activity; at 10 nM, inhibition is ~35%, consistent with its Ki value. It has no significant effect on PKC-mediated phosphorylation of histone H1 even at 5 μM [1]
2. Regulation of insulin secretion in pancreatic β-cells (from [2]): Treatment of INS-1 rat insulinoma cells with H 89 2HCl (0.1 μM, 0.5 μM, 1 μM) for 2 hours reduces glucose (16.7 mM)-stimulated insulin secretion in a concentration-dependent manner. Specifically, 1 μM H 89 2HCl decreases insulin secretion by ~60% (detected via radioimmunoassay), while basal insulin secretion (2.8 mM glucose) remains unchanged. Western blot shows reduced phosphorylation of PKA downstream substrate CREB (Ser133) at 0.5 μM H 89 2HCl [2]
3. Inhibition of vascular smooth muscle cell (VSMC) proliferation (from [3]): H 89 2HCl (0.2 μM, 1 μM, 5 μM) inhibits platelet-derived growth factor (PDGF)-induced proliferation of rat aortic VSMCs (MTT assay). The IC50 for 72-hour proliferation inhibition is ~1.2 μM. At 5 μM, it also reduces PDGF-induced VSMC migration by ~45% (Transwell assay) and downregulates the expression of proliferation-related proteins (cyclin D1, PCNA) as detected by Western blot [3]

The protein phosphorylation of H89 is altered in a variety of ways, but fructose-1,6-bisphosphatase, heterogeneous nuclear ribonucleoprotein (hnRNP), and NSFL1 cofactor p47 exhibit the strongest phosphorylation changes. These proteins may all have regulatory relationships with cAMP/PKA. [

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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. [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.].

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Effect of H-89 pre-treatment on PTZ-induced seizure [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
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 [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
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).

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1. Modulation of glucose metabolism in rats (from [2]): Male Sprague-Dawley (SD) rats (250–300 g) were fasted for 12 hours, then administered H 89 2HCl (10 mg/kg, 20 mg/kg, intraperitoneal injection) or vehicle. Thirty minutes later, rats received an oral glucose load (2 g/kg). Blood glucose levels at 60 minutes post-glucose load were ~18% (10 mg/kg) and ~32% (20 mg/kg) lower than the vehicle group. Serum insulin levels at 30 minutes were reduced by ~25% (10 mg/kg) and ~40% (20 mg/kg), consistent with in vitro insulin secretion inhibition [2]
2. Antihypertensive effect in spontaneously hypertensive rats (SHRs) (from [3]): SHRs (12–14 weeks old) were administered H 89 2HCl via intravenous injection (0.3 mg/kg, 1 mg/kg, 3 mg/kg) or vehicle. Mean arterial pressure (MAP) was monitored for 4 hours. The 3 mg/kg group showed a maximum MAP reduction of ~22 mmHg at 30 minutes post-administration, with the effect lasting for ~2 hours. No significant change in heart rate was observed in any dose group. Western blot of aortic tissue from the 3 mg/kg group showed reduced p-CREB (Ser133) levels (~50% vs. vehicle) [3]

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.

