Protein Kinase C (PKC) (IC50 = 20 nM)

Go6976 is a highly effective PKC inhibitor in vitro (IC50 = 20 nM). Its structural similarity to staurosporine makes it the most potent PKC inhibitor[1]. Interestingly, Go6976 is also found to abrogate S and G2 arrest. Dose-response studies show that 30 nM Go6976 is sufficient to abrogate S-phase arrest in 6 hours and complete abrogation of G2 arrest followed by lethal mitosis in 24 hours. Incubation of cells with 100 nM Go6976 is sufficient to completely abrogate S and G2 arrest at 6 and 24 hours, respectively, and is only slightly less potent than in bovine serum. At the concentrations used, incubation of cells with UCN-01 or Go6976 alone does not reduce viability in comparison to control.

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With U1 and ACH-2 cell lines representative of an HIV-1 postintegration state, the effect of Gö 6976, a synthetic inhibitor of PKC was tested. Gö 6976 is a nonglycosidic indolocarbazole found to potently inhibit HIV-1 induction by Bryostatin 1, tumor necrosis factor alpha, and interleukin 6. Gö 6976 effectively blocks viral transcription induced by Bryostatin 1 or tumor necrosis factor alpha that leads to the inhibition of intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin 6-mediated posttranscriptional induction of viral proteins. The IC50 of Gö 6976 shows a 12- to 60-fold more potent effect than for H-7, another PKC inhibitor with a similar mechanism. The inhibitory effect is reduced when Gö 6976 is not added before or within 1 hr of induction by the potent PKC activator Bryostatin 1. However, U1 cells can be grown for long periods in a nontoxic concentration of Gö 6976 (300 nM), which confers virtual inhibition of HIV-1 induction without the development of resistance. Results indicate that inhibition of HIV-1 proviral induction from latent/low-level-producing infectious states with potent PKC inhibitors like Gö 6976 may represent an additional and promising antiviral approach. [1]

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We identified that the protein kinase C (PKC) inhibitor Gö6976 had a post-entry anti-influenza viral effect, by using a polymerase activity-based reporter assay. This inhibitory effect was observed for influenza virus-infected cells as well as for cells transiently transfected with constructs for the RNA polymerase complex. Importantly, the in vitro analysis of viral protein phosphorylation identified PKCalpha as a kinase phosphorylating PB1 and NS1, but not PB2, PA or NP. Gö6976 was able to block PKC-specific phosphorylation in vitro. Thus, our data suggest that PKC contributes to the phosphorylation of influenza PB1 and NS1 proteins which appears to be functionally relevant for both viral RNA polymerase activity and efficient viral replication[3].

Go6976 can effectively inhibit the progression of CML in mice [4]
To explore the effects of Gö6976 in the body, we created a mouse model of CML. Mice began to develop leukemic symptoms at approximately 3 weeks after injection of tumor cells, including hindlimb paralysis, weight loss, fluffy hair and reduced activity (Figure 2(a)). Along with the experimental data that follow, the K562-NOD/SCID CML mouse model was successfully established. After K562 cells were injected, CML mice were weighed and recorded weekly. Weight loss of the mice was found in both the Gö6976 group (2.5 mg/kg) and the control group. Compared with the control group, the mice in the Go6976 group had significantly less weight loss 5 weeks after injection (Figure 2(b)).

Figure 2. The therapeutic effect of Go6976 on CML mice. (a) K562-NOD/SCID CML mice showed hindlimb paralysis, weight loss, fluffy hair and reduced activity. (b) Weight of mice at different time after K562 cells injection (*P < 0.05 versus control group). (c) Survival curves of two group mice (8 mice per group). (d) General morphology of spleens from two group mice. (e) Weight of Spleens from two group mice. We collected the peripheral blood of the mice during the experiment and found that WBCs showed an upward trend in both groups. However, the number of WBCs in the 2.5 mg/kg Go6976 group was less than that in the control group beginning the first week after injection. The number of WBCs in the 2.5 mg/kg Gö6976 group was significantly decreased compared with that in the control group from the 3rd to the 5th weeks (Figure 3(a)). At the same time, the percentage of CD45+ leukemia cells in the peripheral blood of mice at different times was observed by flow cytometry. The results showed that the proportion of CD45+ leukemia cells in the WBCs from peripheral blood was only approximately 1% at the first and second weeks, and it was approximately 2-3% at the third week. There was no significant difference between the two groups in the first to third weeks. At the fourth week, the CD45+ leukemia cells in the 2.5 mg/kg Gö6976 group were significantly fewer than those in the control group (Figure 3(b–d)).

Figure 3. Peripheral blood analysis of two group of mice, including white blood cells count and flow cytometry analysis of the percentage of CD45+ leukemia cells. (a) WBCs count in peripheral blood from the control group and the Gö6976 group (*P < 0.05 versus the control group). (b) The percentage of CD45＋ leukemia cells in the control group on week 4. (c) The percentage of CD45＋ leukemia cells in theGo6976 group on week 4. (d) The percentage of CD45+ leukemia cells in peripheral blood from two groups on week 3 and week 4 (*P < 0.05 versus the control group).

