Bovine brain PKC (IC50 = 10 nM); PKCβII (IC50 = 16 nM); PKCβI (IC50 = 17 nM); PKCα(IC50 = 20 nM); PKCγ(IC50 = 20nM); FDGFR (IC50 = 65 μM)

P47 phosphorylation produced by α-phosphatase is inhibited by bisindolylmaleimide I hydrochloride (5 μM) [1]. μM) can lower GSK-3 activity in internal substances of adipocytes to 25.1±4.3%[3]. Exosome and microvesicle (EMV) release from PC3 cells is inhibited by bisindolylmaleimide I hydrochloride (10 μM, 24 hours) [4]. Fluorouracil's 5-Cytotoxicity is enhanced by bisindolylmaleimide I hydrochloride (10 μM, 24 hours) [4].

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Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways. [1]

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Here reserchers report that the widely used protein kinase C inhibitors, bisindolylmaleimide I (GF 109203X) and IX, are potent inhibitors of glycogen synthase kinase-3 (GSK-3). Bisindolylmaleimide I and IX inhibited GSK-3 in vitro, when assayed either in cell lysates (IC(50) 360 nM and 6.8 nM, respectively) or in GSK-3beta immunoprecipitates (IC(50) 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 microM) and IX (2 microM) reduced GSK-3 activity in total cell lysates, to 25.1+/-4.3% and 12.9+/-3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 microM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK-3, and that this may explain some of the previously reported insulin-like effects on glycogen synthase activity.[3]

In mice in the mechanically ventilated (MV) group, elevated levels of NLRP3, P-PKCɑ, and PKCɑ are reduced by intraperitoneal injection of bisindolylmaleimide I hydrochloride (0.02 mg/kg) [5]. Bisindolylmaleimide I (0–20 mg/kg, i.p.) lowers the average frequency of shrew vomiting caused by quinpirole [6].

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Streptozotocin (STZ)-induced chronic hyperglycemia has a detrimental effect on neurovascular coupling, linked to increased PKC-mediated phosphorylation and PKC isoform expression changes. Here, we sought to determine whether: 1) selective PKC-α/β/γ inhibitor, GF 109203X, could reverse the effects of chronic hyperglycemia on cerebrovascular reactivity; 2) pancreatic islet transplantation could prevent the development of cerebrovascular impairment seen in a rat model of Type 1 Diabetes. We studied the effect of GF109203X in diabetic (DM), non-diabetic (ND), and transplanted (TR) Lewis rats during either sciatic nerve stimulation (SNS) or the topical applications of the large-conductance Ca2+-operated K+(BKCa) channel opener, NS1619, or the K+ inward rectifier (Kir) channel agonist, KCl. Pial arteriole diameter changes were monitored using a closed cranial window in vivo microscopy technique. The pial arteriole dilatory response associated with SNS was decreased by ~45%, when comparing DM vs either ND or TR rats. Also, pial arteriolar dilations to topical KCl and NS1619 were largely attenuated in DM rats, but not in ND or TR animals. These responses were completely restored by the acute application of GF109203X to the brain surface. The PKC inhibitor had no effect on vascular responses in normoglycemic and TR animals. In conclusion, DM-associated chronic impairment of neurovascular coupling may be readily reversed by a PKC-α/β/γ inhibitor or prevented via pancreatic islet transplantation. We believe that specific PCK isoforms (α/β/γ) are mechanistically linked to the neurovascular uncoupling seen with hyperglycemia. [2]

