From the CNRS, Cell Cycle Group, Station Biologique,
BP 74, Roscoff 29682 Cedex, Bretagne, France, the
§ Department of Chemistry, Division of Food Chemistry and
Environmental Toxicology, University of Kaiserslautern,
Erwin-Schrödinger-Strasse 52, Kaiserslautern 67663, Germany, the
¶ Laboratory of Molecular and Cellular Neuroscience, The
Rockefeller University, New York, New York 10021, and the
Max-Planck Unit for Structural Molecular Biology, Notkestrasse
85, Hamburg D-22603, Germany
Received for publication, March 22, 2000, and in revised form, September 15, 2000
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ABSTRACT |
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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-3 Indigoïds are bis-indoles derived from various natural
sources by fermentation, oxidation, and dimerization in the presence of
light. The colorful indirubin (1) and indigo (5) originate from the dimerization of colorless precursors, indoxyl and
isatin (4) (see Fig. 1). These indoles are released during
the fermentation process from conjugates, the nature of which depends
on the plant (indican, isatan B) or mollusc (indoxylsulfate) species
from which the dyes are prepared (see Fig.
1). The use of indigoïds as
textile dyes dates back to the Bronze age ( (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 disease-specific 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7000), but indigo
(now synthetic) remains the most abundantly produced dye in the world
(blue jeans, denims, etc.) (1, 2). Indigo-producing plants have also
been used in traditional Chinese medicine (3-5). A well-studied
example is Danggui Longhui Wan, a mixture of 11 herbal medicines
traditionally utilized against certain types of leukemias. Only one of
these ingredients, Qing Dai (Indigo naturalis), a
dark blue powder originating from various indigo-producing plants, was
found to carry the antileukemic activity (6). Although it is mostly
constituted of indigo, a minor constituent, indirubin, was identified
as the active component by the Chinese Academy of Medicine (7-9).
Preclinical studies performed with indirubin, and more soluble
analogues, confirmed that these compounds exhibit good antitumor
activity and only minor toxicity (10-14). Clinical trials showed that
indirubin has a definite efficiency against chronic myelocytic leukemia
(5, 15-21).
View larger version (27K):
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Fig. 1.
The bis-indoles indigo (5), indirubin (1),
and isoindigo (40) are derived from the dimerization of indoxyls and
isatins, which are themselves derived from the hydrolysis of either
indican and isatans (plants) or indoxyl sulfates (molluscs).
Several mechanisms of action have been brought forward to explain the antimitotic and antitumoral properties of indirubins (22-24). We recently reported that indirubins are potent inhibitors of cyclin-dependent kinases (CDKs)1 (25), a family of key cell cycle regulators (26-28). Indirubins act by competing with ATP for binding to the catalytic site of the kinase. The kinase selectivity study showed that indirubins have a strong affinity for CDKs (IC50 values in the range of 50-100 nM) (25). Nevertheless, they are not totally devoid of activity toward a few kinases (IC50 values in the 1-10 µM range) (25). This rather loose selectivity, when compared with the high specificity of purine inhibitors of CDKs, led us to continue to investigate the selectivity of indirubins as kinase inhibitors.
We report here that indirubins are very potent inhibitors
(IC50 values in the 5-50 nM range) of glycogen
synthase kinase-3 (GSK-3
). This kinase is an essential element of
the WNT signaling pathway (29). It is involved in multiple
physiological processes, including cell cycle regulation by controlling
the levels of cyclin D1 (30) and
-catenin (31), dorsal-ventral
patterning during development (31-33), insulin action on glycogen
synthesis (34, 35), axonal outgrowth (36), HIV-1 Tat-mediated
neurotoxicity (37), among others. Furthermore, GSK-3
and CDK5 are
responsible for most of the abnormal hyperphosphorylation of the
microtubule-binding protein tau observed in the paired helical
filaments, which are diagnostic for Alzheimer's disease (AD) (38, 39).
It was recently demonstrated that conversion of p35, the regulatory
subunit of CDK5, to a truncated form, p25, deregulates CDK5 activity
and promotes neurodegeneration (40). We here show that indirubins are
very potent inhibitors of CDK5/p25. Furthermore, indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo
at Alzheimer's disease-specific sites. Indirubins may thus constitute
a lead compound in the study and treatment of neurodegenerative
disorders involving abnormal phosphorylation of tau ("taupathies").
