Indirubin-3'-oxime

Indirubin-3'-monoxime is a potent GSK-3β inhibitor, and weakly inhibits 5-Lipoxygenase, with IC50s of 22 nM and 7.8-10 µM, respectively; Indirubin-3'-monoxime also shows inhibitory activities against CDK5/p25 and CDK1/cyclin B, with IC50s of 100 and 180 nM.

In Vitro:Indirubin-3'-monoxime inhibits GSK-3β by competing with ATP, with Ki of 0.85 μM, and Km of 110 μM. Indirubin-3'-monoxime also inhibits tau phosphorylation by GSK-3β, with an IC50 value of around 100 nM. Indirubin-3'-monoxime completely inhibits the phosphorylation of the AT100 epitope[1]. Indirubin-3'-monoxime inhibits vascular smooth muscle cell (VSMC) proliferation with IC50 of ∼2 µM. Indirubin-3'-monoxime blunts migration of VSMC stimulated with the PDGF. Indirubin-3'-monoxime interferes with the migratory response in VSMC, and also suppresses the production of pro-migratory LT in monocytes. Moreover, Indirubin-3'-monoxime inhibits 5-lipoxygenase (5-LO) product synthesis in monocytes and neutrophils, with the same potency (IC50=5.0±1.1 and 3.7±1.2 µM, respectively). Indirubin-3'-monoxime is an inhibitor of 5-LO, with IC50 of 7.8-10 µM in cell-free assay[3].

In Vivo:Indirubin-3'-monoxime (0.1, 0.2 and 0.4 mg/kg, i.p) dose dependently reverses the cognitive impairment and combats the elevated oxidative stress markers in HFD fed mice. Indirubin-3'-monoxime also dose dependently lowers the serum glucose, TGs, TC and insulin levels, and improves the β-cell functioning in HFD fed mice. Moreover, Indirubin-3'-monoxime treatment significantly decreases HOMA-IR levels compared to HFD group. Indirubin-3'-monoxime (0.4 mg/kg) significantly attenuates the increased EL in the HFD group[2].

References:
[1]. Leclerc S, et al. Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors? J Biol Chem. 2001 Jan 5;276(1):251-60. [2]. Sharma S, et al. Neuroprotective role of Indirubin-3'-monoxime, a GSKβ inhibitor in high fat diet induced cognitive impairment in mice. Biochem Biophys Res Commun. 2014 Oct 3;452(4):1009-15. [3]. Blazevic T, et al. Indirubin-3'-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration. Cardiovasc Res. 2014 Mar 1;101(3):522-32.

Induction of cell death in pancreatic ductal adenocarcinoma by indirubin 3'-oxime and 5-methoxyindirubin 3'-oxime in vitro and in vivo.[Pubmed:28347789]

Cancer Lett. 2017 Jul 1;397:72-82.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis. To identify potential effective therapeutic drugs for PDAC, we established a screening system based on spheroid formation using 170#3 mouse PDAC cells with or without fibroblasts. We found that indirubin 3'-oxime (Indox) and 5-methoxyindirubin 3'-oxime (5MeOIndox) inhibited PDAC cell proliferation. Furthermore, PDAC xenograft growth was also inhibited in BALB/c nu/nu mice after administration of Indox and 5MeOIndox. Both phosphorylated CDK1 and cyclin B1 levels in 170#3 cells were significantly reduced by treatment with Indox and 5MeOIndox in vitro and in vivo. Cell cycle analysis revealed that 5MeOIndox, but not Indox, induced G2/M arrest. Annexin V-propidium iodide double-staining analysis demonstrated that Indox induced abundant non-apoptotic cell death of 170#3 cells, while 5MeOIndox predominantly induced early apoptosis, indicating that the cytotoxicity of 5MeOIndox is lower than that of Indox. These results suggest that one mechanism of 5MeOIndox is to induce G2/M arrest of PDAC cells via inhibition of CDK1/cyclin B1 levels, thereby leading to apoptosis. Our findings suggest 5MeOIndox as a potential useful anticancer agent in PDAC.

