How did you become involved in this research? 

“One of the first molecules, roscovitine, is currently entering phase 2 clinical trials against breast and lung cancer.”

This work has an old history!  During a post-doctoral stay in 1978 at the Hopkins Marine Station (the Stanford University marine laboratory), in David Epel's laboratory, I started working on the changes in protein phosphorylation which occur as Urechis (a marine worm) oocytes are fertilized and re-enter M phase and cell divisions.  I then joined Pierre Guerrier's group at the CNRS in the "Station Biologique de Roscoff", France, where I investigated the role of protein phosphorylation during the G2/M transition of another very convenient marine model, the starfish oocyte.  At that time I was ignoring that I had been working for many years, in both models, on CDK1/cyclin B, the fundamental cell cycle regulator which was to be discovered later.  In 1985, I joined Edwin Krebs' and Bennett Shapiro's groups at the University of Washington, Department of Biochemistry.  There, with Steve Pelech, I extensively characterized the so-called M-phase specific Histone H1 kinase, a kinase activated in all cells as they enter the M phase of the cell division cycle.  In 1988, in collaboration with David Beach at Cold Spring Harbor, we identified the catalytic subunit of this kinase as CDC2, now better known as CDK1.  A year later, we identified the associated regulatory subunit as cyclin B, and in 1990, demonstrated that the CDK1/cyclin B complex was activated by subtle changes in phosphorylation of both subunits.  As this fundamental research was carried out, I became interested in using this essential cell-cycle regulator and others as molecular targets for the identification of new antimitotic agents of potential therapeutic interest.  In 1991, we described a simple CDK1 screening method, similar to the assays now being widely used by many pharmaceutical companies in their efforts for detecting protein kinase inhibitors.  My group also participated in the race through the identification, characterization, and optimization of a few families of chemical inhibitors of cyclin-dependent kinases: olomoucine, roscovitine, purvalanol, paullones, indirubins, hymenialdisine, aloisines, meridianins, etc.  These inhibitors have generated considerable interest not only in various fields of cell biology but also in the medical area, because of their potential applications against cancer, neurodegenerative disorders, viral infections, unicellular parasites, etc.  [1].  One of the first molecules, roscovitine, is currently entering phase 2 clinical trials against breast and lung cancer.  It is also in phase 1 against glomerulonephritis [2]. 

Indirubin, a bis-indole derived from various indigo-producing plants, was identified as a CDK1/CDK2 inhibitor while we were screening a collection of chemicals described as active ingredients of traditional Chinese medicine recipes.  In collaboration with the chemistry group of Gerhard Eisenbrand and the crystallography group of Jane Endicott, indirubins were extensively characterized as ATP-competitive inhibitors of CDKs [3].  This provided the first clue to explain the well-demonstrated anti-proliferative and anti-leukemia activity of indirubins [4].  Given the relatedness of CDK1 and CDK5, it was not surprising that the brain-expressed CDK5/p25 was also strongly inhibited by indirubins [5].  While investigating the kinase selectivity of indirubins and their structure/activity relationship, we discovered that besides CDKs, indirubins were very potent inhibitors of glycogen synthase kinase 3 (GSK3), a family of kinases with multiple cellular functions.  Surprisingly indirubins were about 10 fold more potent at inhibiting GSK-3 compared to CDKs.  We thus tested a selection of CDK inhibitors and discovered that many, but not all, were in fact very potent inhibitors of GSK-3 [5].  This has since been confirmed by other groups using other CDK inhibitors.  In the continuation of our work on indirubins we have investigated two new natural sources of indirubins: the Tyrean purple dye produced by certain mollusks, in collaboration with Leandros Skaltsounis [6] and bacteria expressing mutant forms of cytochrome P450, in collaboration with Fred Guengerich [7].  These studies provided new indirubin analogs with various potency and selectivity [8]. Indirubins have now been co-crystallized with CDK2, CDK2/cyclin A, CDK5, GSK-3 and PfPK5, the homolog of CDK1 in Plasmodium falciparum.