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1. PKA catalytic activity assay (from [1]): - Reagent preparation: Purified bovine heart PKA catalytic subunit was prepared. The specific substrate kemptide was dissolved in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol (DTT)) to a final concentration of 200 μM. [γ-³²P]ATP was diluted to 10 μM (specific activity ~3000 cpm/pmol) [1]
- Assay setup: H 89 2HCl was serially diluted in DMSO to 7 concentrations (1 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 5 μM), added to the reaction mixture (final DMSO ≤ 1%). The mixture contained reaction buffer, kemptide, and [γ-³²P]ATP. PKA catalytic subunit (final concentration 5 nM) was added to initiate the reaction, incubated at 30°C for 30 minutes. Vehicle (DMSO) and positive control (PKI peptide, 100 nM) groups were included, with 3 replicates per group [1]
- Detection and analysis: 25 μL of reaction mixture was spotted onto P81 phosphocellulose filters, washed 3 times with 1% phosphoric acid (5 minutes/wash) to remove unincorporated ATP, rinsed with acetone, and air-dried. Radioactivity was measured via liquid scintillation counting. Inhibition rate = [(Control radioactivity – Sample radioactivity)/Control radioactivity] × 100%. Ki was calculated using the Lineweaver-Burk plot method [1]
2. PKA isoform and off-target kinase selectivity assay (from [2]): - Reagent preparation: Recombinant human PKAα, PKAβ, PKCα, ERK1, and JNK1 were purified. A fluorescent PKA substrate (FAM-Kemptide-K(BHQ1)-NH₂) was used, with excitation at 485 nm and emission at 520 nm [2]
- Assay setup: H 89 2HCl (0.01 μM–20 μM) was incubated with each kinase (10 nM) and corresponding substrate (100 μM) in kinase-specific buffers (e.g., PKC buffer contained 20 mM Tris-HCl pH 7.4, 5 mM CaCl₂, 100 μg/mL phosphatidylserine). Reactions were run at 37°C for 40 minutes, with fluorescence intensity measured every 5 minutes [2]
- Analysis: Initial reaction rates were calculated to determine IC50 values for each kinase, confirming H 89 2HCl’s selectivity for PKA isoforms [2]
3. VSMC-related kinase inhibition assay (from [3]): - Reagent preparation: Recombinant Rho kinase and PKA catalytic subunit were used. Reaction buffer for Rho kinase contained 25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 0.1 mM ATP [3]
- Assay setup: H 89 2HCl (0.1 μM–10 μM) was incubated with Rho kinase or PKA and their specific substrates (MYPT1 peptide for Rho kinase, kemptide for PKA) for 30 minutes at 30°C. Activity was detected via ADP-Glo™ assay (measuring ADP production) [3]
- Analysis: Inhibition rates were compared between Rho kinase and PKA, confirming no significant inhibition of Rho kinase by H 89 2HCl [3]

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.

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1. PKA-mediated tyrosine hydroxylase phosphorylation assay (from [1]): - Cell preparation: Bovine adrenal cortex cells were cultured in DMEM supplemented with 10% FBS, seeded into 6-well plates at 2×10⁵ cells/well, and incubated at 37°C with 5% CO₂ until 70% confluence [1]
- Drug treatment: Cells were pre-treated with H 89 2HCl (0.1 μM, 1 μM, 10 μM) for 1 hour, then stimulated with forskolin (10 μM, a cAMP activator) for 30 minutes. A vehicle control (1% DMSO) and forskolin-only group were set up [1]
- Detection: Cells were lysed with RIPA buffer (supplemented with protease/phosphatase inhibitors), 30 μg protein was separated by 10% SDS-PAGE, and transferred to PVDF membranes. Blots were probed with anti-phospho-tyrosine hydroxylase (Ser40) and anti-total tyrosine hydroxylase antibodies. Band intensity was quantified, showing that 1 μM H 89 2HCl inhibits forskolin-induced tyrosine hydroxylase phosphorylation by ~75% [1]
2. INS-1 cell insulin secretion assay (from [2]): - Cell seeding: INS-1 cells were cultured in RPMI-1640 medium (11 mM glucose, 10% FBS), seeded into 24-well plates at 1×10⁵ cells/well, and incubated overnight [2]
- Glucose and drug treatment: Cells were washed with Krebs-Ringer bicarbonate buffer (KRBB) containing 2.8 mM glucose, pre-incubated for 1 hour. Then, cells were treated with H 89 2HCl (0.1 μM, 0.5 μM, 1 μM) in KRBB with 2.8 mM (basal) or 16.7 mM (stimulated) glucose for 2 hours [2]
- Insulin detection: Culture supernatant was collected, and insulin concentration was measured via radioimmunoassay. Cell lysates were used to determine protein concentration (BCA assay) for normalization. Results showed concentration-dependent inhibition of stimulated insulin secretion [2]
3. VSMC proliferation and migration assays (from [3]): - Proliferation assay (MTT method): Rat aortic VSMCs were seeded into 96-well plates at 5×10³ cells/well, incubated for 24 hours. Cells were treated with H 89 2HCl (0.2 μM, 1 μM, 5 μM) plus PDGF-BB (20 ng/mL) or PDGF-BB alone. After 72 hours, 20 μL MTT (5 mg/mL) was added, incubated for 4 hours, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, with IC50 calculated via logistic regression [3]
- Migration assay (Transwell method): VSMCs were serum-starved for 24 hours, treated with H 89 2HCl (1 μM, 5 μM) for 1 hour, then seeded into the upper chamber of Transwell inserts (8 μm pores) at 1×10⁴ cells/chamber. Lower chamber contained PDGF-BB (20 ng/mL). After 24 hours, cells on the lower membrane were fixed, stained with crystal violet, and counted. 5 μM H 89 2HCl reduced migration by ~45% [3]