The Small Molecule Indirubin-3'-Oxime Inhibits Protein Kinase R: Antiapoptotic and Antioxidant Effect in Rat Cardiac Myocytes.[Pubmed:26571010]

Pharmacology. 2016;97(1-2):25-30.

Double-stranded, RNA-dependent protein kinase R (PKR) is a serine/threonine protein kinase activated by various stress signals. It plays an important role in inflammation, insulin sensitivity and glucose homeostasis. Increased PKR activity has been observed in obese humans as well as in obese diabetic mice. Indirubin-3'-oxime (I3O) is an effective inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3-beta. However, the effects of I3O on PKR activity/expression in cultured rat cardiomyocytes have not been reported. We investigated whether I3O attenuates the effects of high glucose on PKR, oxidative stress and apoptotic gene markers. Quantitative PCR and western blotting were used to measure protein and mRNA, respectively. High glucose treatment caused significant increase in the PKR protein/mRNA expression, which was attenuated by co-treatment with I3O. High glucose-treated, cultured cardiomyocytes developed a significant increase in mRNA expression for c-Jun-N-terminal kinase, caspase-3 and NF-kB, which were all attenuated by pretreatment with I3O. There was also a significant increase in reactive oxygen species generation in high glucose-treated, cultured cardiomyocytes, which was attenuated by pretreatment with I3O. In conclusion, I3O may have a preventive role against the deleterious effects of high glucose in the heart.

Validation of a liquid chromatography-triple quadrupole mass spectrometric method for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma and its application to microdose clinical trial.[Pubmed:26139072]

Biomed Chromatogr. 2016 Mar;30(3):323-9.

A liquid chromatography-triple quadrupole mass spectrometric (LC-MS/MS) method was developed and validated for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma to support a microdose clinical trial. The method consisted of a liquid-liquid extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray(TM) for analysis. d3 -AGM-130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10-2000 pg/mL for AGM-130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was

5-Nitro-5'-hydroxy-indirubin-3'-oxime (AGM130), an indirubin-3'-oxime derivative, inhibits tumor growth by inducing apoptosis against non-small cell lung cancer in vitro and in vivo.[Pubmed:26342773]

Eur J Pharm Sci. 2015 Nov 15;79:122-31.

This study examined the anti-tumor effects of AGM130, a novel indirubin-3'-oxime derivative in A549 human non-small cell lung cancer cells. AGM130 significantly inhibited the proliferation and arrested the cell cycle of G2/M phase. Induction of apoptosis was detected in AGM130-treated A549 cells. The protein levels of Cytochrome c release, Bax, cleaved caspases and PARP were increased in AGM130 treated cells, whereas Bcl-2 levels were decreased. AGM130 inhibited Insulin-like growth factor 1 receptor (IGF1R), AKT/mTOR signaling and inactivated mitogen-activated protein kinases (MAPK). AGM130 also induced slight autophagy as pro-survival function and autophagy inhibition by chloroquine (CQ) induced necrosis. In vivo tumor xenograft model, AGM130 dose-dependently suppressed transplanted A549 cell tumor growth and induced the expression of proliferative cell nuclear antigen (PCNA). AGM130 also increased TUNEL positive apoptotic cell populations and the induction of glandular differentiation with mucin pool compared with vehicle-treated control in tumor tissue. These results suggest that AGM130 is an effective novel indirubin-3'-oxime derivative of anti-cancer drug and may be an attractive candidate for non-small cell lung cancer therapy.

The specificities of protein kinase inhibitors: an update.[Pubmed:12534346]

Biochem J. 2003 Apr 1;371(Pt 1):199-204.

We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM).

Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?[Pubmed:11013232]

J Biol Chem. 2001 Jan 5;276(1):251-60.

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.[Pubmed:10559866]

Nat Cell Biol. 1999 May;1(1):60-7.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Indirubin-3'-monoxime is a potent GSK-3β inhibitor, and weakly inhibits 5-Lipoxygenase, with IC50s of 22 nM and 7.8-10 µM, respectively; Indirubin-3'-monoxime also shows inhibitory activities against CDK5/p25 and CDK1/cyclin B, with IC50s of 100 and 180 nM.

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