PKC/protein kinase C

Effects of Ro 31-8820, H89, Ro 31-6045 and H85 on in vitro activity of S6K [2]
Both the bisindolylmaleimide (Ro 31-8220 and Ro 31-6045) and isoquinolinesulfonamide analogues (H89 and H85) were designed to compete with ATP for binding to the active site of kinases. In order to determine whether all the inhibitors directly affected S6K activity, epitope-tagged wild-type S6K was expressed in serum-stimulated HEK293 cells, immuno-purified and assayed for activity at varying doses of each inhibitor (Fig. 3 ). While Ro 31-8220, H89 and H85 all inhibited S6K in vitro, Ro 31-6045 had no effect up to 100 μM, indicating this compound may be acting via inhibition of an upstream signalling pathway.

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Effects of Ro 31-6045 on PDK1 signalling [2]
PDK1-catalysed phosphorylation of T229 in the activation loop of S6K is the best characterised activating mechanism for the kinase. PDK1 also activates other AGC kinase family members including Akt, PKA and PKC via phosphorylation of homologous residues. While PKA and PKC have been shown to be resistant to Ro 31-6045 up to 100 μM, it is theoretically possible that Ro 31-6045 inhibits PDK1 and the resistant kinases require lower levels of PDK1 activity. In order to test this possibility, a detailed dose–response curve of the effects of Ro 31-6045 on Akt, the kinase most closely related to S6K, was carried out (Fig. 4A ). Akt was weakly inhibited with an IC50>50 μM, 10-fold higher than for S6K. Furthermore, Ro 31-6045 had no effect on the activity of PDK1 exogenously expressed in HEK293 cells at concentrations up to 100 μM (Fig. 4B), consistent with the resistance of PKA and PKC and indicating that Ro 31-6045 does not act via PDK1.

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We have used radioligand binding studies to determine the affinities of seven bisindolylmaleimide analogues, six of which are selective inhibitors of protein kinase C, at human muscarinic M1-M4 receptors. The compounds were most potent at M1 receptors, and Ro-31-8220 was the most potent analogue, with a Kd of 0.6 microM at M1 receptors. The weakest compounds, bisindolylmaleimide IV and bisindolylmaleimide V, had Kd values of 100 microM. If it is necessary to use protein kinase C inhibitors at concentrations of 10 microM or more in studies involving muscarinic receptors then bisindolylmaleimide IV may be the most appropriate inhibitor to use [1].

The mitogen-stimulated protein kinase p70(s6k)/p85(s6k) (S6K) plays an essential role in cell proliferation and growth, with inhibitors of the S6K signalling pathway showing promise as anti-tumour therapeutics. Here, we report that the bisindolylmaleimide derivative Ro 31-6045, previously reported to be inactive as a kinase inhibitor, inhibited S6K activity in vivo with an IC50=8 microM. Structure/function analysis using mutant forms of S6K indicates that Ro 31-6045 inhibition is independent of the upstream activator mTOR. Ro 31-6045 will prove useful in elucidating the complex activation mechanism of S6K and its independence from mTOR will allow confirmation of functional data obtained using the mTOR inhibitor rapamycin [2].

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Effects of Ro 31-8220, H89, Ro 31-6045 and H85 on in vivo activation of S6K [2]
In order to search for selective inhibitors of S6K signalling we have investigated the in vivo potency of two commonly used inhibitors of AGC family kinases, the protein kinase C (PKC) inhibitor Ro 31-8220 and the protein kinase A (PKA) inhibitor H89 that have been shown to inhibit S6K in vitro. Their inactive analogues Ro 31-6045 and H85 were incorporated as specificity controls (Fig. 1 ). Ro 31-8220 and H89 potently inhibited EGF-induced activation of S6K in fibroblasts with IC50 values of 600 nM and 3 μM respectively. Surprisingly, Ro 31-6045 also inhibited S6K with IC50=8 μM and H85 with IC50=25 μM. None of these compounds inhibited the related kinase Akt at 5 μM, a concentration that significantly inhibited S6K activation (Fig. 2A,B ). Interestingly, both Ro 31-8220 and H89 reproducibly enhanced Akt activity.

