PKC-α (IC50 = 5 nM); PKC-βII (IC50 = 14 nM); MAPKAP-K1b (IC50 = 3 nM); MSK1 (IC50 = 8 nM); S6K1 (IC50 = 15 nM);PKC-βI (IC50 = 24 nM); PKC-ε (IC50 = 24 nM); PKC-γ (IC50 = 27 nM); Rat Brain PKC (IC50 = 23 nM); GSK3β (IC50 = 38 nM)

Ro 31-8220 是一种强效 PKC 抑制剂，对 PKCα、PKCβI、PKCβII、PKCγ、PKCε 和大鼠脑 PKC 的 IC50 值分别为 5、24、14、27、24 和 23 nM[1]。此外，Ro 31-8220 强烈抑制 MSK1、S6K1、GSK3β 和 MAPKAP-K1b（IC50 分别为 3、8、15 和 38 nM）； MKK3、MKK4、MKK6 和 MKK7 不受影响。此外，电压依赖性 Na+ 通道直接被 Ro 31-8220[2] 抑制。在小脑颗粒神经元中，Ro 31-8220 (1 μM) 对对氧磷诱导的神经元细胞死亡具有神经保护作用，抑制对氧磷诱导的 caspase-3 活性，并降低对氧磷引起的磷酸 PKC pan 水平的增加 [3]。

在小鼠中，Ro 31-8220（6 mg/kg/d，皮下注射）的半衰期为 5.7 小时，且耐受性良好。经过六周的治疗后，用 Ro 31-8220 治疗的 MLP−/− 小鼠在缩短分数方面表现出显着的改善，但 WT 小鼠没有表现出任何变化[4]。

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药理抑制PKCα可恢复MLP−/−小鼠的心功能[4]
由于传统的肌力药物慢性治疗与心力衰竭患者的不良结果相关，因此我们在这里研究了慢性给予Ro-31-8220超过4-6周对MLP - / -心力衰竭小鼠的影响。在研究开始时和6周后，通过超声心动图评估所有小鼠的心室功能。Ro-31-8220（或对照品）每天注射一次，剂量为6mg /kg/day， s.q。Ro-31-8220化合物被用于Ro-32-0432化合物的所有体内研究，只是因为与大规模合成相关的费用问题以及在小鼠中长期使用所需的量。Ro-31-8220在大鼠体内的药动学分析 药物的半衰期为5.7小时，6小时时血浆浓度比PKCα的IC50高100倍（见讨论）。该剂量的Ro-31-8220在小鼠中耐受性良好，在6周内没有观察到的有害影响，也不影响体重。在6周的时间内，注射了载体或Ro-31-8220的野生型小鼠在分数缩短或任何其他心室尺寸测量方面没有变化（图5A和表1）。相比之下，与载体治疗或治疗前的基线值相比，6周注射Ro-31-8220的MLP - / -小鼠在分数缩短方面表现出显著的恢复（图5A，表1）。图5A所示的研究是在6个月大的野生型和MLP - / -小鼠中进行的。尽管在4周的治疗中，老龄MLP−/−小鼠（14个月）与给药小鼠相比，在部分缩短方面也有类似的改善（图5B，表1）。野生型和MLP−/−小鼠也接受了分离的、工作的心脏准备，显示长期给药Ro-31-8220的MLP−/−小鼠的心脏收缩力显著增加。与野生型小鼠相当（表2）。在舒张功能（- dP/dt）和左心室压力方面也观察到类似的恢复（表2）。
虽然Ro-31-8220在4周或6周的慢性给药期间挽救了MLP - / -小鼠的心脏收缩性能，但它没有改变心脏重量，也没有通过超声心动图评估逆转心室扩张，也没有改善组织病理学（数据未显示）。这些观察结果表明，与慢性Ro-31-8220治疗相关的心脏功能的部分增加可能涉及对收缩力本身的急性影响。事实上，在MLP−/−小鼠中注射Ro-31-8220仅3天，与载药治疗相比，分数缩短得到改善（图5C）。

蛋白激酶C（PKC）同工酶家族被认为在许多不同的细胞类型中介导了广泛的信号转导途径。一系列双吲哚基马来酰亚胺已被评估为传统PKC家族（PKCsα、β、γ）成员的抑制剂，以及新的Ca（2+）非依赖性PKC家族PKCε的代表性抑制剂。与吲哚咔唑星孢菌素相比，所有研究的双吲哚基马来酰亚胺对PKCα的选择性都比其他检测的同工酶低。此外，带有构象限制侧链的双吲哚基马来酰亚胺作为PKCε抑制剂的活性较低。其中最引人注目的是Ro 32-0432，它对PKCα的选择性是PKCε的10倍，对PKCβI的选择性是PKC-ε的4倍[2]。

