Inhibition of γ-secretase by the CK1 inhibitor IC261 does not depend on CK1δ

Article abstract of DOI:10.1016/j.bmcl.2010.02.110

CK1 and γ-secretase are interesting targets for therapeutic intervention in the treatment of cancer and Alzheimer's disease. The CK1 inhibitor IC261 was reported to inhibit γ-secretase activity. The question is: Does CK1 inhibition directly influence γ-se

Full text of DOI:10.1016/j.bmcl.2010.02.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2958–2963
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of
c-secretase by the CK1 inhibitor IC261 does not depend on CK1d
Nicole Höttecke a, Miriam Liebeck a, Karlheinz Baumann b, Robert Schubenel b, Edith Winkler c,
Harald Steiner c, Boris Schmidt a,
*
a Clemens Schöpf-Institute of Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstr. 22, D-64287 Darmstadt, Germany
b F. Hoffmann-La Roche Ltd, Pharmaceuticals Division, Preclinical Research CNS, Bldg. 70/345, CH-4070 Basel, Switzerland
c German Center for Neurodegenerative Diseases (DZNE) and Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Schillerstr. 44, 80336 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
CK1 and
Alzheimer’s disease. The CK1 inhibitor IC261 was reported to inhibit
is: Does CK1 inhibition directly influence -secretase activity? Therefore we analyzed the SAR of 15 ana-
logues and their impact on -secretase activity. The most active compounds were investigated on CK1d
activity. These findings exclude a direct influence of CK1don -secretase, because any change in the sub-
-secretase inhibition is still exerted by
c
-secretase are interesting targets for therapeutic intervention in the treatment of cancer and
Received 15 January 2010
Revised 26 February 2010
Accepted 27 February 2010
Available online 4 March 2010
c-secretase activity. The question
c
c
c
stitution pattern of IC261 diminished CK1 inhibition, whereas
c
Keywords:
Alzheimer’s disease
several analogues.
Ó 2010 Elsevier Ltd. All rights reserved.
c
-Secretase
Casein kinase 1
Amyloid b secretion
Alzheimer’s disease is a devastating illness, which robs patients
of the ability to manage their lives on their own. This illness is
accompanied by protein aggregates in the brain composed of the
amyloid-b-peptide (Ab), which are called amyloid plaques.1 The
amyloid-b-peptide is generated by the subsequent degradation of
the amyloid precursor protein (APP), a type I transmembrane pro-
larity of IC261 with the known, potent GSM (Sulindac-S (2), and
Sulindac-sulfon (3)) stimulated us to investigate the oxoindole-
backbone of IC261 common to many kinase inhibitors and the po-
tential influence of CK1e/d inhibitors on
c-secretase activity).
H4-cells do neither express constitutively active CK1
e
-271 nor
do they overexpress CK1e, which were postulated to be the regu-
tein, by two aspartyl proteases, the b-secretase and the
c-secre-
lating CK1 isoforms.6 Actually, Ab secretion from the utilized H4
tase. The -secretase is promising target for therapeutic
c
a
cells responded to IC261 treatment five times stronger, suggesting
intervention as it liberates various Ab-peptides with a length of
38, 40, or 42 amino acids.2 The toxicity depends on the length:
Ab42 is the most toxic species while Ab38 is regarded to be non-
toxic as increased production of Ab38 does not diminish cellular
an CK1
analysis (SAR) towards
activity was carried out by systematical variation of the oxoindole
substitution utilizing the CK1/IC261 co-crystallized structure (PDB:
1EH4). (Table 1). The CK1 isoforms differ in the primary structure
e
independent effect. A dual structure–activity relationship
-secretase activity in H4-cells and CK1d
c
viability. Several
c-secretase inhibitors (GSI), which decrease total
Ab levels, and several
c-secretase modulators (GSM), which shift
of the C-terminal non-catalytic domain. However, CK1d and CK1e
the cleavage-site to the non-toxic Ab38, have been identified so
do not differ in the ATP-binding-site for IC261, thus a cell free
CK1d activity assay guided the structure–activity relationship for
both CK1 isoforms. This approach is commonly employed for the
far.3–5
Flajolet et al.6 reported IC261 (1) (Scheme 1), a presumably
selective ATP-competitive casein kinase 1
e
(CK1
e
) inhibitor, which
development of GSK3a/b inhibitors.