We here also show that indirubin-3'-monoxime inhibits phosphorylation of DARPP-32 by CDK5. DARPP-32 is a striatum protein acting downstream of dopamine action, which is either phosphorylated on Thr-34 by cAMP-dependent protein kinase (PKA) (it then acts as
a phosphatase 1 inhibitor) or on Thr-75 by CDK5 (it then becomes a PKA
inhibitor) (41). Finally, we also show that many, but not all, CDK
inhibitors are potent GSK-3
inhibitors. Whether the
antimitotic/antitumoral properties of indirubins (and other CDK
inhibitors) derive from their dual inhibitory effects on GSK-3
and
CDKs remains to be determined.
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EXPERIMENTAL PROCEDURES |
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Chemistry
Indigo (2) (Fluka), isatin (3) (Fluka),
5,5',7,7'-indigotetrasulfonic acid potassium salt (25)
(Fluka), 5,5',7-indigotrisulfonic acid potassium salt (26)
(Fluka), indigo carmine (27) (Fluka), 5-chloroisatin
(30) (Lancaster), 5-fluoroisatin (29) (Aldrich),
5-bromoisatin (31) (Fluka), 5-methylisatin (32)
(Sigma), isatin-5-sulfonic acid sodium salt dihydrate (33)
(Fluka), 5-nitroisatin (34) (Acros), 1-methylisatin
(35) (Acros), 1-phenylisatin (36) (Lancaster),
indoxyl acetate (43) (Fluka), 5-bromoindoxyl acetate
(44) (Fluka), and other solvents and reagents were obtained
from commercial suppliers. They were at least of reagent grade and were
used without further purification. Indirubin (1),
5-iodoindirubin (4), 5-bromoindirubin (5),
5-chloroindirubin (6), 5-fluoroindirubin (7), 5-methylindirubin (8), 5-nitroindirubin (9),
indirubin-5-sulfonic acid (sodium salt) (10),
5'-bromoindirubin (11), 5,5'-dibromoindirubin
(12), 5'-bromoindirubin-5-sulfonic acid (sodium salt)
(13), indirubin-3'-monoxime (14), 5-iodoindirubin-3'-oxime (15), 6-iodoindirubin
(16), 1-methylindirubin (17), 1-phenylindirubin
(18), 3'-hydroxyiminoindirubin-5-sulfonic acid (sodium salt)
(19), indirubin-5-sulfonamide (20),
indirubin-5-sulfonic acid dimethylamide (21),
indirubin-5-sulfonic acid (2-hydroxyethyl)amide (22),
indirubin-5-sulfonic acid bis-(2-hydroxyethyl)amide (23),
indirubin-5-sulfonic acid methylamide (24), 5-iodoisatin
(28), isatin-5-sulfonic acid dimethylamide (37),
isatin-5-sulfonic acid bis-(2-hydroxyethyl)amide (38),
6-iodoisatin (39), isoindigo (40), 2,2'-bi-indole
(41), 3,3'-diphenyl-2,2'-bi-indole (42), isatin-5-sulfonamide (45), isatin-5-sulfonic acid
(2-hydroxyethyl)amide (46), isatin-5-sulfonic acid
methylamide (47), and 2-hydroxyimino-N-(3-iodophenyl)acetamide (48)
were synthesized and purified as described in the Supplementary
material section. Synthesis reactions involving oxygen or
moisture-sensitive compounds were performed under a dry argon
atmosphere. All reaction mixtures and column chromatographic fractions
were analyzed by thin layer chromatography on plates (Alugram Sil
G/UV254, purchased from Macherey & Nagel). Column
chromatography was carried out using Silica Gel 60 (0.063-0.2 mm,
Macherey & Nagel). Melting points of the non-indigoïd compounds
were determined on a Büchi 510 melting point apparatus and were
uncorrected. Melting points over 260 °C were determined on a Wagner
and Munz Kupferblock. Elemental analyses were performed using a 2400 CHN elemental analyzer (PerkinElmer Life Sciences). Unless otherwise
indicated, NMR spectra were recorded at room temperature.
1H NMR spectra were recorded at 400 MHz, 13C
NMR spectra at 100 MHz on a Bruker AMX 400, using tetramethylsilane, or
Me2SO ( = 39.4 ppm) as internal standard.
J values are reported in hertz. Apparent multiplicities were
designated as s, singlet; d, doublet; dd, double doublet; t, triplet;
pt, pseudo-triplet; q, quartet; m, multiplet; b, broad. Mass spectra
were taken in the positive ion mode under electron impact (EI 70 eV)
using a Finnigan MAT 90 mass spectrometer. Gas
chromatography/mass spectrometry was performed using a Hewlett-Packard,
5890 Series II gas chromatograph on a 25-m fused silica column
(Hewlett-Packard HP-5, I.D. = 0.25 mm; 0.25 µm) and a
Hewlett-Packard, HP 5971A mass-selective detector with the following
temperature program: 80 °C (4 min), 25 °/min, 320 °C (16.4 min).
All compounds were dissolved and stored as 10 mM stock solutions in Me2SO. They were diluted in aqueous buffers just prior use.
Biochemical Reagents
Sodium orthovanadate, EGTA, EDTA, RNase A, Mops,
-glycerophosphate, phenylphosphate, sodium fluoride,
glutathione-agarose, dithiothreitol (DTT), bovine serum albumin (BSA),
nitrophenylphosphate, leupeptin, aprotinin, pepstatin, soybean trypsin
inhibitor, benzamidine, and histone H1 (type III-S) were obtained from
Sigma Chemical Co. [
-32P]ATP (PB 168) was obtained
from Amersham Pharmacia Biotech.
The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE) was synthesized by the Peptide Synthesis Unit, Institute of Biomolecular Sciences, University of Southampton, UK.
AT-8, AT-180, and AT-100 antibodies were obtained from Innogenetics, SA, Ghent, Belgium, PHF-1 was a gift from Dr. P. Davies (Bronx, NY), and K9JA was obtained from Dako (Hamburg, Germany).
Buffers
Homogenization Buffer--
60 mM
-glycerophosphate, 15 mM
p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1 mM
NaF, 1 mM phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 10 µg/ml soybean trypsin inhibitor, and 100 µM benzamidine.
Buffer A-- Buffer A consisted of 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl, pH 7.5, 50 µg/ml heparin.
Buffer C-- Buffer C consisted of homogenization buffer but 5 mM EGTA, no NaF, and no protease inhibitors.
Tris-buffered Saline-Tween 20 (TBST)-- This buffer consisted of 50 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20.
Hypotonic Lysis Buffer (HLB)-- HLB buffer consisted of 50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 10% glycerol, 1% Nonidet-P40, 5 mM DTT, 1 mM EGTA, 20 mM NaF, 1 mM orthovanadate, 5 µM microcystin, and 100 µg/ml each of leupeptin, aprotinin, and pepstatin.
Kinase Preparations and Assays
Kinase activities were assayed in Buffer A or C (unless otherwise stated), at 30 °C, at a final ATP concentration of 15 µM. Blank values were subtracted, and activities were calculated as picomoles of phosphate incorporated for a 10-min incubation. The activities are usually expressed in percentage of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethyl sulfoxide. In a few cases phosphorylation of the substrate was assessed by autoradiography after SDS-PAGE (see below).
GSK-3
GSK-3 was expressed in and purified from insect Sf9
cells (42). It was assayed, following a 1/100 dilution in 1 mg/ml BSA, 10 mM DTT, with 5 µl of 40 µM GS-1 peptide
as a substrate, in buffer A, in the presence of 15 µM
[
-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume
of 30 µl. After 30-min incubation at 30 °C, 25-µl aliquots of
supernatant were spotted onto 2.5- × 3-cm pieces of Whatman P81
phosphocellulose paper, and, 20 s later, the filters were washed
five times (for at least 5 min each time) in a solution of 10 ml of
phosphoric acid/liter of water. The wet filters were counted in the
presence of 1 ml of ACS (Amersham Pharmacia Biotech) scintillation fluid.