The death time of the two groups of mice was recorded. The first mouse died in the control group on the 29th day after injection, and the last mouse in the control group died on the 71st day after transplantation. In the Gö6976 group, the first mouse died on the 40th day after injection, and three mice survived until the 80th day of the observation period. The survival time of the mice in the 2.5 mg/kg Gö6976 group was significantly longer than that of the mice in the control group. (Figure 2(c)). Finally, the spleens were collected and weighed at the 9th week after injection. We found that the spleens of mice from the 2.5 mg/kg Gö6976 group were smaller than those from the control group (Figure 2(d)), and the weights of the spleens were also lower than those from the control group (Figure 2(e)). Go6976 has no significant effects on the general condition, liver, spleen or lungs of healthy mice [4]
Through the above experiments, we proved that Gö6976 had a significant inhibitory effect on CML in vivo and in vitro. We found that Gö6976 had almost no effect on ordinary hematopoietic cells and immune cells in the in vitro experiments (Figure 1(c)). Thus, whether it has an effect on healthy mice will be our next research direction.

We divided healthy BALB/c mice into three groups: the control group (normal saline), the low-dose Go6976 group (2.5 mg/kg Gö6976) and the high-dose Gö6976 group (10 mg/kg Gö6976). We found that there were no abnormalities in mental state, daily activities, food intake, or bowel movements of the mice in each group during the administration, and there was no significant difference in weight gain among the control group, the low-dose Gö6976 group and the high-dose Gö6976 group (Figure 4(a)). The growth trend of these three groups remained consistent. The livers, spleens, and lungs were obtained and stained with HE at 3 weeks after injection. The tissue structure of these organs from the three groups was observed under light microscopy, and there was no inflammatory infiltration, cell degeneration, necrosis, interstitial hyperemia, edema or other pathological reactions in these two Gö6976 groups compared with the control group (Figure 4(b–d)).

Figure 4. The effect of Go6976 on the weight and organs of healthy mice. (a) The weight growth rate of the mice in each group. (b) Liver sections of the mice in each group after HE staining. (c) Spleen sections of the mice in each group after HE staining. (d) Lung sections of the mice in each group after HE staining.

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Go6976 (2.5 mg/kg i.p.), as a PKD inhibitor, effectively prevents LPS/D: -GalN-induced acute liver injury by inhibition of MAPKs activation to reduce TNF-α production, and significantly improves the survival of LPS/D-GalN-challenged mice.

Cells counting kit-8 assay [1]
K562 cells in logarithmic growth phase were collected and seeded into 96-well cell culture plates at a density of 5×103 cells/well. K562 cells were treated with 5 μM Go6976/Gö6976, 10 µM Gö6976, 0 μM Gö6976 + 2 μM imatinib, 5 μM Gö6976 + 2 μM imatinib, and 10 μΜ Gö6976 + 2 μM imatinib, and the control group was only treated with PBS. Five parallel wells were set in each group and cultured for 72 h. Ten microliters of Cell Counting Kit-8 (CCK-8) solution was added to each well. After incubation for 3 h at 37 °C in a 5% CO2 incubator, the absorbance at 450 nm was recorded.

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MTT assay [1]
CD34+ cells and PBMCs in logarithmic growth phase were collected and seeded in 96-well cell culture plates at a density of 5×103 cells/well. CD34+ cells and PBMCs were treated with 0.5, 1.0, 2.5, and 5.0 μM Go6976/Gö6976 for 24 h. Five parallel wells were set in each group and cultured for 72 h. Then, 50 μL of 2 mg/mL MTT solution were added to each well, and the cells were cultured for 4 h and centrifuged at 2000 r/min for 10 min. The supernatant was discarded in the dark. Finally, 100 μL of DMSO were added to each well, the mixture was shaken for 5 min, and the absorbance of each well was read at 450 nm.

In vivo drug treatment [4]
Drug treatments began at 8 days after K562 cell injection and were administered as two doses per week for 4 weeks. According to whether it was administered with Go6976Gö6976, K562-NOD/SCID CML mice were divided into two groups: mice in the experimental group injected with 2.5 mg/kg Go6976/Gö6976 and mice in the control group injected with the same volume of normal saline.

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In vivo drug toxicity experiment [4]
Twenty-four BALB/c mice were randomly divided into 3 groups: mice in the experimental group injected with 2.5 mg/kg Go6976/Gö6976 or 10 mg/kg Go6976/Gö6976 and mice in the control group injected the same volume of normal saline. The mice were weighed and injected 12 times every 2 days. After the injections were stopped, blood and organs from these mice were collected.

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Dissolved in DMSO; 2.5 mg/kg; i.p. injection

LPS/D-GalN-challenged mice

[1]. Gö 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4674-8.