is also an equipotent inhibitor to the CK1d-isoform (CK1d)
This structure guided the variation of the compounds aiming
either at enhanced interaction with CK1 or to exclude interaction
with CK1 (R3 = Me, 15). We chose the 5-cloro substituted oxoindole
as backbone as it is supposed to show enhanced metabolic stabil-
ity,10 which is commonly used in medicinal chemistry, for exam-
ple, Pfizer’s Carprofen (4). All compounds were tested in the
cellular Ab generation assay.11 The four most potent GSIs were sub-
sequently investigated on their CK1d activity to investigate the
(IC50 = 2.57 l
M in cells).19 IC261 exerts rather weak GSI activity
in comparison to reported potent GSIs.6–9 IC261 causes a signifi-
cant reduction of Ab40 (68%) and Ab42 (61%) levels in N2a cells
overexpressing constitutively active CK1e-271 within 5–50 lM
concentration at 3 h after incubation. The reported increase of
Ab40 secretion by approximately 35% (Fig. 2A in Ref. 6) under over-
expression of constitutively active CK1d in N2a cells and the simi-
influence of CK1d on the c-secretase. A further aim of this investi-
gation was the identification of selective GSIs or even GSMs, void
off cross-activity on CK1d or related kinases.
* Corresponding author. Tel.: +49 6151 163075; fax: +49 6151 163278.
E-mail address: schmidt_boris@t-online.de (B. Schmidt).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.110

N. Höttecke et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2958–2963
2959
Scheme 1. Structural similarity of IC261 and the GSM of the Sulindac-series (2, 4).
Table 1
Compounds 1, 5–16
Entry
Compds
Code
R1
R2
R3
Ref.
1
2
3
4
5
6
7
8
9
10
11
12
13
1
5
6
7
8
BSc3930 IC261
BSc3926
BSc3921
BSc3923
BSc3922
BSc3928
BSc3914
BSc3929
BSc3944
BSc3890
BSc3925
BSc3927
Sunitinib
2,4,6-Trimethoxy-benzene
2-Methoxybenzene
2-Fluorobenzene
2-Nitrobenzene
2-Benzene sulfonic acid
Benzo[d][1,3]dioxole
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
6
12
—
13
—
14
—
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
9
10
11
12
13
14
15
16
3,5-Difluorobenzene
4-Benzeneacetamide
—
4-Chloropropoxy-benzene
2,3,4-Trimethoxy-benzene
3,4,5-Trimethoxy-benzene
2,4,6-Trimethoxy-benzene
N-(2-(Diethylamino)-ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide
—
a
15
16
a
a
Commercially available.
The Knoevenagel condensation of these IC261-derivatives uti-
1418 (Scheme 3, see Supplementary data) to be complete within
2 days in methanol solution, which compares to the assay condi-
tions: buffered H2O, 24 h. Thus the cellular data are obtained for
E/Z mixtures regardless of the purity of the initial isomer.
lizes an oxoindole-derivative, a respective aldehyde and piperidine
as a base. The reaction is carried out under microwave irradiation
at 100 °C for 30 min to provide the products in moderate to good
yields.17 The Z-isomer was enriched in the subsequent re-crystalli-
zation. The proportion of the E/Z isomers was analyzed by HPLC–
MS signal integration and the HPLC signals were definitely as-
signed to the molecular mass. (Scheme 2, see Supplementary data)
The assignment of the two isomers to the two HPLC signals was
established by 1H NMR-spectroscopy.
