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)

Effective against PKCα, PKCβI, PKCβII, PKCγ, PKCε, and rat brain PKC, Ro 31-8220 mesylate has IC50 values of 5, 24, 14, 27, 24, and 23 nM, respectively[1]. Furthermore, Ro 31-8220 demonstrates no effect on MKK3, MKK4, MKK6, and MKK7 but significantly inhibits MAPKAP-K1b, MSK1, S6K1, and GSK3β (IC50s, 3, 8, 15, and 38 nM, respectively). Furthermore, voltage-dependent Na+ channel suppression is a direct effect of Ro 31-8220[2]. Ro 31-8220 (1 μM) inhibits the increase in phospho-PKC pan levels brought on by paraoxons, decreases the neuroprotective effects of paraoxons on cerebellar granule neurons, and suppresses the activity of caspase-3 that is triggered by paraoxons[3].

In mice, Ro 31-8220 (6 mg/kg/d, sc) has a half-life of 5.7 hours and is well tolerated. After six weeks of therapy, MLP−/− mice treated with Ro 31-8220 exhibit a substantial rescue in fractional shortening, but WT mice show no change[4].

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Pharmacologic inhibition of PKCα restores cardiac function in MLP−/− mice [4]
Since chronic treatment with traditional inotropes is associated with adverse outcomes in heart failure patients, here we investigated the affects of chronic Ro-31-8220 administration over 4–6 weeks in MLP−/− heart failure mice. All mice were assessed for ventricular performance by echocardiography at the beginning of the study and 6 weeks later. Ro-31-8220 (or vehicle) was injected once a day at a dosage of 6 mg/kg/day, s.q. The Ro-31-8220 compound was employed for all in vivo studies over the Ro-32-0432 compound only because of issues related to expense surrounding large-scale synthesis and the quantity that was needed for long-term use in mice. Pharmacokinetic analysis of Ro-31-8220 in the rat showed a half-life of 5.7 hours and plasma concentrations of drug were 100-fold greater than the IC50 for PKCα at 6 hrs (see discussion). This dosage of Ro-31-8220 was well tolerated in the mouse and had no observable detrimental effects over 6 weeks, nor was body-weight affected. Wildtype mice injected with vehicle or Ro-31-8220 showed no change in fractional shortening or any other measures of ventricular dimensions over the 6-week period (Fig 5A, and Table 1). In contrast, MLP−/− mice showed a dramatic rescue in fractional shortening after 6 weeks of Ro-31-8220 compared with vehicle treatment or baseline values before treatment (Fig 5A, Table 1). The study shown in Figure 5A was performed in 6-month old wildtype and MLP−/− mice, although a similar improvement in fractional shortening was observed in aged MLP−/− mice (14 months) compared with vehicle-treated mice over four-weeks of treatment (Fig 5B, Table 1). Wildtype and MLP−/− mice were also subjected to isolated, working heart preparation, demonstrating a significant increase in cardiac contractility in MLP−/− mice chronically treated with Ro-31-8220, comparable to wildtype mice (Table 2). A similar rescue in diastolic function (−dP/dt) and left ventricular pressure developed was also observed (Table 2).
While Ro-31-8220 rescued cardiac contractile performance in MLP−/− mice over 4 or 6 weeks of chronic administration, it did not alter heart weight, or reverse cardiac chamber dilation as assessed by echocardiography, or improve histo-pathology (data not shown). These observations suggest that part of the increase in cardiac performance associated with chronic Ro-31-8220 treatment might involve an acute influence on contractility itself. Indeed, injection of Ro-31-8220 in MLP−/− mice for only 3 days improved fractional shortening compared with vehicle treatment (Fig 5C).

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[2].

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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A neurotoxic concentration of paraoxon (200 μM) is added to the granule cell cultures for the indicated time on day in vitro (DIV) 8. The following drugs are added to the granule cell cultures prior to or after paraoxon exposure on DIV 8: Ro-81-3220 (1 μM) is added 15 min prior to or 3 h after the addition of paraoxon. TPA (0.1 μM) is added 15 min prior to the addition of paraoxon[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.