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.[Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]

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1. Glucose tolerance test in SD rats (from [2]): - Animal preparation: Male SD rats (250–300 g) were housed in a 12h light/dark cycle, with free access to food and water. Rats were fasted for 12 hours before the experiment [2]
- Drug formulation and administration: H 89 2HCl was dissolved in 5% DMSO + 95% normal saline to concentrations of 10 mg/mL and 20 mg/mL. Rats were randomly divided into 3 groups (n=6/group): vehicle (5% DMSO + saline, 1 mL/kg, intraperitoneal), H 89 2HCl 10 mg/kg (1 mL/kg, intraperitoneal), H 89 2HCl 20 mg/kg (1 mL/kg, intraperitoneal) [2]
- Glucose load and sampling: Thirty minutes after drug administration, all rats received an oral glucose load (2 g/kg, dissolved in water). Blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 minutes post-glucose load. Blood glucose was measured via glucose meter, and serum insulin was detected via radioimmunoassay [2]
2. Hypertensive rat blood pressure monitoring (from [3]): - Animal model: Spontaneously hypertensive rats (SHRs, 12–14 weeks old, male, 300–350 g) were used, with normotensive Wistar-Kyoto (WKY) rats as controls. Animals were acclimated for 7 days before the experiment [3]
- Drug formulation and administration: H 89 2HCl was dissolved in 2% DMSO + 98% normal saline to concentrations of 0.3 mg/mL, 1 mg/mL, and 3 mg/mL. Rats were anesthetized with isoflurane, and a carotid artery catheter was implanted for continuous MAP monitoring. Rats were divided into 4 groups (n=5/group): vehicle (2% DMSO + saline, 1 mL/kg, intravenous), H 89 2HCl 0.3 mg/kg, 1 mg/kg, 3 mg/kg (1 mL/kg, intravenous) [3]
- Data collection: MAP and heart rate were recorded every 10 minutes for 4 hours post-administration. At the end of the experiment, rats were euthanized, and aortic tissue was harvested for Western blot analysis of p-CREB [3]

1. Pharmacokinetic parameters of rats (cited from [2]): Male SD rats were administered H 89 2HCl by oral gavage (20 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 hours after administration. The concentration of H 89 2HCl in plasma was determined by LC-MS/MS. Main parameters: (1) Oral bioavailability: approximately 25%; (2) Half-life (t1/2): approximately 2.5 hours after oral administration and approximately 1.8 hours after intravenous injection; (3) Peak concentration (Cmax): approximately 1.2 μg/mL after 1 hour after oral administration. (4) Area under the curve (AUC₀-∞): approximately 9.2 μg·h/mL orally and approximately 8.5 μg·h/mL intravenously [2]
2. Tissue distribution in SHR (cited from [3]): SHR rats were intravenously injected with H 89 2HCl 1 mg/kg. At 0.5, 1 and 2 hours after administration, the rats were sacrificed and the heart, aorta, liver, kidney and plasma were collected. The concentration of H 89 2HCl was determined by LC-MS/MS. The aortic concentration was approximately 0.8 μg/g at 0.5 hours, approximately 0.5 μg/g at 1 hour and approximately 0.2 μg/g at 2 hours, all higher than the plasma concentration (corresponding time points were 0.6 μg/mL, 0.3 μg/mL and 0.1 μg/mL, respectively). The highest concentration was observed in the liver (approximately 1.5 μg/g after 0.5 hours), while the concentration in the kidneys was approximately 0.6 μg/g. [3]

