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?

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.     

Biosynthesis of new indigoid inhibitors of protein kinases using recombinant cytochrome P450 2A6

Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H-indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3alpha/beta were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3alpha/beta as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3alpha/beta, dimethyl indirubin 5,5'-dicarboxylate (IC50 of 51 nM), was modified by chemical reactions. One product, indirubin 5,5'-dicarboxylic acid 5-methyl ester, inhibited GSK-3alpha/beta with an IC50 of 14 nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5'-dicarbonitrile was also modified to the corresponding 3'-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10 nM towards GSK-3alpha/beta, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.     

Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations

Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.     

Expression and activity of cyclin-dependent kinases and glycogen synthase kinase-3 during NT2 neuronal differentiation

In the presence of retinoic acid undifferentiated NT2 cells turn into terminally differentiated hNT (or NT2N) neurons within 5 weeks. We have used this in vitro cellular model to investigate the changes in expression and activity of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) during this neuronal differentiation process. We here show that CDK1/2 protein level and kinase activity sharply decrease during the NT2-->hNT transition. In contrast, the activity of CDK5/p35 dramatically increases, probably as a result of an enhanced expression of p35 in a stable CDK5 level background. GSK-3 activity increases modestly during the differentiation of hNT cells, and this event correlates with enhanced expression of each of the three GSK-3 isoforms. Pharmacological inhibitors of CDKs and GSK-3 lead to a dose-dependent decrease in cell viability.     

Pyrazolo[3,4-b]quinoxalines. A new class of cyclin-dependent kinases inhibitors

Protein kinases are involved in most physiological processes and in numerous diseases. Therefore, inhibitors of protein kinases have therefore a wide therapeutic potential. While screening for inhibitors of cyclin-dependent kinases (CDK's) and glycogen synthase kinase-3 (GSK-3), we identified pyrazolo[3,4-b]quinoxalines as sub-micromolar inhibitors of CDK1/cyclin B. A preliminary structure-activity relationship study suggests that this family of compounds can be optimized to inhibit CDK's and GSK-3. Compounds were tested for their anti-proliferative activity and the results show that several of them displayed a significant inhibitory effect on CDK1/cyclin B. The most active compound (1) was also tested against the brain kinases CDK5/p25 and GSK-3, and proved to be a good inhibitor of both of them. On the contrary, none of the compounds showed any activity in the CDC25 phosphatase assay. As an additional approach, affinity chromatography on immobilized pyrazolo[3,4-b]quinoxalines will be used to identify the intracellular targets of this family of compounds.     

Triazolo[1,5-a]pyrimidines as novel CDK2 inhibitors: protein structure-guided design and SAR

Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.     

Synthesis of novel 5-substituted indirubins as protein kinases inhibitors

In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we synthesized and evaluated new 5-substituted indirubins. The effects of 34 indirubin derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3, as well as on SH-SY5Y human neuroblastoma cell survival, were investigated.     

Structure-based design of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors to activate Wnt/β-catenin pathway

Deregulation of Wnt/β-catenin pathway is closely related to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), and glycogen synthase kinase 3β (GSK-3β), the central negative regulator of Wnt pathway, is regarded as an important target for these diseases. Here, we report a series of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors by rational-design and synthesis, which show high selectivity against GSK-3β over Cyclin-dependent kinase 2 (CDK2), and significantly activate the cellular Wnt/β-catenin pathway. The structure–activity relationship of these GSK-3β inhibitors was also explored by in silico molecular docking.     

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer's disease and other neurodegenerative 'taupathies'. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3beta. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3beta in Alzheimer's disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.     

The CDK4/6 inhibitor palbociclib synergizes with irinotecan to promote colorectal cancer cell death under hypoxia

Hypoxia is an inherent impediment to cancer therapy. Palbociclib, a highly selective inhibitor for CDK4/6, has been tested in numerous clinical trials and has been approved by the FDA. We previously reported that CDK inhibitors can destabilize HIF1α regardless of the presence of hypoxia and can sensitize tumor cells to TRAIL through dual blockade of CDK1 and GSK-3β. To translate this knowledge into a cancer therapeutic strategy, we investigated the therapeutic effects and molecular mechanisms of CDK inhibition against colon cancer cells under normoxia and hypoxia. We found that palbociclib sensitizes colon cancer cells to hypoxia-induced apoptotic resistance via deregulation of HIF-1α accumulation. In addition to inhibition of cell proliferation, we observed that palbociclib promotes colon cancer cell death regardless of the presence of hypoxia at a comparatively high concentration via regulating ERK/GSK-3β signaling and GSK-3β expression. Furthermore, palbociclib synergized with irinotecan in a variety of colon cancer cell lines with various molecular subtypes via deregulating irinotecan-induced Rb phosphorylation and reducing HIF-1α accumulation under normoxia or hypoxia. Collectively, our findings provide a novel combination therapy strategy against hypoxic colon cancer cells that may be further translated in the clinic.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.     