CDK1/Cyclin B--
CDK1/cyclin B was extracted in homogenization
buffer from M phase starfish (Marthasterias glacialis)
oocytes and purified by affinity chromatography on
p9CKShs1-Sepharose beads, from which it was eluted by free
p9CKShs1 as described previously (43, 44). The kinase
activity was assayed in buffer C, with 1 mg/ml histone H1, in the
presence of 15 µM [-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 µl. After 10-min
incubation at 30 °C, 25-µl aliquots of supernatant were spotted
onto P81 phosphocellulose papers and treated as described above.
CDK/p25-- CDK5/p25 was reconstituted by mixing equal amounts of recombinant mammalian CDK5 and p25 expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified by affinity chromatography on glutathione-agarose (vectors kindly provided by Dr. J. H. Wang). (p25 is a truncated version of p35, the 35-kDa CDK5 activator.) Its activity was assayed in buffer C as described for CDK1/cyclin B.
In Vitro and in Vivo Tau Phosphorylation
Cells and Viruses-- Sf9 cells (Invitrogen, San Diego, CA) were grown at 27 °C in monolayer culture Grace's medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and 50 µg/ml gentamicin and 2.5 µg/ml amphotericin. BaculoGold was obtained from PharMingen (San Diego, CA), pVL1392 was obtained from Invitrogen.
Tau Transfection-- The gene for htau23, the shortest human tau isoform, was excised from the bacterial expression vector pNG2 (45) with XbaI and BamHI and inserted into the baculovirus transfer vector pVL1392 cut with the same restriction endonucleases. The BaculoGold system was used to construct the tau baculovirus-containing vector. The BaculoGold DNA is a modified type of baculovirus containing a lethal deletion. Cotransfection of the BaculoGold DNA with a complementing baculovirus transfer vector rescued the lethal deletion of this virus DNA and reconstituted viable virus particles carrying the htau23 coding sequence. Plasmid DNA used for transfections was purified using Qiagen cartridges (Hilden, Germany). Sf9 cells grown in monolayers (2 × 106 cells in a 60-mm cell culture dish) were cotransfected with baculovirus DNA (0.5 µg of BaculoGold DNA) and with vector derivatives of pVL1392 (2 µg) using a calcium phosphate coprecipitation method. The presence of recombinant protein was examined in the infected cells 5 days post-infection by SDS-PAGE and Western blotting.
Treatment of Sf9 Cells with Kinase Inhibitors-- To determine the effects of aminopurvalanol and indirubin-3'-monoxime on tau phosphorylation, Sf9 cells infected with baculovirus expressing htau23 protein were treated 36 h post-infection (when cells have already expressed levels of tau sufficient for the outgrowth of cell processes (46)) with 20 µM inhibitors for 3 h before being harvested.
Tau Western Blotting-- Sf9 cells were infected with recombinant virus at a multiplicity of infection of 1-5. Cell lysates were prepared in hypotonic lysis buffer (HLB). After 15-min centrifugation at 16,000 × g, the supernatant was recovered and its NaCl concentration raised to 500 mM. It was then boiled for 10 min and recentrifuged at 16,000 × g for 15 min. Proteins (3 µg) were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and Western-blotted with the following antibodies: AT-8 (1:2000), AT-180 (1:1000), AT-100 (1:1000), PHF-1 (1:600), and polyclonal anti-tau antibody K9JA. The immunostaining was visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech, Braunschweig, Germany).
Tau Phosphorylation--
Tau phosphorylation in vitro
was performed using purified GSK-3 and recombinant human tau-32
(provided by Dr. M. Goedert) as a substrate. After 30-min
incubation in the presence of various indirubin-3'-monoxime
concentrations, under the GSK-3
assay conditions described
above, the kinase reaction was stopped by addition of Laemmli sample
buffer. Tau was resolved by 10% SDS-PAGE, and its phosphorylation
level was visualized by autoradiography.
In Situ Inhibition of CDK5 in the Striatum
Adult mouse brain striatal slices were prepared using standard
methodology (47). Following equilibration in Krebs' bicarbonate buffer
oxygenated with continuous aeration (95% O2/5%
CO2), slices were treated with various concentrations of
indirubin-3'-monoxime or 10 µM roscovitine for 60 min or
were left in Krebs' bicarbonate buffer for the same period of time.