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To test the effects of the PKC inhibitor GF 109203X/bisindolylmaleimide I on PKC and NLRP3, male C57BL/6 mice (7 weeks old, 19 ~ 23 g) were randomly divided into four groups: control group(C), bisindolylmaleimide I-pretreated group(B), MV group, and bisindolylmaleimide I-pretreated + MV (B + MV) group. The mice were pretreated with bisindolylmaleimide I through intraperitoneal injection (0.02 mg/kg) 1 h before MV. MV was performed at a high tidal volume (30 ml/kg). To explore the ameliorative effect of EX on VILI, the mice were randomly divided into C group, MV group, EX group and EX + MV group and subjected to either MV or 5 weeks of EX training. After ventilation, haematoxylin-eosin (HE) staining and wet/dry weight ratio was used to assess lung pathophysiological changes. PKCɑ, P-PKCɑ, ASC, procaspase-1, caspase-1, pro-IL-1β, IL-1β, NLRP3 and occludin (tight junction protein) expression in lung tissues was determined by Western blotting. The level of IL-6 in alveolar lavage fluid was determined by ELISA. Results: NLRP3, P-PKCɑ, and PKCɑ levels were inceased in MV group, but GF 109203X/bisindolylmaleimide I treatment reversed these changes. Inhibition of PKC production prevented NLRP3 activation. Moreover, MV increased ASC, procaspase-1, caspase-1, pro-IL-1β, and IL1β levels and decreased occludin levels, but EX alleviated these changes. HE staining and lung injury scoring confirmed an absence of obvious lung injury in C group and EX group. Lung injury was most severe in MV group but was improved in EX + MV group. Overall, these findings suggest that MV activates the NLRP3 inflammasome by activating PKCɑ and inducing occludin degradation, while Exercise attenuates NLRP3 inflammasome and PKCɑ activation. Besides, exercise improves cyclic stretch-induced degradation of occludin. Conclusion: PKC activation can increase the level of NLRP3, which can lead to lung injury. Exercise can reduce lung injury by inhibiting PKCɑ and NLRP3 activation. Exercise maybe a potential measure for clinical prevention of VILI.[5]

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With its five receptor subtypes (D1-5), dopamine is implicated in a myriad of neurological illnesses. Dopamine D2 receptor-based agonist therapy evokes nausea and vomiting. The signaling mechanisms by which dopamine D2 receptors evoke vomiting remains unknown. Phosphatidylinositol 3-kinases (PI3K)- and protein kinase C (PKC)-related signaling cascades stimulate vomiting post-injection of various emetogens in emetically competent animals. This study investigated potential mechanisms involved in dopamine D2 receptor-mediated vomiting using least shrews. We found that vomiting evoked by the selective dopamine D2 receptor agonist quinpirole (2 mg/kg, i.p.) was significantly suppressed by: i) a dopamine D2 preferring antagonist, sulpiride (s.c.); ii) a selective PI3K inhibitor, LY294002 (i.p.); iii) a PKCαβII inhibitor, GF 109203X (i.p.); and iv) a selective inhibitor of extracellular signal-regulated protein kinase1/2 (ERK1/2), U0126 (i.p.). Quinpirole-evoked c-fos immunofluorescence in the nucleus tractus solitarius (NTS) was suppressed by pretreatment with sulpiride (8 mg/kg, s.c.). Western blot analysis of shrew brainstem emetic loci protein lysates revealed a significant and time-dependent increase in phosphorylation of Akt (protein kinase B (PKB)) at Ser473 following a 30-min exposure to quinpirole (2 mg/kg, i.p.). Pretreatment with effective antiemetic doses of sulpiride, LY294002, GF 109203X, or U0126 significantly reduced quinpirole-stimulated phosphorylation of emesis-associated proteins including p-85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473). Our results substantiate the implication of PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt signaling pathways in dopamine D2 receptor-mediated vomiting. Potential novel antiemetics targeting emetic proteins associated with these signaling cascades may offer enhanced potency and/or efficacy against emesis.[6]

Assay of PKC is arrayed by measuring 32Pi transferred from [γ-32Pi] ATP to lysine-rich histone type Ill-s. The reaction mixture (80 μL) contains 50 mM Tris-HCI, pH 7.4. 100 μM CaCl2, 10 mM MgCI2, 37.5 μg/mL histone type Ill-s, l0 μM [γ-32Pi] ATP (1250cpm/pmol), 31 μM bovine brain phosphatidylserine and 0.5 μM 1,2 sn-dioleylglycerol. 15 μL of purified PKC (final concentration in assay 0.38 μg/mL) is added to the incubation mixture. After 10 minutes, the reaction is stopped by addition of at 30 μL of casein 30 mg/mL and 0.9 mL of 12% trichlomacetic acid [1].