IC261 (1) is a competitive ATP-binding-site inhibitor. The inter-
action with this binding-site was reported by Mashhoon et al.
based on the co-crystallization (PDB: 1EH4) of CK1 with IC261.19
1 features both a hydrogen-bond-donor in form of the indole-
amin and three methoxy-substituents as hydrogen-bond-acceptors,
which can be divided in two o- and one p-substitution. The structure
analysis suggests two hydrogen-bonds of the indole-amine with
The isomerization of the pure Z-isomer to the equilibrium of E-
and Z-isomers was monitored by HPLC–MS for 9 (Scheme 2), 1H
NMR-spectroscopy and 2-dimensional-NMR-spectroscopy for
Asp86 and Leu88 19
o-methoxy-group and Lys41 and an intermolecular interaction with
.
Notable interactions were assigned to the
Scheme 2. E/Z isomerization in solution.

2960
N. Höttecke et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2958–2963
ent may cause these minor changes in potency of 5. A complete
loss of activity was observed for those derivatives which lacked
o-methoxy-groups. This observation demonstrates the essential
role of the o-methoxy-group which fits exactly into the ATP-bind-
ing-site. This assumption was confirmed by introduction of an
benzo[d][1,3]dioxole (9) which did not display any significant
activity on Ab-levels. This group bears oxygen in the m-position,
but has an additional methylene bridge to another oxygen in the
p-position. The activity loss can be explained by sterical hindrance
of the dioxole group, which may displace Asp154 thus disrupting
essential hydrogen-bonds. Secondary, m-substitution is detrimen-
tal to activity, this was additionally tested by two fluorines (10) in
the m-positions, which displayed surprisingly increased Ab42 gen-
eration of up to 112% at a concentration of 5
lM. This may be due
to inverse modulation; such a switch from straight to inverse
c-
secretase modulation on
previously.6,20
a common scaffold was observed
Two additional hydrogen-bonds are formed by the p-substi-
tuted methoxy-group with Ser22 and Asp154. Ser22 exerts the role
of a gate keeper in CK1: Entry of the ATP in the binding-site causes
a conformational change into the closed form by Ser22, which locks
the binding-site and prevents ATP-diffusion out of the binding-site.
This in turn stabilizes CK1 in the active conformation. The ability of
1 to form a hydrogen-bond with Ser22 locks the ATP-binding-site
permanently. The position of Ser22 is fixed in the closed form of
the pocket and thereby prevents the replacement of IC261 by ATP.
The interaction of a p-substituent with Ser22 was investigated
by the introduction of both sterically demanding and functional
groups (11, 12).21–23 These groups should prevent the conforma-
tional change into the closed form of the ATP-binding-site. The
FDA-approved kinase inhibitor Sunitinib (16) was included into
the investigation for the same rational. 11 did not show any effect
Scheme 3. E/Z isomerization of 14 analyzed by 1H NMR-spectroscopy.
a benzene hydrogen and interactions of the p-methoxy-group with
Ser22 and Asp154 (Fig. 1).
The symmetric substitution pattern with two o-methoxy-
groups enables interactions of both rotamers. Both the E-isomer
purity and the atrop-isomerism found in the co-crystal were cap-
tured from the equilibrium conformations in solution. The influ-
on Ab levels, but an unexpected inverse modulation of the c-secre-
ence of the o-substitution was determined by
a derivative
tase was observed for 12, which decreased the Ab38 levels and in-
creased Ab42 levels (Fig. 2). The inverse modulation by 12 as well as
10 indicates the mechanism to be different from total inhibition of
Ab generation. The moderate inhibition of Ab secretion (Ab38 = 26%,
Ab40 = 53%, Ab42 = 26%) by 16 was not observed in the purified c-
secretase assay. This indicates an indirect, potentially upstream
bearing just one o-methoxy-group (5) and several derivatives
where the methoxy-groups were replaced by fluorine (6), nitro
(7) or sulfonic acid (8).