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Effects of Ro 31-6045 on in vivo activation of S6K mutants [2]
In order to further delineate the mechanism of action of Ro 31-6045 we determined the effect of this inhibitor on the activity of some S6K structure/function mutants. S6K is activated by a complex mechanism involving wortmannin-sensitive activation of PI3K and signalling via the rapamycin-sensitive kinase mTOR. A mutant form of S6K with the hydrophobic motif phosphorylation site and three autoinhibitory sites substituted with acidic residues (dED3E) is resistant to inactivation by both wortmannin and rapamycin. Deleting portions of the C- and N-termini of the kinase results in a mutant (ΔNΔC) that remains resistant to rapamycin but sensitive to wortmannin, indicating a clear difference between the two processes. As both mutants are phosphorylated on T229, the target site of the PI3K-dependent activator PDK1, it is likely that this double deletion mutant reveals a novel signalling pathway. Because these mutants apparently distinguish the two activating pathways, each was tested for its sensitivity to inhibition by Ro 31-6045 (Fig. 5A ). Like wortmannin, Ro 31-6045 failed to significantly inhibit the activity of the constitutively active dED3E mutant at concentrations that reduced the activity of both the double truncation mutant and wild-type enzyme by 80%. Western blot analysis of these extracts using the phospho-T389 antibody revealed that the inhibition of wild-type and ΔNΔC by Ro 31-6045 was accompanied by the reduction of Thr-389 phosphorylation to levels similar to baseline (Fig. 5B), consistent with the inhibitor targeting phosphorylation of this key regulatory site. Indeed, substitution of T389 with glutamic acid renders ΔNΔC totally resistant to Ro 31-6045 (Fig. 5A), confirming T389 phosphorylation as the critical target for inhibition.

S6K, Akt and PDK1 activity assays [2]
Endogenous S6K activity was assayed as described previously using 40S ribosomes as substrate. Myc-tagged p70s6k was immunoprecipitated and assayed as described previously. Akt activity was assayed as described previously using RPRAATF peptide as substrate. The results are expressed in units of activity per mg protein lysate or as % of control values. One unit of activity results in the transfer of 1 pmol of 32Pi to the respective substrate per minute. Autophosphorylation activity of myc-tagged PDK1 was determined essentially as reported previously. Briefly, myc-PDK1 was immunoprecipitated from 250 μg of extracts from transiently transfected HEK293 cells and the washed immunocomplexes incubated for 20 min at 30°C in a total volume of 20 μl of 25 mM Tris–HCl pH 7.5, 0.5 mM dithiothreitol, 0.5 mM benzamidine, 50 μM [γ-32P]ATP (5000 cpm/pmol) and 10 mM MgCl2. The reaction was stopped by boiling in SDS–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and proteins resolved by SDS–PAGE. Phosphorylation of PDK1 was visualised by autoradiography of the dried gel and quantitated by liquid scintillation counting of excised bands.

Cell culture and preparation of cell extracts [2]
HEK293 and Balb/c 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum at 37°C in a 5% CO2 atmosphere. Balb/c 3T3 cells were plated at 2×106 per 10 cm plate for 24 h, then made quiescent by culturing for 24 h, in DMEM containing no serum. Serum-starved cells were rinsed twice with HEPES buffer solution containing 120 mM NaCl and 20 mM HEPES, pH 7.4. Cells were then treated at 37°C with 5 nM EGF for 5 min. Alternatively, the cells were pretreated for 20 min with varying concentrations of Ro 31-8220, H89, Ro 31-6045 or H85 as indicated. Following stimulation, the cells were rinsed twice with ice cold phosphate-buffered saline (PBS) and lysed in a buffer containing 50 mM Tris, pH 7.5, 120 mM NaCl, 1% Nonidet-P40, 1 mM EDTA, 50 mM NaF, 40 mM β-glycerophosphate, 0.1 mM sodium vanadate, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride. Cell extracts were collected with a plastic scraper, and cleared by centrifugation at 4°C for 15 min at 12 000×g. Protein concentration was measured by the method of Bradford, with bovine serum albumin as standard. Aliquots of the supernatant were frozen in liquid nitrogen and stored at −70°C.