1-Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3 beta

Kenpaullone derivatives with a modified parent ring system were synthesized in order to develop kinase inhibitors with enhanced selectivity. Among the novel structures, 1-azakenpaullone was found to act as a selective GSK-3beta versus CDK1 inhibitor. The charge distribution within the 1-azakenpaullone molecule is discussed as a possible explanation for the enhanced GSK-3beta selectivity of 1-azakenpaullone compared to other paullone derivatives.     

p27 Kip1 nuclear localization and cyclin-dependent kinase inhibitory activity are regulated by glycogen synthase kinase-3 in human colon cancer cells

The cellular mechanisms regulating intestinal differentiation are poorly understood. Sodium butyrate (NaBT), a short-chain fatty acid, increases p27 Kip1 expression and induces cell cycle arrest associated with intestinal cell differentiation. Here, we show that treatment of intestinal-derived cells with NaBT induced G0/G1 arrest and intestinal alkaline phosphatase, a marker of differentiation, activity and mRNA expression; this induction was attenuated by inhibition of glycogen synthase kinase-3 (GSK-3). Moreover, treatment with NaBT increased the nuclear, but not the cytosolic, expression and activity of GSK-3beta. NaBT decreased cyclin-dependent kinase CDK2 activity and induced p27 Kip1 expression; inhibition of GSK-3 rescued NaBT-inhibited CDK2 activity and blocked NaBT-induced p27 Kip1 expression in the nucleus but not in the cytoplasm. In addition, we demonstrate that NaBT decreased the expression of S-phase kinase-associated protein 2 (Skp2), and this decrease was attenuated by GSK-3 inhibition. Furthermore, NaBT increased p27 Kip1 binding to CDK2, which was completely abolished by GSK-3 inhibition. Overexpression of an active form of GSK-3beta reduced Skp2 expression, increased p27 Kip1 in the nucleus and increased p27 Kip1 binding to CDK2. Our results suggest that GSK-3 not only regulates nuclear p27 Kip1 expression through the downregulation of nuclear Skp2 expression but also functions to regulate p27 Kip1 assembly with CDK2, thereby playing a critical role in the G0/G1 arrest associated with intestinal cell differentiation.     

N-terminal cleavage of GSK-3 by calpain: a new form of GSK-3 regulation

Although GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins, here we describe N-terminal proteolysis as a novel way to regulate GSK-3. When brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of approximately 40 and 30 kDa, a proteolytic process that was inhibited by specific calpain inhibitors. Interestingly, instead of inhibiting this enzyme, GSK-3 truncation augmented its kinase activity. When we digested recombinant GSK-3 alpha and GSK-3beta protein with calpain, each isoform was cleaved differently, yet the truncated GSK-3 isoforms were still active kinases. We also found that lithium, a GSK-3 inhibitor, inhibits full-length and cleaved GSK-3 isoforms with the same IC(50) value. Calpain removed the N-terminal ends of His-tagged GSK-3 isoenzymes, and exposing cultured cortical neurons with ionomycin, glutamate, or N-methyl-d-aspartate led to the truncation of GSK-3. This truncation was blocked by the calpain inhibitor calpeptin, at the same concentration at which it inhibits calpain-mediated cleavage of NMDAR-2B and of p35 (the regulatory subunit of CDK5). Together, our data demonstrate that calpain activation produces a truncation of GSK-3 that removes an N-terminal inhibitory domain. Furthermore, we show that GSK-3 alpha and GSK-3beta isoenzymes have a different susceptibility to this cleavage, suggesting a means to specifically regulate these isoenzymes. These data provide the first direct evidence that calpain promotes GSK-3 truncation in a way that has implications in signal transduction, and probably in pathological disorders such as Alzheimer disease.     

Proliferative and androgenic effects of indirubin derivatives in LNCaP human prostate cancer cells at sub-apoptotic concentrations