Slices were homogenized by sonication in boiling 1% SDS and 50 mM NaF. Protein concentrations were determined by the BCA
method using a BSA standard curve. Equal amounts of protein (80 µg)
were subjected to SDS-PAGE using a 15% acrylamide gel,
electrophoretically transferred to nitrocellulose membrane, and
immunoblotted with a phosphorylation state-specific antibody that
selectively detects DARPP-32 phosphorylated at Thr-75 (41).
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RESULTS |
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Indirubins Inhibit GSK-3 and CDK5/p25--
In the course of
studying the CDK inhibitory properties of indirubin, we synthesized a
series of indole derivatives and dimers (Table
I). While further investigating the
kinase inhibition selectivity of indirubin-3'-monoxime, the indirubin
used in our cellular studies, we noticed that this compound was a
powerful inhibitor of GSK-3
(see below). Our collection of
indoles/bis-indoles was further evaluated for inhibition against
purified GSK-3
, CDK5/p25, and CDK1/cyclin B. Kinase activities were
assayed with an appropriate substrate (GSK-3
, GS1 peptide; CDKs,
histone H1) in the presence of 15 µM ATP and increasing
concentrations of compounds. IC50 values were calculated
from the dose-response curves and are presented in Table
II. The GSK-3
and CDK inhibition activity was limited to the indirubins family. Neither indigo nor
isatin, and their derivatives, displayed a significant effect on any of
the three kinases. To compare the effects of active compounds on
GSK-3
and CDKs, the IC50 values toward each enzyme were
plotted against the IC50 values for the other two kinases (Fig. 2). This analysis shows that the
efficacies of indirubins toward CDK1 and CDK5 are closely related,
whereas the efficacies toward GSK-3
and CDKs are less so. This
probably reflects the closer evolutionary proximity between CDK1 and
CDK5 compared with that between GSK-3
and CDKs (48). The
dose-response curves for (a) the most active compound on the
three kinases (5-iodo-indirubin-3'-monoxime), (b) the most
GSK-3
-selective compound (5,5'-dibromoindirubin), (c) the
most CDK-selective compound (5-sulfonic acid-indirubin-3'-monoxime), and (d) the most frequently used compound in the studies of
indirubins' cellular effects (indirubin-3'-monoxime) are presented in
Fig. 3.
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Indirubins Act by Competition with ATP at the Catalytic
Site--
To investigate the mechanism of indirubins' action on
GSK-3, kinetic experiments were performed by varying both ATP levels (0.1, 0.15, 0.25, and 0.5 mM) and indirubin-3'-monoxime
concentrations (0, 0.5, 1, 1.5, and 2 µM) (Fig.
4). Double-reciprocal plotting of the
data suggests that indirubin-3'-monoxime acts as a competitive inhibitor of ATP binding. The apparent Ki was 0.85 µM. The apparent Km was 110 µM. Because recombinant GSK-3
was used throughout this
study, this preparation is likely to contain an unknown proportion of
inactive, misfolded enzyme as well as some proteolytic degradation
fragments. Therefore, we feel that Km and
Ki values are only estimates.
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Indirubins Inhibit in Vitro and in Vivo Tau Phosphorylation by
GSK-3--
To confirm the inhibitory effects of indirubins on
GSK-3
activity (assessed with a peptide substrate) we tested
indirubin-3'-monoxime on the phosphorylation of a more physiological
substrate, the microtubule-binding protein tau. Bacterially expressed
recombinant human tau was indeed phosphorylated in vitro by
GSK-3
, and this phosphorylation was inhibited in a
dose-dependent manner by indirubin-3'-monoxime, with an
IC50 value of around 100 nM (Fig. 5,
A and B).
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We next investigated the effect of indirubin-3'-monoxime on the phosphorylation of human tau23 expressed in Sf9 cells (Fig. 5C). Cells were left untreated (control) or exposed to 20 µM indirubin-3'-monoxime or aminopurvalanol, a CDK-selective inhibitor. Htau23 was resolved by SDS-PAGE followed by immunoblotting with various antibodies (Fig. 5D).