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GSK-3 activity assay [3]
GSK-3 activity was measured in cell lysates and in GSK-3β immunoprecipitates. GSK-3β was immunoprecipitated from cell lysates by tumbling with 4 μl of anti-GSK-3β monoclonal antibody and 3.75 mg protein A-Sepharose for 2 h at 4°C. The resulting immunoprecipitates were washed three times in kinase assay buffer (20 mM HEPES, pH 7.5, 20 mM β-glycerophosphate and 1 mM EDTA) and finally resuspended in 300 μl of kinase assay buffer containing 0.1% mercaptoethanol and 2.5 μM cAMP-dependent protein kinase inhibitor peptide (IP20). The activity of GSK-3 was measured in duplicate in 20 μl of cell lysate or 20 μl of GSK-3β immunoprecipitate using the synthetic peptide substrate RRAAEELDSRAGS(P)PQL (0.71 mg/ml) [14] in the absence or in the presence of the GSK-3 inhibitor, lithium chloride (50 mM). The assay was terminated after 15 min incubation with [γ- 32P]ATP by spotting onto P81 ion-exchange paper. The paper was washed four times in 0.6% phosphoric acid and bound radioactivity quantified by scintillation counting. Phosphorylation of peptide by adipocyte lysates and by GSK-3β immunoprecipitates was essentially completely inhibited by lithium chloride. The average activity of GSK-3 in the extracts was 1220±144 pmol peptide phosphorylated/min/g dry weight of adipocytes (n=11). The average activity of GSK-3β in immunoprecipitates was 276±54 pmol peptide phosphorylated/min/g dry weight of adipocytes (n=11).

Researchers further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways [1].

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Microvesicle (MV) release from tumour cells influences drug retention, contributing to cancer drug resistance. Strategically regulating MV release may increase drug retention within cancer cells and allow for lower doses of chemotherapeutic drugs. The contribution of exosomes to drug retention still remains unknown. Potential exosome and MV (EMV) biogenesis inhibitors, tested on human prostate cancer (PC3) cells for their capacity to inhibit EMV release, were also tested on PC3 and MCF-7 (breast cancer) cells for improving chemotherapy. Agents inhibiting EMV release most significantly, whilst maintaining cell viability, were chloramidine (Cl-amidine; 50 µM) and GF 109203X/bisindolylmaleimide-I (10 µM). Apoptosis mediated by the chemotherapy drug 5-fluorouracil (5-FU) was significantly enhanced in PC3 cells in the presence of both these EMV inhibitors, resulting in a 62% (Cl-amidine + 5-FU) and 59% (bisindolylmaleimide-I + 5-FU) decrease in numbers of viable PC3 cells compared to 5-FU alone after 24 h. For MCF-7 cells, there were similar increased reductions of viable cells compared to 5-FU treatment alone ranging from 67% (Cl-amidine + 5-FU) to 58% (bisindolylmaleimide-I + 5-FU). Using combinatory treatment, the two EMV inhibitors further reduced the number of viable cancer cells tested. Neither inhibitor affected cell viability. Combining selected EMV inhibitors may pose as a novel strategy to enhance the efficacy of chemotherapeutic drug-mediated apoptosis. [4]

