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

在添加福司可林前 1 小时用 H-89 (30 M) 预处理细胞，可显着且剂量依赖性地抑制 orskolin 诱导的蛋白质磷酸化。 [1] H89 抑制的其他激酶包括 S6K1、MSK1、PKA、ROCKII、PKB 和 MAPKAP-K1b，IC50 值分别为 80、120、135、270、2600 和 2800 nM。 [2] [3] 一些细胞受体和离子通道，包括 Kv1.3 K+ 通道、1AR 和 2AR，对 H89 也有活性。 [4]在添加毛喉素前 1 小时用 H-89 (30 M) 预处理细胞，可显着且剂量依赖性地抑制毛喉素诱导的蛋白质磷酸化。 [1] H89 抑制的其他激酶包括 S6K1、MSK1、PKA、ROCKII、PKBα 和 MAPKAP-K1b，IC50 值分别为 80、120、135、270、2600 和 2800 nM。 [2] [3] 一些细胞受体和离子通道，包括 Kv1.3 K+ 通道、β1AR 和 β2AR，对 H89 也具有活性。 [4]

H89 的蛋白质磷酸化以多种方式发生改变，但果糖 1,6-二磷酸酶、异质核核糖核蛋白 (hnRNP) 和 NSFL1 辅因子 p47 表现出最强的磷酸化变化。这些蛋白可能都与cAMP/PKA有调节关系。

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在经过 PTZ 治疗的动物中，H-89（0.2 mg/100g，腹腔注射）显着增加了癫痫潜伏期和阈值。 H-89 显着提高癫痫潜伏期和癫痫阈值，并抑制布克拉地辛 (300 nM) 的致癫痫作用，剂量为 0.05 和 0.2 mg/100 g，腹膜内注射 [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]。

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H-89预处理对PTZ诱发癫痫的影响[Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
图2A和B显示了用不同剂量的H-89（0.05、0.1和0.2 mg/100 g，i.p.，30分钟）预处理对PTZ（0.5%w/v i.v）诱导的癫痫发作的影响。与对照组相比，以0.2 mg/100克的剂量给药H-89显著增加了癫痫发作的潜伏期和阈值（***p<0.001）。与对照组动物相比，其他两种剂量的H-89（0.05和0.1 mg/100 g）在癫痫发作潜伏期和阈值方面没有观察到显著差异（图2A和B）。

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己酮可可碱和H-89联合预处理对PTZ诱导的小鼠癫痫发作的影响[Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
属于该组合组的所有动物在PTZ输注前45分钟接受PTX作为第一组分，30分钟接受H-89作为第二组分。与对照组相比，接受PTX 50 mg/kg和H-89 0.2 mg/kg以及PTX 100 mg/kg和H-890.2 mg/100 g的组在癫痫发作潜伏期和阈值方面存在显著差异（***P<0.001）（图4A和B）。PTX（50和100 mg/kg）给药显著减弱了H-89（0.2 mg/100 g）对癫痫发作阈值和潜伏期的影响（*P<0.05）（图4A和B）。

cAMP 依赖性蛋白激酶活性在最终体积为 0.2 mL 的反应混合物中进行测定，其中包含 50 mM Tris-HCl (pH 7.0)、10 mM 醋酸镁、2 mM EGTA、1 μM cAMP 或不含 cAMP，3.3 -20 μM [γ-32P]ATP (4 × 105 cpm)、0.5 μg 酶、100 μg 组蛋白 H2B 和每种化合物，如所示。

测定细胞内cAMP 的水平。培养 48 小时后，PC12D 细胞在含有 30 μM H-89 的测试培养基中生长 1 小时，然后暴露于含有 10 μM 毛喉素和 30 μM H-89 的全新培养基中。添加 0.5 ml 6% 三氯乙酸，同时用橡胶警察刮下细胞并进行超声处理。加入2ml石油醚，混合，3000rpm离心10分钟，提取三氯乙酸。吸出顶层后，残留样品溶液用于分析。

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.]

[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 salt prepared from N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide and two equivalents of hydrogen chloride. It has a role as an EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitor. It contains 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]

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

88.7

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: 5 mg/mL (9.63 mM) in 10% DMSO + 90% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.75 mg/mL (5.30 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.75 mg/mL (5.30 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.5 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100μL 25.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: ≥ 2.5 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 7 中的溶解度: ≥ 0.55 mg/mL (1.06 mM) (饱和度未知) in 1% DMSO 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 8 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。