AT100 recognizes tau phosphorylated at Thr-212 and Ser-214; this
reaction is highly specific for Alzheimer tau but occurs in Sf9
cells as well, provided both sites are phosphorylated (49). The epitope
is formed by sequential phosphorylation, first of Thr-212 by GSK-3,
then of Ser-214 by PKA (49). Indirubin-3'-monoxime completely inhibits
the phosphorylation of the AT100 epitope. The phosphorylation of these
two sites may be indirectly dependent on CDK5, because aminopurvalanol,
which is a very poor inhibitor of GSK-3
(Table
III) or PKA (50), completely abolishes
the AT100 epitope phosphorylation.
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AT8, AT180, and PHF-1 are specific for different phosphorylated SP or
TP motifs, respectively, Ser-202 and Thr-205, Thr-231 and Ser-235, and
Ser-396 and Ser-404. The primary target of GSK3 is the PHF-1 site,
followed by the AT-8 site (51). Indirubin-3'-monoxime moderately
inhibits the phosphorylation of the PHF-1 epitope. Both inhibitors
completely inhibit phosphorylation at the AT-8 epitope and partially at
the AT180 epitope. These sites are efficiently phosphorylated by CDKs
(e.g. cdk5) and to a lesser extent by GSK-3
.
Altogether the results suggest that indirubin-3'-monoxime inhibits both
the CDKs and GSK3.
Indirubins Inhibit DARPP-32 Phosphorylation by CDK5 in
Vivo--
The neuronal protein DARPP-32 has been recently identified
as a physiological substrate of CDK5/p25 (41). When phosphorylated by
this kinase on Thr-75, DARPP-32 becomes an inhibitor of PKA. Phosphorylation on this site does not occur in p35/
tissue (41). To assess the ability of indirubin-3'-monoxime to inhibit
CDK5 in the brain, slices of striatum (an area of the brain enriched in
DARPP-32) were prepared and treated with different concentrations of
indirubin-3'-monoxime or roscovitine (Fig.
6). Homogenates from these slices were
then probed with a phosphorylation state-specific antibody that only
detects DARPP-32 phosphorylated at Thr-75. Indirubin-3'-monoxime was
able to inhibit phosphorylation of DARPP-32 in situ and in
the same range of concentrations previously found to be effective for
roscovitine (41).
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Many CDK Inhibitors Are Potent Inhibitors of
GSK-3--
Intrigued by the efficiency of indirubins to inhibit
both CDKs and GSK-3
, we decided to test the effects of reported CDK inhibitors on purified GSK-3
(Table III). The results confirmed the
strong selectivity of the olomoucine/roscovitine/purvalanol series,
which was essentially inactive on GSK-3
. The non-selective isopentenyladenine was active on both CDKs and GSK-3
.
Butyrolactone-I was inactive on GSK-3
. Both staurosporine and its
7-hydroxy analogue (UCN-01), two rather non-selective kinase
inhibitors, were found to inhibit GSK-3
quite efficiently (Table
III; Fig. 7). The GSK-3
inhibitory
properties of UCN-01 are particularly interesting, in view of the
development of this compound as an anticancer agent (phase II trials).
UCN-01 has been reported to inhibit various kinases, including protein
kinase C (52), CDK2 (53), and the chk1 kinase (54). The well-studied
flavopiridol is also in phase II as an antitumor agent (55). It
inhibits both CDK1/2 and CDK4 (56). We here report that it also
inhibits GSK-3
at similar concentrations (Table III; Fig. 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).
Finally, the fungi-derived CDK inhibitor toyocamycin (60) has also some
GSK-3
inhibitory activity. In summary, except for butyrolactone and
the purines, all reported CDK inhibitors are potent inhibitors of
GSK-3
.
