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Cellular Effects of Paullones

Paullones have been tested as potential anti-tumour agents. They indeed inhibit mammalian cell proliferation in culture (Schultz et al. 1999; Zaharevitz et al. 1999; Gussio et al. 2000) with an accumulation of cells both in G1 and G2, as would be expected from an inhibition of both CDK1 and CDK2. However, the fact that paullones inhibit CDKs in vivo still remains to be demonstrated. Paullones also inhibit the proliferation of Leishmania mexicana (Knockaert et al. 2002). Two potential targets were purified from this unicellular parasite using gwennpaullone agarose beads, and one of them was identified as mitochondrial MDH (Knockaert et al. 2002), the other as a new MAP kinase-like protein (J. Mottram, personal communication).

A recent study using the leukaemia Jurkat cell line showed that alster-paullone perturbs mitochondrial membrane potential, induces activation of several caspases (caspase-9, then caspase-8 and caspase-3) and triggers ap-optosis (Lahusen et al. 2003). The molecular mechanisms beyond this apop-tosis effect are still unclear. Whether inhibition of mitochondrial MDH plays a role in the mitochondrial effects of alsterpaullone, and the subsequent induction of apoptosis, remains to be determined.

Paullones are rather potent inhibitors of the nervous system kinase CDK5 in vitro. To evaluate whether this was true in vivo, we investigated the effects of alsterpaullone on the phosphorylation of DARPP-32 on threonine 75, a CDK5-selective site (Bibb et al. 1999), using isolated brain striatum slices. Alsterpaullone inhibited DARPP-32 threonine 75 phosphorylation in a dose-dependent manner, indicating its ability to target CDK5 in a cellular setting (Leost et al. 2000).

As described above, GSK-3a and GSK-3b are also two major targets of paullones in vitro. To demonstrate that they are also in vivo targets, we have analysed the effects of kenpaullone and alsterpaullone on SH-SY5Y cells in culture (Fig. 7). As expected, kenpaullone and alsterpaullone, even more potently, induce an accumulation of b-catenin, a direct consequence of its stabilisation by dephosphorylation. Increased dephospho-b-catenin (detected with an antibody that cross-reacts with b-catenin only when it is not phosphory-

Kcnpaullonc Alsterpaullone iL 2_ 5 20 2_ 5 20 0

- dephospho-ß-catenin

- loading control

Fig. 7 Paullones are selective GSK-3 inhibitors in cell cultures. SH-SY5Y cells were untreated (0) or exposed to 0.5-20 mM kenpaullone or alsterpaullone for 24 h. Proteins were then separated by SDS-PAGE followed by Western blotting with antibodies directed (top to bottom) against b-catenin, dephospho-b-catenin, phospho-tyrosine 276 (GSK-3a)/-ty-rosine 216 (GSK-3b), phospho-serine 9 GSK-3 and a loading control (non-specific band detected with the dephospho-b-catenin). (Courtesy of Dr. Xiaozhou P. Ryan)

Kcnpaullonc Alsterpaullone iL 2_ 5 20 2_ 5 20 0

- dephospho-ß-catenin

- loading control

Fig. 7 Paullones are selective GSK-3 inhibitors in cell cultures. SH-SY5Y cells were untreated (0) or exposed to 0.5-20 mM kenpaullone or alsterpaullone for 24 h. Proteins were then separated by SDS-PAGE followed by Western blotting with antibodies directed (top to bottom) against b-catenin, dephospho-b-catenin, phospho-tyrosine 276 (GSK-3a)/-ty-rosine 216 (GSK-3b), phospho-serine 9 GSK-3 and a loading control (non-specific band detected with the dephospho-b-catenin). (Courtesy of Dr. Xiaozhou P. Ryan)

lated on GSK-3 specific sites) is also seen with both paullones. Finally, both paullones inhibit the phosphorylation of GSK-3a and GSK-3b on tyrosine 276 and tyrosine 216, respectively. Phosphorylation of these sites is directly involved in GSK-3 activation, and their inhibition by paullones (through a yet-unknown mechanism) further contributes to GSK-3 inhibition.

Recently, the GSK-3 inhibitory property of kenpaullone has been used to support data demonstrating that GSK-3a regulates the production of amy-loid-b peptides (Phiel et al. 2003). Amyloid-b peptides derive from the amyloid precursor protein through the proteolytic action of b- and g-secretases. Amyloid-b peptides accumulate and aggregate in Alzheimer's disease, and the formation of amyloid plaques is thought to play a major role in the development of the disease (De Strooper and Woodgett 2003). Very interestingly, both lithium (millimolar concentrations), a classical inhibitor of GSK-3, and kenpaullone (micromolar concentrations), but not roscovitine (inactive on GSK-3), inhibit the production of amyloid-b peptides in cell lines (Phiel et al. 2003). Depletion of GSK-3a by RNAi together with overexpression studies confirm that GSK-3a is required for the generation of amy-loid-b peptides.

Hyperphosphorylation of the microtubule-binding protein tau, and its subsequent aggregation in paired helical filaments (neurofibrillary tangles) is also a landmark of Alzheimer's disease (De Strooper and Woodgett 2003). GSK-3 and CDK5 are two major kinases implicated in abnormal tau hyper-phosphorylation. Using Sf9 cells overexpressing human tau, we found that alsterpaullone is able to inhibit tau phosphorylation on epitopes which represent major GSK-3 phosphorylation sites detected in Alzheimer's disease (cross-reacting with PHF-1 and AT100 antibodies) (Leost 2000).

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