Animal/Disease Models: Quinpirole-treated shrews[2]
Doses: 0-20 mg/kg
Route of Administration: ip
Experimental Results: diminished quinpirole-injection) diminished the mean frequency of quinpirole-induced vomiting in shrews[ 6]. Induces vomiting. Blocks quinpirole-mediated ERK1/2 phosphorylation in the shrew brainstem. In the typical neurovascular coupling experiment, 1 hour after the anesthesia switch from isoflurane to fentanyl, and regular aCSF suffusion (equilibration period), the rats were subjected to one or two sciatic nerve stimulation episodes followed by NS1619 (10 and 50 µM) or K+ (KCl 6 and 12 mM) suffusions under the cranial window for 5 min at each concentration. In the DM and ND groups, after a recovery period of at least 5 min, we initiated a suffusion of GF 109203X(also known as Bisindolylmaleimide I or Gö 6850). GF109203X, at 20 nM, tends to favor the inhibition of PKC-α (IC50 of 8 nM). However, such concentration is thought to inhibit also PKC-βI, -βII and -γ (IC50 of 18, 20, and 21 nM, respectively), but not other PKC isoforms, e.g. δ, ε and ζ (IC50 of 210, 132 and 5800 nM, respectively). Therefore, all the conventional PKC isoforms might be similarly affected by GF109203X. Pial arteriolar diameter changes, relative to baseline, after 40 min suffusion of GF109203X (20 nM) under the cranial window were modest and not significantly different when comparing non-diabetic and diabetic rats (-2 ± 3% and -3 ± 6 %, respectively; P > 0.05). Forty minutes later, a second sciatic nerve stimulation was imposed, followed by re-evaluation of NS1619 and K+-induced dilations. [2]

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Seven-week-old male C57BL/6 N mice weighing 19–23 g were fed for 1 week to allow them to adapt to the environment. The mice were kept in a constant temperature of 25 °C under a 12-hour light/dark cycle and were fed a standard diet of pellets and water. To determine the relationship between PKC activation and NLRP3, the mice were randomly divided into the following four groups (n = 6 in each group): control (C) group, GF 109203X/bisindolylmaleimide I-pretreated (0.02 mg/kg)(B) group, mechanical ventilation (MV) group and bisindolylmaleimide I-pretreated (1 h) and mechanically ventilated (B + MV) group. Mechanical ventilation was not conducted in C and B groups. The other two groups were mechanically ventilated for 4 h using an ALC-V8 animal ventilator. The ventilation parameters were set as follows: tidal volume 30 ml/kg, respiratory rate 60 times/min, I/E ratio of 1:2, no positive end-expiratory pressure, fraction of inspired oxygen 21% and room temperature 25 °C.[5]

[1]. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771-81.

[2]. Impairment of neurovascular coupling in Type 1 Diabetes Mellitus in rats is prevented by pancreatic islet transplantation and reversed by a semi-selective PKC inhibitor. Brain Res. 2017 Jan 15;1655:48-54.

[3]. The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity. FEBS Lett. 1999 Nov 5;460(3):433-6.

[4]. Chloramidine/Bisindolylmaleimide-I-Mediated Inhibition of Exosome and Microvesicle Release and Enhanced Efficacy of Cancer Chemotherapy. Int J Mol Sci. 2017 May 9;18(5):1007.

[5]. Aerobic exercise alleviates ventilator-induced lung injury by inhibiting NLRP3 inflammasome activation. BMC Anesthesiol. 2022 Dec 1;22(1):369.

[6]. Signal transduction pathways involved in dopamine D2 receptor-evoked emesis in the least shrew (Cryptotis parva). Auton Neurosci. 2021 Jul;233:102807.