Certain indirubin derivatives are potent cyclin-dependent kinase (CDK) and glycogen synthase kinase (GSK-3β) inhibitors and may be effective against various cancers. We evaluated the effects of aloisine A, alsterpaullone, aminopurvalanol, indirubin-3′-oxime, 6-Br-indirubin-3′-oxime, kenpaullone, olomoucine and roscovitine on cell proliferation, prostate-specific antigen (PSA) expression, androgen receptor (AR) activation, and GSK-3β and β-catenin expression in androgen-dependent LNCaP human prostate cancer cells. Effects were also evaluated in MDA-kb2 human breast cancer cells containing an AR-responsive luciferase construct. Steroid-deprived LNCaP cells were exposed to indirubins ± dihydrotestosterone (DHT, 0.1 nM) and cell proliferation was assessed by MTT assay after 120 h. PSA expression was determined by real-time quantitative RT-PCR after 24 h. Cytoplasmic and nuclear GSK-3β/β-catenin expression and phosphorylation status was determined by Western blotting. Effects on MDA-kb2 luciferase expression were determined after 24 h using Steady-Glo (Promega). Indirubin-3′-oxime, 6-Br-indirubin-3′-oxime, alsterpaullone and kenpaullone increased LNCaP cell proliferation and PSA expression (0.03–1 μM; apoptosis occurred >1 μM), whereas aminopurvalanol significantly (p < 0.05) reduced DHT-stimulated PSA expression (31%) at 1 nM. The other indirubin derivatives had no effect. The same was observed for induction of AR-dependent MDA-kb2 luciferase expression. Kenpaullone (1, 3 μM) decreased the active- and increased the inactive form of cytoplasmic GSK-3β, and increased nuclear AR and β-catenin accumulation. Flutamide (10 μM), unexpectedly, also strongly increased nuclear β-catenin accumulation. Indirubin derivatives that were potent GSK-3β inhibitors (relative to CDK1) stimulated LNCaP cell proliferation and other androgenic responses, suggesting (in a cancer treatment context) these compounds may increase AR-dependent prostate cancer growth if not used within an appropriate therapeutic dose-range.     

Multitargeted drug development: Discovery and profiling of dihydroxy substituted 1-aza-9-oxafluorenes as lead compounds targeting Alzheimer disease relevant kinases

Alzheimer disease (AD) turned out to be a multifactorial process leading to neuronal decay. So far merely single target structures which attribute to the AD progression have been considered to develop specific drugs. However, such drug developments have been disappointing in clinical stages. Multitargeting of more than one target structure determines recent studies of developing novel lead compounds. Protein kinases have been identified to contribute to the neuronal decay with CDK1, GSK-3β and CDK5/p25 being involved in a pathological tau protein hyperphosphorylation. We discovered novel lead structures of the dihydroxy-1-aza-9-oxafluorene type with nanomolar activities against CDK1, GSK-3β and CDK5/p25. Structure–activity relationships (SAR) of the protein kinase inhibition are discussed within our first compound series. One nanomolar active compound profiled as selective protein kinase inhibitor. Bioanalysis of a harmless cellular toxicity and of the inhibition of tau protein phosphorylation qualifies the compound for further studies.     

Substrate Competitive GSK-3 Inhibitors strategy and Implications

Glycogen synthase kinase-3 (GSK-3) is a highly conserved protein serine/threonine kinase ubiquitously distributed in eukaryotes as a constitutively active enzyme. Abnormally high GSK-3 activity has been implicated in several pathological disorders, including diabetes and neuron degenerative and affective disorders. This led to the hypothesis that inhibition of GSK-3 may have therapeutic benefit. Most GSK-3 inhibitors developed so far compete with ATP and often show limited specificity. Our goal is to develop inhibitors that compete with GSK-3 substrates, as this type of inhibitor is more specific and may be useful for clinical applications. We have employed computational, biochemical, and molecular analyses to gain in-depth understanding of GSK-3's substrate recognition. Here we argue that GSK-3 is a promising drug discovery target and describe the strategy and practice for developing specific substrate-competitive inhibitors of GSK-3.     

Synthesis of 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine derivatives as CDK inhibitors and cytotoxic agents

We here report the synthesis and biological evaluation of new 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine designed as potential CDK inhibitors. Indole, 5-hydroxyindole, and phenol derivatives were used to generate three substitutions of the pyridine. The resulting skeletons were successively exploited to introduce various dimethylaminoalkyl side chains by Williamson type reactions. The synthesis includes Stille or Suzuki type reactions, which were realized on the 3,5-dibromopyridine. The preparation and the use of stannylindoles in mono or bis cross-coupling reactions were also described and each step was optimized and detailed. Kinase assays were realized and shown that nude compounds 7, 18, and 25 inhibited CDK1 in the 0.3–0.7 micromolar range with a good selectivity over GSK-3. Cytotoxicity against CEM human leukemia cells was evaluated with IC₅₀ values in the 5–15 micromolar range. Precise structure–activity relationships were delineated. Molecular modeling and docking solutions were proposed to complete the studies and to explain the observed SAR in the CDK assays.     

6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis

Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3′-oxime (6-BIO), 6-Br-indirubin-3′acetoxime and 6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC₅₀) values ?1.2 μM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3β, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3β and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals.