Paullones, a Series of Cyclin-Dependent Kinase Inhibitors:  Synthesis, Evaluation of CDK1/Cyclin B Inhibition, and in Vitro Antitumor Activity

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135

Numerous inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3 (GSK-3) are being developed in view of their potential applications against cancers and neurodegenerative disorders. Among these, paullones constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors. However, their actual intracellular targets remain to be identified. To address this issue we have immobilized a paullone, gwennpaullone, on an agarose matrix. Extracts from various cell types and tissues were screened for proteins interacting with this matrix. This approach validated GSK-3␣ and GSK-3␤ as major intracellular paullone targets and also mitochondrial, but not cytoplasmic, malate dehydrogenase (MDH). Mitochondrial MDH was indeed inhibited by micromolar concentrations of paullones. Mitochondrial MDH was the major paullone-binding protein in the parasitic protozoon Leishmania mexicana, and paullones inhibited growth of the parasite. This simple batchwise affinity chromatography approach constitutes a straightforward method for the identification of intracellular targets of this particular class of novel anti-mitotic compounds. It has revealed an unexpected target, mitochondrial MDH, the inhibition of which may participate in the pharmacological effects of paullones.

“…Paullones were initially identified as CDK inhibitors (11,12). However, during extensive selectivity studies, we found that paullones were also excellent inhibitors of GSK-3 (13).…”

Section: Discussionmentioning

confidence: 99%

Section: Synthesis and Immobilization Of Paullonesmentioning

confidence: 99%

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Intracellular Targets of Paullones

Knockaert¹,

Wieking²,

Schmitt³

et al. 2002

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135

“…7). The recently described paullones (57,58) were also found to be excellent GSK-3␤ inhibitors. 2 Another recently described CDK inhibitor derived from marine sponges, hymenialdisine, is equally efficient on GSK-3␤ and CDKs (59).…”

Section: Indirubins Inhibit In Vitro and In Vivo Tau Phosphorylation mentioning

confidence: 96%

Indirubins Inhibit Glycogen Synthase Kinase-3β and CDK5/P25, Two Protein Kinases Involved in Abnormal Tau Phosphorylation in Alzheimer's Disease

Leclerc¹,

Garnier²,

Hoessel³

et al. 2001

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674

497

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-3 ␤ (GSK-3 ␤). Testing of a series of indoles and bis-indoles against GSK-3 ␤, 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␤'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 ␤, 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 diseasespecific 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␤. To which extent these GSK-3␤ 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 ␤ to be described.

“…Previous reports have found that, although lithium has no effect on PKA activity (72), indirubin-3-monoxime has a 300-fold greater effect on GSK-3 than PKA (73), and alsterpaullone has an undetermined effect on PKA. Interestingly, however, both indirubin-3-monoxime and alsterpaullone are also able to inhibit cyclin-dependent kinases (73,74). Therefore, as a negative control we also tested the cyclin-dependent kinase inhibitor roscovitine, which has no effect on PKA activity and has a 1000-fold greater effect on cyclin-dependent kinases than GSK-3 (71, 75), and found that it was unable to significantly block this PKA effect.…”

Section: Pka and Gsk-3 Synergize To Completely Block Ionomycininducedmentioning

confidence: 99%

Protein Kinase A Negatively Modulates the Nuclear Accumulation of NF-ATc1 by Priming for Subsequent Phosphorylation by Glycogen Synthase Kinase-3

Sheridan¹,

Heist

Beals

et al. 2002

104

The nuclear localization and transcriptional activity of the NF-ATc family of transcription factors, essential to many developmental, differentiation, and adaptation processes, are determined by the opposing activities of the phosphatase calcineurin, which promotes nuclear accumulation of NF-ATc, and several kinases, which promote cytoplasmic accumulation. Many reports suggest that protein kinase A (PKA) negatively modulates calcineurin-mediated NF-ATc activation. Here we show that overexpression of PKA causes phosphorylation and cytoplasmic accumulation of NF-ATc1 in direct opposition to calcineurin by phosphorylating Ser-245, Ser-269, and Ser-294 in the conserved serine-proline repeat domain, and that mutation of these serines blocks the effect of PKA. Activation of endogenous PKA is similarly able to promote phosphorylation of these sites on NFATc1 in two lymphoid cell lines. We further show that a complete block of NF-ATc1 nuclear localization by PKA requires a second kinase activity that can be supplied by glycogen synthase kinase-3 (GSK-3), and that mutation of either the PKA phosphorylation sites or the upstream GSK-3 sites prevents the effect of PKA. Thus, we propose that PKA functions cooperatively as a priming kinase for further phosphorylation by GSK-3 to oppose calcineurin-mediated nuclear accumulation and transcriptional activity of NF-ATc1 and that, through this mechanism, PKA may be an important modulator of many NF-ATc-dependent processes.