Bim-1 is a member of indoles.
In conclusion, the dysregulation of cerebral perfusion accompanying chronic hyperglycemia, in rat models of type 1 diabetes mellitus, is well-documented. The above pathophysiologic process has been shown to include loss of BKCa and Kir channel-mediated vasodilating functions as well as a significant decrease in the pial arteriolar dilations evoked by somatosensory activation, via sciatic nerve stimulation, in streptozotocin-treated diabetic rats. Present results also suggested that the restoration and maintenance of normoglycemia, via the endogenous production of insulin from transplanted pancreatic islets, are able to prevent the compromise of neurovascular coupling observed in diabetic animals. Moreover, the acute topical administration of an inhibitor of the classical PKC isoforms, GF109203X, was capable of reversing the diabetic cerebrovascular impairments. Both of these neuroprotective mechanisms might have significant clinical implications.[2]

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Several groups have reported that bisindolylmaleimide IX activates JNK in cells in a PKC-independent manner. Activation of JNK by insulin is blocked by wortmannin in CHO cells expressing the insulin receptor and is likely, therefore, to be downstream of PI3 kinase activation. This raises the possibility that inhibition of GSK-3 activity may lead, presumably indirect, to the activation of JNK. This hypothesis is consistent with the observation that the bisindolylmaleimide IX- and insulin-stimulated JNK activation in rat adipocytes are not additive. It requires rigorous testing, particularly as bisindolylmaleimide IX is known to inhibit other protein kinases, such as MAPKAP kinase and p70S6 kinase. However, it should be noted that these particular kinases are unlikely to be involved as insulin and bisindolylmaleimide IX have opposite effects on their activity.
One of the substrates of JNK is c-Jun, which forms part of the activating protein-1 complex (AP-1 complex), and is phosphorylated by JNK on two regulatory sites Ser-63 and Ser-73. Phosphorylation of these sites transactivates c-Jun, and may also explain the increased c-jun expression induced by bisindolylmaleimide IX. Stimulation of AP-1 activity in response to bisindolylmaleimide IX is likely, therefore, to be the result of increased c-Jun synthesis and/or phosphorylation of c-Jun on Ser-63 and Ser-73 by increased JNK activity. However, GSK-3 phosphorylates c-Jun on three sites in a region proximal to the DNA-binding domain (residues 227–252), resulting in decreased c-Jun DNA binding and transcriptional activity. Indeed, transfection experiments have shown that AP-1 activity is inhibited by co-expression of GSK-3. Inhibition of GSK-3 activity by bisindolylmaleimide IX might therefore abolish this negative restraint, thereby increasing c-Jun/AP-1 activity.
In summary, we have demonstrated that both bisindolylmaleimide I and IX are potent and direct inhibitors of GSK-3. Our results raise the possibility that some of the insulin-like effects of bisindolylmaleimide IX, in particular the activation of glycogen synthase, may be the result of the ability of these compounds to inhibit GSK-3.[3]

C25H24N4O2.HCL

448.9446

448.167

C, 66.88; H, 5.61; Cl, 7.90; N, 12.48; O, 7.13

176504-36-2

Bisindolylmaleimide I;133052-90-1

6419775

Orange to red solid powder

4.719

3

3

6

32

748

0

XRAMWNCMYJHGGH-UHFFFAOYSA-N

InChI=1S/C25H24N4O2.ClH/c1-28(2)12-7-13-29-15-19(17-9-4-6-11-21(17)29)23-22(24(30)27-25(23)31)18-14-26-20-10-5-3-8-16(18)20;/h3-6,8-11,14-15,26H,7,12-13H2,1-2H3,(H,27,30,31);1H

3-[1-[3-(Dimethylamino)propyl]indol-3-yl]-4-(1H-indol-3-yl)pyrrole-2,5-dione Hydrochloride

BIS-I HCl; GF-109203X HCl; GO-6860 HCl; 176504-36-2; Bisindolylmaleimide I HCl; Bisindolylmaleimide I, HCl; Bisindolylmaleimide I (hydrochloride); Bisindolylmaleimide I, Hydrochloride; 1H-Pyrrole-2,5-dione, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-, hydrochloride (1:1); GF 109203X hydrochloride; DTXSID00423558; BISI HCl; GF109203X HCl; GO6860 HCl; BIS I HCl; GF 109203X HCl; GO 6860 HCl;

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)

DMSO : ~62.5 mg/mL (~139.22 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.63 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.)