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)

With IC50s of 5, 24, 14, 27, 24, and 23 nM for PKCα, PKCβI, PKCβII, PKCγ, PKCε, and rat brain PKC, respectively, Ro 31-8220 is a strong inhibitor of PKC[1]. Furthermore, Ro 31-8220 strongly inhibits MSK1, S6K1, GSK3β, and MAPKAP-K1b (IC50s: 3, 8, 15, and 38 nM, respectively); MKK3, MKK4, MKK6, and MKK7 are unaffected. Moreover, voltage-dependent Na+ channels are directly suppressed by Ro 31-8220[2]. In cerebellar granule neurons, Ro 31-8220 (1 μM) is neuroprotective against paraoxon-induced neuronal cell death, inhibits paraoxon-induced caspase-3 activity, and lowers the increase in phospho-PKC pan levels caused by paraoxon [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 show ed 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.

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

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)