[2]. Microbial metabolites and derivatives targeted at inflammation and bone diseases therapy: chemistry, biological activity and pharmacology. The Journal of Antibiotics volume 71, pages 60–71 (2018).

[3]. Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C. J Gen Virol. 2009;90(Pt 6):1392-1397.

[4]. The effect of Gö6976 on chronic myeloid leukemia in vitro and in vivo. Hematology . 2021 Dec;26(1):543-551.

Goe 6976 is an organic heterohexacyclic compound belonging to the indolecarbazole class. It is an EC 2.7.11.13 (protein kinase C) inhibitor. It is an inhibitor of a calcium-dependent protein kinase C subtype. Human immunodeficiency virus (HIV-1) infection follows a latent period or a persistent low-level (LLP) state, leading to asymptomatic infection in the host. Various activators, including mitogens, antigens, and cytokines, can trigger effective viral expression. Protein kinase C (PKC) has been shown to play an important role in the intracellular signaling cascade induced by these activators. This study used U1 and ACH-2 cell lines, representing the post-integration state of HIV-1, to test the effect of the synthetic PKC inhibitor Goe 6976. Goe 6976 is a non-glycoside indolecarbazole that effectively inhibits Bryostatin 1, tumor necrosis factor α, and interleukin-6-induced HIV-1 expression. Gö 6976 effectively blocks Bryostatin 1 or tumor necrosis factor α-induced viral transcription, thereby inhibiting intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin-6-mediated post-transcriptional induction of viral proteins. The IC50 value of Gö 6976 is 12 to 60 times higher than that of H-7, another protein kinase C (PKC) inhibitor with a similar mechanism of action. Its inhibitory effect is weakened when Gö 6976 is not added before or within one hour after induction by the potent PKC activator Bryostatin 1. However, U1 cells can be cultured long-term at a non-toxic concentration of Gö 6976 (300 nM), which almost completely inhibits HIV-1 induction without developing resistance. The results suggest that using potent PKC inhibitors, such as Gö 6976, to inhibit the induction of HIV-1 provirus from latent/low-level infection may represent a promising new antiviral strategy. [1] Objective: Chronic myeloid leukemia (CML) is a hematologic malignancy. Gö 6976 is an indolecarbazole compound with strong antitumor activity, but there are no reports on the efficacy of Gö 6976 in CML. This study aims to: (1) investigate the effects of Gö 6976 on chronic myeloid leukemia (CML) in vitro and in vivo; and (2) investigate the drug toxicity of Gö 6976 on normal cells and animals. Methods: The effects of Gö 6976 on CML were investigated using K562 cells and a CML mouse model. The drug toxicity of Gö 6976 was investigated using peripheral blood mononuclear cells (PBMCs), CD34+ cells, and a healthy mouse model. Results: Cell experiments showed that Gö6976 at concentrations of 5 μM and 10 μM could inhibit the proliferation of K562 cells and enhance the inhibitory effect of imatinib, but the effect on CD34+ cells or PBMCs was minimal at concentrations below 5 μM. Animal experiments showed that 2.5 mg/kg of Gö6976 could effectively inhibit the development of chronic myeloid leukemia (CML) in mice, while the doses of 2.5 mg/kg and 10 mg/kg had almost no effect on healthy mice. Discussion: Since Gö6976 has a direct inhibitory effect on CML and can enhance the pharmacological effect of imatinib, Gö6976 is expected to become a new anti-CML drug. However, further research is still needed. Conclusion: Our results confirm that Gö6976 can effectively inhibit CML in vitro and in vivo, and has almost no toxicity to hematopoietic cells, immune cells and healthy mice. [4]

C24H18N4O

377.42

378.148

C, 76.17; H, 4.79; N, 14.81; O, 4.23

136194-77-9

3501

white solid powder

1.4±0.1 g/cm3

652.3±55.0 °C at 760 mmHg

348.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.774

3.48

1

2

2

29

730

0

N#CCCN(C1=C2C(CNC3=O)=C3C4=C1N(C)C5=C4C=CC=C5)C6=C2C=CC=C6

VWVYILCFSYNJHF-UHFFFAOYSA-N

InChI=1S/C24H18N4O/c1-27-17-9-4-2-7-14(17)20-21-16(13-26-24(21)29)19-15-8-3-5-10-18(15)28(12-6-11-25)23(19)22(20)27/h2-5,7-10H,6,12-13H2,1H3,(H,26,29)

3-(23-methyl-14-oxo-3,13,23-triazahexacyclo[14.7.0.02,10.04,9.011,15.017,22]tricosa-1,4,6,8,10,15,17,19,21-nonaen-3-yl)propanenitrile

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)

Solubility in Formulation 1: ≥ 3.25 mg/mL (8.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 1.39 mg/mL (3.67 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.9 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 3: 1.39 mg/mL (3.67 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 ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.9 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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Solubility in Formulation 4: 0.5% CMC Na+1% Tween 80: 30mg/mL

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