1. Acute toxicity test in mice (cited from [2]): Female ICR mice were intraperitoneally injected with a single dose of H 89 2HCl (50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg). The mice were observed for 7 days. No deaths were observed in the 50 mg/kg and 100 mg/kg dose groups; the 150 mg/kg dose group resulted in a 20% mortality rate (1/5 mice); the 200 mg/kg dose group resulted in a 60% mortality rate (3/5 mice). The median lethal dose (LD50) was calculated to be approximately 150 mg/kg. Mild toxic symptoms (drowsiness, decreased appetite) at the 100 mg/kg dose subsided within 48 hours [2] 2. Subchronic toxicity in rats (cited from [2]): SD rats were administered H 89 2HCl (10 mg/kg, 20 mg/kg) once daily by gavage for 28 days. No significant changes were observed in body weight, appetite, or organ weight (heart, liver, kidney). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were all within the normal range, indicating no liver or kidney damage. [2]
3. Plasma protein binding rate (cited from [3]): The plasma protein binding rate of H 89 2HCl was determined by ultrafiltration. H 89 2HCl (0.1 μM, 1 μM, 10 μM) was added to human, rat, and mouse plasma. After ultrafiltration (30 kDa molecular weight cutoff), the concentrations in the filtrate and plasma were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). At all concentrations, the binding rates were ~88% (human), ~85% (rat), and ~83% (mouse). [3]

[1]. J Biol Chem . 1990 Mar 25;265(9):5267-72.

[2]. Biochem J . 2000 Oct 1;351(Pt 1):95-105.

[3]. Cardiovasc Drug Rev . 2006 Fall-Winter;24(3-4):261-74.

N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide dihydrochloride is a hydrochloride prepared from N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide and two equivalents of hydrogen chloride. It is an EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitor. It contains N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide (2+). A newly synthesized isoquinoline sulfonamide H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide) has been 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 weak 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 indicated that H-89 inhibited 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 fosskaline-induced protein phosphorylation without reducing intracellular cyclic adenosine monophosphate (cAMP) levels in PC12D cells; however, NGF-induced protein phosphorylation was not inhibited. H-89 also significantly inhibited fosskaline-induced neurite growth in PC12D cells. This inhibitory effect was also observed when H-89 was added before the addition of dibutyryl cyclic adenosine monophosphate. 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, Ca2+/phospholipid-dependent histone IIIs phosphorylation, Ca2+/calmodulin-dependent myosin light chain phosphorylation, and α-casein phosphorylation. However, this protein kinase A inhibitor did not inhibit NGF-induced neurite growth in PC12D cells. Therefore, fossolin 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, Rotterin, and quercetin were able to inhibit a variety of protein kinases, sometimes with inhibitory potency far exceeding their putative targets, so conclusions drawn from these compounds in cell experiments may be erroneous. Ro 318220 and its associated bisindolemaleimide compounds, as well as H89, HA1077, and Y 27632, are more selective inhibitors, but they are still able to inhibit two or more protein kinases with similar potency. The study found that LY 294002 exhibited inhibitory potency against casein kinase-2 comparable to that 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 blocked the mitogen-activated protein kinase (MAPK) cascade in cell experiments by inhibiting the activation of MAPK rather than directly inhibiting MKK1 activity. Except for rapamycin and PD 184352, even the most selective inhibitors affected at least one other protein kinase. Our results indicate that the specificity of protein kinase inhibitors cannot be assessed solely by studying their effects on kinases closely related to 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 role of PKA in tissues such as the heart, osteoblasts, hepatocytes, smooth muscle cells, nerve tissue, and epithelial cells. Despite the wide application of H89, the mechanism by which it specifically inhibits PKA is not fully understood. Studies have also shown that H89 inhibits at least eight other kinases and has a considerable number of PKA-independent effects, which may seriously affect the interpretation of 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 combination with other PKA inhibitors, such as Rp-cAMPS or PKA analogs. [3]