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DISCUSSION |
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Indirubins Inhibit GSK-3--
We have previously described the
inhibition of CDKs by indirubin and analogues (25). In this article, we
further investigated CDK5 inhibition and we also report that GSK-3
is an excellent target for indirubins. Other indigoïds are
inactive (Table II). CDK5 is very closely related to CDK1 and CDK2
(73% and 75% identity, respectively) (61). GSK-3
is one of the
evolutionarily closest enzymes to the CDK family (42). We have
confirmed that indirubin-3'-monoxime inhibits GSK-3
by competing
with ATP for binding to the catalytic site (Fig. 4). The crystal
structure of CDK2/indirubin-3'-monoxime shows that the inhibitor binds,
through three hydrogen bonds, to the backbone atoms of Glu-81 and
Leu-83, two residues located in the ATP-binding pocket of the enzyme
(25). The corresponding amino acids are Glu and Cys in CDK5, and Asp
and Val, in GSK-3
. These observations support the idea that
indirubins bind to GSK-3
as they do to CDKs. This is further
supported by the structure-activity relationship study (Table II, Fig.
2): good CDK inhibitors are good GSK-3
inhibitors. There are two
noticeable exceptions, 5,5'-dibromoindirubin (12), which is
quite active on GSK-3
but poorly active on CDKs, and
5-SO3Na-indirubin-3'-monoxime (19), which is
over 10-fold less active on GSK-3
than on CDKs (Fig. 2). Although the 5-SO3Na substitution on indirubin-3'-monoxime
stimulates the inhibitory activity toward CDKs, it does not seem to
operate the same way with GSK-3
. In contrast, a 5-iodo substitution
is very favorable for inhibitory activity toward GSK-3
. These two
exceptions suggest that it might be possible to obtain indirubin
derivatives highly selective for either GSK-3
or CDKs. The synthesis
of compounds specific for either CDK1 or CDK5 appears to be less likely.
Selectivity of Indirubins--
Although indirubins have a strong
affinity for CDKs, we had previously noticed that they were slightly
less selective than purines (25). The identification of GSK-3 as a
very sensitive target of indirubins raises some questions about the
range of cellular targets of these compounds. We intend to purify and
identify the indirubin-binding proteins, from various tissues, by
affinity chromatography on immobilized indirubin-3'-monoxime. The
crystal structures of indirubins in complex with CDK2 provides precious information with respect to the orientation of the two indole rings
within the ATP-binding pocket. Carbons 6' and 7' clearly point toward
the outside of the kinase catalytic site and are accessible to solvent.
This is where a linker could be attached to tether the inhibitor to a
solid matrix while maintaining free access of the inhibitor to its
kinase targets. Using this approach with purvalanol, based on the
CDK2/purvalanol crystal structure (62), we have recently been able to
identify the intracellular targets of purvalanol in a variety of cells
and tissues (63).
Selectivity of Other CDK Inhibitors--
Most reported CDK
inhibitors are powerful GSK-3 inhibitors (Table III). This
observation has several important consequences: First, the range and
identity of the exact cellular targets of these compounds must be
seriously evaluated. We believe that the affinity chromatography
approach described above is the most straightforward approach available
at present. We are currently developing it with purines, indirubins,
paullones, and hymenialdisine. Most of these compounds have been
crystallized in the ATP-binding pocket of CDK2 (64), and this will
allow proper orientation of the inhibitors on the matrix. Second,
previously published papers on the cellular effects of these CDK
inhibitors need to be re-evaluated. In the case of flavopiridol,
several authors (65, 66) had convincingly suggested that it might act
on targets other than CDKs while inducing apoptosis. Third, it remains
to be determined whether GSK-3
inhibition is a favorable or a
negative property of these compounds in their potential use as
antitumor agents, as well as for any other application, including
neuroprotection. This is an important question, because it will orient
the search for and optimization of these inhibitors either toward
highly CDK- or GSK-3
-selective compounds or toward dual-specificity agents. In the case of antitumor properties, GSK-3
inhibition (expected to favor cell division) (30), and CDK inhibition (expected to
arrest the cell cycle) (67, 68) may turn out to create an
"intracellular conflict of interest," which might only be solved by
the induction of apoptosis. This is clearly a desired effect in cancer
therapy. However, GSK-3
inhibition may reduce or even mask
therapeutically interesting properties of CDK inhibitors. Alternatively, GSK-3
inhibitors might be more efficient in cells if
devoid of CDK inhibitory properties.