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Transient transfections [2]
HEK293 cells were plated at 1×106 per 10 cm plate and 24 h after plating were transfected with p70s6k expression constructs tagged with the myc epitope using the calcium phosphate method as described previously or with myc epitope-tagged PDK1. The constructs used were wild-type (2B4); constitutively active, rapamycin and wortmannin resistant (dED3E) with T389 and T421 mutated to glutamic acid and S411, S418, and S424 mutated to aspartic acid residues; the rapamycin resistant, wortmannin-sensitive double truncation mutant (ΔNΔC) missing residues 1–54 and 389–502 and ΔNΔC with T389 mutated to glutamic acid (ΔNΔC389E). Twenty-four hours after transfection, the cells were serum-starved for 24 h. Following serum-starvation the cells were rinsed with HEPES buffer and stimulated with 20% serum for 20 min. Alternatively the cells were pretreated with varying concentrations of Ro 31-8220, Ro 31-6045, H89 or H85 as indicated. Following stimulation the cells were washed and harvested as above.

[1]. Muscarinic interactions of bisindolylmaleimide analogues. Eur J Pharmacol. 1998 Nov 6;360(2-3):281-4.

[2]. Ro 31-6045, the inactive analogue of the protein kinase C inhibitor Ro 31-8220, blocks in vivo activation of p70(s6k)/p85(s6k): implications for the analysis of S6K signalling. FEBS Lett. 2002 May 22;519(1-3):135-40.

Ro 31-6045 is a member of indoles.

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In addition to aiding in the analysis of S6K signalling, Ro 31-6045 may provide a lead compound for development of S6K signalling-specific inhibitors of therapeutic importance. Maintenance of a sustained elevated rate of protein synthesis is a fundamental requirement for tumour formation. Over the last year analogues of the S6K signalling pathway antagonist, rapamycin have been tested in pre-clinical and phase I studies with preliminary evidence of anti-tumour effects being observed in renal cell carcinoma and non-small cell lung cancer. In fact, a specific role for S6K in breast cancer has been inferred from the observation that the S6K gene is amplified and the protein overexpressed in a number of breast cancer cell lines and 8% (68/698) of primary tumours.
In summary, we have identified the bisindolylmaleimide derivative Ro 31-6045 as an in vivo inhibitor of S6K. Structure/function analysis using mutant forms of S6K indicates that Ro 31-6045 targets T389 phosphorylation independent of the upstream activator mTOR. This inhibitor will prove useful in elucidating the complex activation mechanism of S6K, and in combination with a drug resistant mutant of S6K (dED3E), this reagent will allow rapamycin-independent analysis of S6K signalling in cell-based systems. In addition, Ro 31-6045 may provide a lead in the development of S6K-specific inhibitors of tumour growth. [2]

C21H15N3O2

341.362704515457

341.116

C, 73.89; H, 4.43; N, 12.31; O, 9.37

113963-68-1

2400

Orange to red solid powder

1.5±0.1 g/cm3

655.7±55.0 °C at 760 mmHg

271°C

350.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.794

3.65

2

2

2

26

604

0

O=C1C(C2=CNC3C=CC=CC2=3)=C(C(N1C)=O)C1=CNC2C=CC=CC1=2

SWAWYMIKGOHZMR-UHFFFAOYSA-N

InChI=1S/C21H15N3O2/c1-24-20(25)18(14-10-22-16-8-4-2-6-12(14)16)19(21(24)26)15-11-23-17-9-5-3-7-13(15)17/h2-11,22-23H,1H3

3,4-bis(1H-indol-3-yl)-1-methylpyrrole-2,5-dione

bisindolylmaleimide v; 113963-68-1; 1-Methyl-3,4-bis(3-indolyl)maleimide; 3,4-di(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione; Ro 31-6045; 3,4-bis(1H-indol-3-yl)-1-methylpyrrole-2,5-dione; 2,3-bis(1H-Indol-3-yl)-N-methylmaleimide; 1H-Pyrrole-2,5-dione, 3,4-di-1H-indol-3-yl-1-methyl-; Ro-31-6045

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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