The paper describing the effects of indirubins on CDK5 and GSK-3, and the frequent sensitivity of both families of kinases to the same inhibitory compounds [5] has generated some interest for several reasons.  I believe that the main one is the clear involvement of CDK5 and GSK-3 in Alzheimer’s disease (brief review in [9]). Both enzymes are indeed involved in the two major hallmarks of this neurodegenerative disease, namely the formation of β-amyloid peptides from the β-amyloid precursor protein and the hyperphosphorylation of the microtubule-binding protein Tau.  The fact that dual-specificity inhibitors could be generated is a very encouraging finding for those who believe that modulating β-amyloid peptide production and Tau hyperphosphorylation might favorably influence the development and outcome of Alzheimer’s disease.  In addition there is an exponentially growing interest for the development of GSK-3 inhibitors for their applications in neurodegenerative diseases and diabetes [10] and potentially in regenerative medicine based on embryonic stem cells [11].  The possibility of developing GSK-3 –selective inhibitors from CDK inhibitors is particularly attractive in this respect.

In summary I hope that our work has stimulated an interest for dual-specificity inhibitors of kinases involved in Alzheimer’s and other neurodegenerative diseases.  We also hope that many years of work on indirubins will ultimately lead to the generation of useful drugs derived from a fascinating family of natural products. 

[1] Knockaert, M., Greengard, P. and Meijer, L., 2002. Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol. Sci. 23, 417-425.

[2] Meijer, L. and Raymond, E., 2003. Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials. Accounts Chem.Res. 36, 417-425.

[3] Hoessel, R., Leclerc, S., Endicott, J., Noble, M., Lawrie, A., Tunnah, P., Leost, M., Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G. and Meijer, L., 1999. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nature Cell Biology 1, 60-67.

[4] Damiens, E., Baratte, B., Marie, D., Eisenbrand, G. and Meijer, L., 2001. Anti-mitotic properties of indirubin-3’-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest. Oncogene 20, 3786-3797.

[5] Leclerc, S., Garnier, M., Hoessel, R., Marko, Bibb, J.A., Snyder, G.L., Greengard, P., Biernat, J., Mandelkow, E.-M., Eisenbrand, G. and Meijer, L., 2001. Indirubins inhibit glycogen synthase kinase - 3b and CDK5/p25, two kinases involved in abnormal tau phosphorylation in Alzheimer’s disease - A property common to most CDK inhibitors ? J. Biol. Chem. 276, 251-260.

[6] Meijer, L., Skaltsounis, A.L., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X.P., Vonica, C.D., Brivanlou, A., Dajani, R., Tarricone, A., Musacchio, A., Roe. S.M., Pearl, L. and Greengard, P., 2003. GSK-3 selective inhibitors derived from Tyrian purple indirubins. Chem. & Biol. 10, 1255-1266.

[7] Guengerich, F.P., Sorrells, J.L., Schmitt, S., Krauser, J.A., Aryal, P. and Meijer, L., 2004. Generation of new protein kinase inhibitors utilizing cytochrome P450 mutant enzymes for indigoid synthesis. J. Med. Chem. 47, 3236-3241.

[8] Polychronopoulos, P., Magiatis, P., Skaltsounis, L., Myrianthopoulos, V., Mikros, E., Tarricone, A., Musacchio, A., Roe, S.M., Pearl, L., Leost, M., Greengard, P. and Meijer, L., 2004. Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase -3 and cyclin-dependent kinases. J. Med. Chem. 47, 935-946.

[9] De Strooper, B. and Woodgett, J. (2003) Alzheimer's disease: Mental plaque removal. Nature 423, 392-393.

[10] Meijer, L., Flajolet, M. and Greengard, P., 2004. Pharmacological inhibitors of glycogen synthase kinase-3. Trends Pharmacol. Sci., in press (September issue).

[11] Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A., 2004. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3 specific inhibitor. Nature Med. 10, 55-63.

Laurent Meijer, Lab Director
CNRS, Cell Cycle Group
Roscoff Marine Biology Institute
Roscoff, France