Angewandte
Chemie
reconfirms that the potency of 1-OH is entirely based on
specific recognition. It is also noteworthy that ligand 2-OH is
almost two orders of magnitude less active than the corre-
sponding ruthenium complex 1-OH, clearly verifying that the
entire assembly is necessary for full activity.
Ruthenium complex 1-OH also shows high selectivity. The
protein kinases Abl (IC50 = 1 mm), CHK-1 (IC50 = 2 mm), Lck
(IC50 = 500 nm), MAPK-1 (IC50 > 3 mm), RSK-1 (25 nm), Src
(IC50 = 4 mm), and Zap-70 (IC50 > 10 mm) are inhibited at
concentrations that are 80 to more than 30000 times higher
relative to GSK-3a. Even the evolutionarily closely related
protein kinase CDK-2 is only inhibited with an IC50 of
200 nm.[9–12]
Next, we investigated whether 1-OH can inhibit GSK-3
inside mammalian cells. GSK-3 is a negative regulator of the
wnt signal transduction pathway that phosphorylates b-
catenin.[13] Phosphorylated b-catenin is unstable and is
degraded rapidly by the proteasome. Wnt signaling inhibits
GSK-3, leading to stabilization of b-catenin protein. b-
Catenin then accumulates and serves as a transcriptional
coactivator through its interaction with the T-cell factor (Tcf)
family of transcription factors. Thus inhibition of GSK-3 by
pharmacological inhibitors or by wnt signaling leads to
increased b-catenin levels and activation of wnt-dependent
transcription.
Scheme 2. Synthesis of ruthenium complex 1-OH and pyridocarbazole
2-OH. a) 2-Acetylpyridine, tBuOH, 4 h reflux (100%). b) Trimethylsilyl
polyphosphate, 1158C, overnight (63%). c) BBr3, CH2Cl2, À608C, then
RT overnight (87%). d) DIEA, DMF, 08C, 40 min, then TBS triflate,
08C, 1 h (71%). e) Li hexamethyldisilazide, THF, À158C, 45 min, then
5 in THF, À158C, 15 min, then RT, 45 min (58%). f) hn, Pyrex filter,
MeCN, 3 h, (78%). g) [Ru(Cp)(CH3CN)2(CO)]PF6, K2CO3, MeCN, 558C,
overnight (86%). h) TBAF, CH2Cl2, RT, 30 min (87%). i) TBAF, CH2Cl2,
RT, 30 min (80%). DIEA=N,N-diisopropylethylamine, TBAF=tetrabu-
tylammonium fluoride, TBS=tert-butyldimethylsilyl.
To test for cellular accumulation of b-catenin as a
response to inhibition of GSK-3 by ruthenium complex 1-
OH, we used human embryonic kidney cells (HEK293T) that
have stably incorporated
a Tcf-luciferase transcription
reporter (OT-Luc cells). This transcription reporter generates
luciferase in response to increased concentrations of b-
catenin.[14] Exposure of OT-Luc cells to varying concentra-
tions of 1-OH over a period of 24 h yields a strong
upregulation of luciferase in the concentration window of
3 mm down to 100 nm (Figure 2A). For example, at a 1-OH
concentration of 300 nm, luciferase activity is enhanced by a
factor of 1180. Intriguingly, at the same time, methylated
control Me1-OH and free ligand 2-OH do not yield any
significant increase in luciferase activity at a concentration of
1 mm (1.6- and 2.4-fold, respectively). As a positive control,
the established selective GSK-3 inhibitor LiCl leads to an
increase of the luminescence signal by a factor of 1190 at a
concentration of 30 mm.[14,15]
tively, approximately an order of magnitude lower than that
of the initial complex 1-H. For comparison, ruthenium
compound 1-OH is at least 100 times more potent against
GSK-3 than staurosporine under our experimental conditions
(IC50 of 40 nm against GSK-3a).[9–12] Methylation of the imide
nitrogen atom (Me1-OH) reduces the activity dramatically by
almost four orders of magnitude (IC50 of 2 mm against GSK-
3a), as demonstrated by the IC50 curves in Figure 1, which
To verify the accumulation of b-catenin directly, we
analyzed the cellular b-catenin concentration by Western
blotting after incubation with 1-OH. Figure 2B demonstrates
an increase in b-catenin in the presence of 1-OH, but not
Me1-OH or 2-OH, at 1 mm. These experiments demonstrate
that ruthenium complex 1-OH crosses the cell membrane and
activates the wnt pathway at low micromolar and even
nanomolar concentrations.
It is also noteworthy that 1-OH does not show any signs of
cytotoxicity at concentrations of 3 mm or less, supporting our
assumption that the kinetically inert ruthenium center is only
an innocent nonreactive bystander. The integrity of complex
1-OH inside the cell is also reinforced by the fact that the
entire ligand assembly is necessary for the activity inside the
cell and that the free ligand 2-OH does not activate the wnt
pathway itself at similar concentrations. This is a remarkable
Figure 1. IC50 curves with GSK-3a obtained by phosphorylation of
phosphoglycogen synthase peptide-2 with [g-32P]ATP: ruthenium com-
*
*
plex 1-OH ( ), ligand 2-OH ( ), and methylated ruthenium complex
!
Me1-OH ( ).
Angew. Chem. Int. Ed. 2005, 44, 1984 –1987
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1985