对硫磷的活性代谢产物对氧磷是一种乙酰胆碱酯酶（AChE）抑制剂，通过凋亡机制杀死培养的小脑颗粒细胞神经元。蛋白激酶C是一种具有多种功能的酶，但其在对氧磷诱导的细胞死亡中的作用尚不清楚。我们发现，神经毒性浓度的对氧磷会增加PKC磷酸化。我们使用PKC激活剂佛波醇12-肉豆蔻酸13-乙酸酯（TPA）测试了PKC是否参与对氧磷诱导的神经元细胞死亡。TPA可增加PKC活性，并将对氧磷的神经毒性作用增强28%。与此形成鲜明对比的是，添加PKC抑制剂Ro-31-8220可以保护30%以上的神经元，否则这些神经元在治疗前或治疗后都会死于对氧磷诱导的神经元细胞死亡，并显著降低磷酸化PKC水平。研究还表明，Ro-31-8220的预处理完全阻断了对氧磷诱导的半胱氨酸天冬氨酸蛋白酶-3活性。这些结果表明，对氧磷神经毒性需要激活蛋白激酶C。[3]

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在体外（DIV）8的指定时间内，将神经毒性浓度的对氧磷（200μM）添加到颗粒细胞培养物中。在DIV 8暴露于对氧磷之前或之后，将以下药物添加到颗粒细胞培养物中：在添加对氧磷前15分钟或后3小时添加Ro-81-3220（1μM）。在加入对氧磷前15分钟加入TPA（0.1μM）[1]。

In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKCalpha-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKCalpha cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKCalpha was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology.[4]

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6 mg/kg/d; s.c.

The affects of long-term Ro 31-8220 administration over 4 to 6 weeks in MLP / heart failure mice are investigated. All mice are assessed for ventricular performance by echocardiography at the beginning of the study and 6 weeks later. Ro 31-8220 (or vehicle) is injected subcutaneouslyonce per day at a dosage of 6 mg/kg/d.

[1]. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294 ( Pt 2):335-7.

[2]. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000 Oct 1;351(Pt 1):95-105.

[3]. Inhibition of protein kinase C protects against paraoxon-mediated neuronal cell death. Neurotoxicology. 2007 Jul;28(4):843-9.

[4]. Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure. Circulation. 2006 Aug 8;114(6):574-82.

Ro 31-8220 is an imidothiocarbamic ester, a member of indoles and a member of maleimides. It has a role as an EC 2.7.11.13 (protein kinase C) inhibitor. It is functionally related to a maleimide.
Ro 31-8220 is a methanesulfonate salt form, a staurosporine analog that inhibits protein kinase C and induces apoptosis.

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The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon.[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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Paraoxon, the active metabolite of parathion, is an acetylcholinesterases (AChE) inhibitor that kills cultured cerebellar granule cell neurons via an apoptotic mechanism. Protein kinase C is an enzyme with diverse functions but its role in paraoxon-induced cell death is unknown. We show that a neurotoxic concentration of paraoxon increases PKC phosphorylation. We tested whether PKC is involved in paraoxon-induced neuronal cell death by using the PKC activator, phorbol 12-myristate 13-acetate (TPA). TPA increases PKC activity and enhances the neurotoxic effect of paraoxon by 28%. In sharp contrast, addition of the PKC inhibitor Ro-31-8220 protects more than 30% neurons that would otherwise die from paraoxon-induced neuronal cell death in either a pretreatment or post-treatment paradigm and markedly reduces phospho-PKC pan levels. We also show that the pretreatment of Ro-31-8220 blocks paraoxon-induced caspase-3 activity completely. These results suggest that activation of protein kinase C is required for paraoxon neurotoxicity. [3]

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Background: The conventional protein kinase C (PKC) isoform alpha functions as a proximal regulator of Ca2+ handling in cardiac myocytes. Deletion of PKCalpha in the mouse results in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, whereas overexpression of PKCalpha in the heart blunts contractility. Mechanistically, PKCalpha directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase. Methods and results: In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKCalpha-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKCalpha cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKCalpha was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Conclusions: Pharmacological inhibition of PKCalpha, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure. [4]

C25H23N5O2S

457.55

457.157

125314-64-9

Ro 31-8220 mesylate;138489-18-6

5083

Typically exists as Pink to red solid at room temperature

1.42g/cm3

5.43

193.95

3

4

7

33

845

0

S(/C(=N/[H])/N([H])[H])C([H])([H])C([H])([H])C([H])([H])N1C([H])=C(C2C(N([H])C(C=2C2=C([H])N(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C23)=O)=O)C2=C([H])C([H])=C([H])C([H])=C12

DSXXEELGXBCYNQ-UHFFFAOYSA-N

InChI=1S/C25H23N5O2S/c1-29-13-17(15-7-2-4-9-19(15)29)21-22(24(32)28-23(21)31)18-14-30(11-6-12-33-25(26)27)20-10-5-3-8-16(18)20/h2-5,7-10,13-14H,6,11-12H2,1H3,(H3,26,27)(H,28,31,32)

3-[3-[4-(1-methylindol-3-yl)-2,5-dioxopyrrol-3-yl]indol-1-yl]propyl carbamimidothioate

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)