Indirubins Inhibit Tau Phosphorylation: Implication in Alzheimer's
Disease--
Three groups of proteins are known to play a major role
in the development of AD: presenilins, the amyloid peptides, and the microtubule-binding protein tau. Mutations in the presenilin genes
are the most common cause of early onset familial AD (69, 70). Amyloid
peptides, derived from proteolytic cleavage of the amyloid
precursor protein, form the extracellular senile neuritic plaques, a
diagnostic feature of AD (69).
Numerous reports describe the abnormal hyperphosphorylation of tau in
AD (38, 39, 70). When hyperphosphorylated, tau aggregates into paired
helical filaments (PHF), which form the typical neurofibrillary
tangles, a hallmark feature of AD. Four recent discoveries have
recently helped in understanding the link between tau
hyperphosphorylation and AD: 1) the existence of tau mutations in
AD-related diseases (39), 2) the functions of tau in regulating
intracellular traffic along microtubules (71), 3) the accumulation of
p25 and increase in CDK5 kinase activity in the brains of AD patients
(40), and 4) the positioning of tau hyperphosphorylation downstream of
presenilins and amyloid- (70). Tau hyperphosphorylation occurs on
more than 20 sites and is essentially carried out by two
proline-directed kinases, GSK-3
and CDK5/p25, and by PKA (38, 39,
70, 72), and indirectly carried out by casein kinase 1, which
phosphorylates and activates CDK5 (73). Presenilin associates with
GSK-3
, and presenilin 1 mutations that cause AD increase the ability of presenilin 1 to bind and activate GSK-3
(74). Exposure of hippocampal neurons to amyloid
peptides leads to GSK-3
stimulation and enhanced tau phosphorylation (75). CDK5 potentiates
GSK-3
-stimulated tau phosphorylation (76). CDK5 is increased in AD
brains (40). Lithium, a recently reported GSK-3
inhibitor
(IC50 in the millimolar range), reduces AD-like tau
phosphorylation in cultured cells (77-80). Altogether, these data
suggest that CDK5 and GSK-3
inhibition can be expected to impact
severely on the hyperphosphorylation of tau observed in AD and possibly
also on the outcome of this disease. Indirubins, and many of the
previously reported CDK inhibitors (flavopiridol, paullones, UCN-01,
hymenialdisine) constitute lead compounds with great potential for the
treatment of AD and other "taupathies."
In summary, we have identified indirubins as very potent inhibitors of
GSK-3 and CDK5, two major kinases involved in tau hyperphosphorylation. Indirubins therefore constitute a promising family of lead compounds, which deserve to be evaluated as therapeutic agents in Alzheimer's disease and other neurodegenerative disorders.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ning Xiuren for his help with Chinese ideograms and the fishermen of the Station Biologique de Roscoff for collecting the starfish. We are grateful to our following colleagues for providing reagents: P. Davies, M. Goedert, and J. H. Wang.
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FOOTNOTES |
---|
* This research was supported by grants from the Association pour la Recherche sur le Cancer (ARC 5343) (to L. M.) and the Conseil Régional de Bretagne (to L. M.), by Grant 0310938 from the German Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (to G. E.), and by United States Public Health Service Grants MH40899 and DA10044 (to P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains experimental procedures on chemistry and
synthesis of indoles and indirubins.
** To whom correspondence should be addressed: Tel.: 33-29-82-92-339; Fax: 33-29-82-92-342; E-mail: meijer@sb-roscoff.fr.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M002466200
2 M. Leost, C. Schultz, A. Link, Y.-Z. Wu, J. Biernat, E.-M. Mandelkow, J. A. Bibb, G. L. Snyder, P. Greengard, D. W. Zaharevitz, R. Gussio, A. Senderovitz, E. A. Sausville, C. Kunick, and L. Meijer, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
CDK, cyclin-dependent kinase;
AD, Alzheimer's disease;
HLB, hypotonic lysis buffer;
PHF, paired helical filaments;
PKA, protein
kinase A (cAMP-dependent kinase);
GSK-3, glycogen
synthase kinase-3
;
EI, electron impact;
Mops, 4-morpholinepropanesulfonic acid;
DTT, dithiothreitol;
BSA, bovine
serum albumin;
PAGE, polyacrylamide gel electrophoresis.
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