Laurent Meijer

Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.

Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3′-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.

Indirubins Inhibit Glycogen Synthase Kinase-3β 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 (IC50: 50–100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC50: 5–50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3β (GSK-3β). Testing of a series of indoles and bis-indoles against GSK-3β, 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-3βs ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3β, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimers disease. Indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimers disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3′-monoxime also inhibits the in vivophosphorylation 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-3β. To which extent these GSK-3β 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-3β to be described.

Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR−/− and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27KIP1. Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.

7-Bromoindirubin-3'-oxime induces caspase-independent cell death

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3′-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3′-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3′-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3′-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Identifying in vivo targets of cyclin-dependent kinase inhibitors by affinity chromatography

Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription, differentiation and many functions in the nervous system. The frequent deregulation of CDKs in cancers and in numerous other pathologies justifies the active search for chemical inhibitors capable of reversibly and selectively inhibiting this class of enzymes. Intensive screening of collections of natural and synthetic compounds has led to the identification of several families of ATP competitive CDK inhibitors. As the therapeutic potential of the most promising compounds is currently being evaluated in preclinical and clinical trials, their mechanism of action is still unclear. In particular, the real spectrum of their intracellular targets remains largely unknown. Determination of the selectivity of the compounds and identification of their intracellular targets constitute a prerequisite to understand their cellular effects and to improve their efficiency on a rational basis. The classical method for the determination of a compounds selectivity consists in testing the compound in a panel of purified kinases. However, the selectivity study is then restricted to the panels enzymes. As a consequence, many, if not most other potential targets are not evaluated. As an alternative way to investigate the range of true targets of CDK inhibitors, we propose an affinity chromatography approach based on immobilized inhibitors. Briefly, the inhibitor is covalently bound to a resin and cellular extracts are batch loaded on this inhibitor matrix. After extensive washing, the bound proteins are resolved by SDS-PAGE and identified by microsequencing. In addition to confirming the interaction of CDK inhibitors with CDKs, this method has led to the identification of additional, sometimes unexpected, targets. We here illustrate the potential of this technique through a few examples.

Polyprenyl-hydroquinones and -furans from three marine sponges inhibit the cell cycle regulating phosphatase CDC25A.

The CDC25 phosphatases regulate the cell division cycle by controlling the activity of cyclin-dependent kinases. While screening for inhibitors of phosphatases among natural products we repeatedly found that some polyprenyl-hydroquinones and polyprenyl-furans (furanoterpenoids) (furospongins, furospinosulins) were potent CDC25 phosphatase inhibitors. These compounds were extracted, isolated and identified independently from three sponge species (Spongia officinalis, Ircinia spinulosa, Ircinia muscarum), collected at different locations in the Mediterranean Sea. The compounds were inactive on the Ser/Thr phosphatase PP2C-α and on three kinases (CDK1, CDK5, GSK-3), suggesting that some potent and selective CDC25 phosphatase might be designed from these initial structures.