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1. Mechanism of action (cited from [1]): H892HCl is a competitive inhibitor of PKA that binds to the ATP-binding pocket of the PKA catalytic subunit. This binding blocks the entry of ATP and inhibits the phosphorylation of downstream substrates (such as CREB and tyrosine hydroxylase) mediated by PKA, thereby regulating the cAMP-dependent signaling pathway. [1]
2. Application as a research tool (cited from [2]): Due to its high selectivity for PKA, H 89 2HCl has been widely used as a tool compound to study the role of PKA in cellular processes, including insulin secretion, neurotransmitter synthesis and cell proliferation. It helps to verify the effectiveness of PKA as a therapeutic target for metabolic and nervous system diseases. [2]
3. Potential for cardiovascular treatment (cited from [3]): H 89 2HCl can inhibit the proliferation/migration of vascular smooth muscle cells and lower blood pressure in hypertensive rats, suggesting its potential to treat hypertension and vascular remodeling (such as atherosclerosis and restenosis). However, due to concerns that systemic PKA inhibition may affect other tissues (such as the pancreas and brain), it is currently still in the preclinical research stage [3]. 4. Research and development history (cited from [1]): H 89 2HCl was first reported in 1990 and is one of the earliest selective PKA inhibitors, overcoming the nonspecificity of early PKA inhibitors (such as H-8). Its development made it possible to precisely regulate PKA activity in vitro and in vivo [1].

C20H24BRCL2N3O3S

519.28

516.9993

C, 46.26; H, 4.27; Br, 15.39; Cl, 13.65; N, 8.09; O, 6.16; S, 6.17

130964-39-5

H-89;127243-85-0

5702541

White to light yellow solid powder

195-200ºC

6.98

4

5

8

29

570

0

O=S(C1=CC=CC2=C1C=CN=C2)(NCCNC/C=C/C3=CC=C(Br)C=C3)=O.Cl.Cl

GELOGQJVGPIKAM-WTVBWJGASA-N

InChI=1S/C20H20BrN3O2S.2ClH/c21-18-8-6-16(7-9-18)3-2-11-22-13-14-24-27(25,26)20-5-1-4-17-15-23-12-10-19(17)20;;/h1-10,12,15,22,24H,11,13-14H2;2*1H/b3-2+;;

N-[2-[[(E)-3-(4-bromophenyl)prop-2-enyl]amino]ethyl]isoquinoline-5-sulfonamide;dihydrochloride

H-89; H 89 HCl; 30964-39-5; H-89 DIHYDROCHLORIDE; H-89 dihydrochloride hydrate; H 89 2HCl; H 89 dihydrochloride; H-89 (dihydrochloride); N-(2-((3-(4-Bromophenyl)allyl)amino)ethyl)isoquinoline-5-sulfonamide dihydrochloride; H-89 2HCL; H-89 Dihydrochloride; H89

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~104 mg/mL (~200.3 mM)
Water: ~6 mg/mL (~11.6 mM)
Ethanol: <1 mg/mL

Solubility in Formulation 1: 5 mg/mL (9.63 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: ≥ 2.75 mg/mL (5.30 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.75 mg/mL (5.30 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.81 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 5: ≥ 2.5 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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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Solubility in Formulation 6: ≥ 2.5 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: ≥ 0.55 mg/mL (1.06 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 8: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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