Mice: 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 iminothiocarbamate belonging to the indole and maleimide classes. It is an EC 2.7.11.13 (protein kinase C) inhibitor. Its function is similar to that of maleimides. Ro 31-8220 is a mesylate form, an asteroidin analog that inhibits protein kinase C and induces apoptosis. The protein kinase C (PKC) isoenzyme family is believed to mediate a wide range of signal transduction pathways in various cell types. A series of bisindolyl maleimides have been evaluated as inhibitors of traditional PKC family members (PKC-α, -β, -γ) as well as novel Ca2+-independent PKC family members representing PKC-ε. Compared to the indole-carbazole compound asteroidin, all studied bisindolyl maleimides showed slightly higher selectivity for PKC-α than other isoenzymes. In addition, bisindolylmaleimide compounds with conformationally restricted side chains showed low inhibitory activity against PKC-ε. The most notable example was Ro 32-0432, which showed 10 times the selectivity for PKC-α and 4 times the selectivity for PKC-βI. [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 investigated their inhibitory effects on a variety of protein kinases. Compounds KT 5720, Rottlerin, and quercetin were found to inhibit a variety of protein kinases, sometimes with inhibitory potency far exceeding their putative targets, so conclusions drawn from cell experiments based on these compounds may be erroneous. Ro 318220 and its associated bisindolemaleimide compounds, as well as H89, HA1077, and Y 27632, while more selective inhibitors, still inhibit two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to 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 MAPK cascade in cellular experiments by inhibiting the activation of mitogen-activated protein kinase (MAPK) kinase (MKK1) 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 suggest that the specificity of a protein kinase inhibitor cannot be assessed solely by studying its effects on kinases closely related to primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays. [2]

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Paraoxon is the active metabolite of phosphorus thiophosphate and an acetylcholinesterase (AChE) inhibitor that kills cultured cerebellar granule cells via apoptosis. Protein kinase C (PKC) is a multifunctional enzyme, but its role in paraoxon-induced cell death remains unclear. We found that neurotoxic concentrations of paraoxon increase PKC phosphorylation. We used the PKC activator phorbol 12-myristate 13-acetate (TPA) to detect whether PKC is involved in paraoxon-induced neuronal cell death. TPA increased PKC activity and enhanced the neurotoxicity of paraoxon by 28%. In stark contrast, the addition of the PKC inhibitor Ro-31-8220 protected more than 30% of neurons from paraoxon-induced neuronal death, both in pretreatment and posttreatment modes, and significantly reduced the overall level of phosphorylated PKC. We also found that pretreatment with Ro-31-8220 completely blocked paraoxon-induced caspase-3 activity. These results suggest that activation of protein kinase C is a necessary condition for paraoxon neurotoxicity. [3]

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Background: Conventional protein kinase C (PKC) isoform α functions as a proximal regulator of Ca2+ treatment in cardiomyocytes. PKCα deficiency in mice leads to increased sarcoplasmic reticulum Ca2+ load, enhanced Ca2+ transients, and increased contractility, while PKCα overexpression in the heart weakens contractility. Mechanistically, PKCα directly regulates Ca2+ treatment by altering the phosphorylation state of inhibitor-1, thereby inhibiting the activity of protein phosphatase-1, which in turn regulates phosphoprotein activity and subsequently modulates sarcoplasmic reticulum Ca2+ ATPase activity. Methods and Results: In this study, we found that short-term inhibition of conventional PKC subtypes using Ro-32-0432 or Ro-31-8220 significantly enhanced cardiac contractility in wild-type mice in vivo or in vitro working heart models, but this phenomenon was not observed in PKCα-deficient mice. Ro-32-0432 also enhanced cardiac contractility in two different in vivo heart failure models. In a mouse model of heart failure caused by muscle lim protein gene deletion, short-term or long-term treatment with Ro-31-8220 significantly enhanced cardiac contractility and restored pumping function. Furthermore, in a rat model of post-myocardial infarction cardiomyopathy, adenovirus-mediated gene therapy using dominant-negative PKCα cDNA rescued heart failure. The study also found that PKCα is the major canonical PKC subtype expressed in the adult heart, suggesting that these findings may be related to human pathophysiology. Conclusion: Pharmacological inhibition of PKCα or the general canonical subtype may be a novel therapeutic strategy to enhance myocardial contractility in patients with certain stages of heart failure. [4]

C25H23N5O2S.CH4O3S

553.65

457.157

C, 56.40; H, 4.92; N, 12.65; O, 14.45; S, 11.58

138489-18-6

Ro 31-8220;125314-64-9

5083

Yellow to orange solid powder

1.4±0.1 g/cm3

1.740

3.95

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.S(C([H])([H])[H])(=O)(=O)O[H]

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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.76 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 20.8 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: ≥ 2.08 mg/mL (3.76 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 20.8 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.)