Just one out of these derivatives (5 Ab38 = 62%, Ab40 = 76%,
Ab42 = 65%) displayed a significant decrease in Ab-levels compara-
ble to the activity of IC261 (Ab38 = 55%, Ab40 = 77%, Ab42 = 77%,
Table 2). The single o-substituent and the absence of a p-substitu-
mode of action.
Figure 1. ATP-binding-site interaction of 1 according Mashhoon et al. (PDB: 1EH4).19

N. Höttecke et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2958–2963
2961
Table 2
Activity of Ab generation of oxindoles at a concentration of 10
l
M in H4-cells
Entry
Compds
Code
CK1d (%)
Ab38 (%)
Ab40 (%)
Ab42 (%)
Tox. (lM)
c log Pc
1
2
3
4
5
6
7
8
9
10
11
12
13
1
5
6
7
8
BSc3930 IC261
BSc3926
BSc3921
BSc3923
BSc3922
BSc3928
BSc3914
BSc3929
BSc3944
BSc3890
BSc3925
BSc3927
Sunitinib
19
55
62
87
77
76
88
88
96
77
65
91
83
88
40
40
2.31
3.12
3.40
3.30
2.40
3.02
3.56
1.73
3.93
2.86
2.86
3.10
2.99
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
91
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
40
94
101
86
9
93
90
10
11
12
13
14
15
16
81a
99
106a
97
112a
97
66
27
54
35
93
46
72
65
123
47
65
65
26
99
20
80
20
40
n.t.b
87
n.t.b
26
53
a
b
c
Activity at a concentration of 5
n.t. = not tested.
Calculated by ChemDraw.
lM.
Figure 2. Modelled ATP-binding-site interaction of 12 (based on PDB: 1EH4) and its dose–response curve.
As the IC261 substitution pattern (2,4,6-trimethoxybenzene)
IC261 and the three most potent derivatives 13, 15 and 12 were
tested for inhibitory activity on CK1d and 42 other kinases to eval-
was found to be important for the activity, it was systematically
varied into 2,3,4- and 3,4,5-trimethoxybenzene (13, 14). These
two derivatives also exert a significant reduction in total Ab.
Although the o-positions of 14 (Ab38 = 54%, Ab40 = 72%,
Ab42 = 65%) are substituted with hydrogens, it is equipotent to 1
(Ab38 = 55%, Ab40 = 77%, Ab42 = 77%). A distinct rise in activity was
observed for 13 (Ab38 = 27%, Ab40 = 66%, Ab42 = 47%) with a meth-
oxy-group in o-, m- and p-position.
uate the influence of CK1 on c
-secretase (Fig. 3).24 Surprisingly only
IC261 shows a significant inhibition of CK1d (81% inhibitory activ-
ity, Table 1, Fig. 3). As soon as the substitution pattern differs from
2,4,6-trimethoxybenzene, no significant CK1d inhibition (>80%)
was observed. This is shown, for example, in the case of 13
(2,3,4-trimethoxybenzene), which bears a methoxy-group in the
m- instead of the second o-position. It exerts an inhibition of a
mere 1% under the same conditions (Fig. 3). In addition, no signif-
icant inhibitory activity on CK1d was found for 15 and 12.
Compounds 12 and 13 are inactive on CK1d yet display signifi-
cant inhibition (98% and 89%) of the tyrosine kinase Flt3, which
plays an important role in leukemia. However, Flt3 is not known
to exert effects on APP metabolism.
The most important interaction was anticipated to be the in-
dole-nitrogen forming two hydrogen-bonds with Asp86 and
Leu88 19
This crucial interaction was challenged by a methyl-sub-
.
stitution of the nitrogen, expecting a 90% loss of activity. Surpris-
ingly, a rise in Ab-inhibitory activity of 15 compared to IC261
was observed. The three Ab-species were significantly decreased
to 35% for Ab38 and to 65% for Ab40 and Ab42 each. Particularly this
observation was the first major evidence against the involvement
of CK1 in our biological assay.
Only the N-methylated indolinone 15 lacked significant inhibi-
tion (inhibitory activity <20%) for all kinases tested, which con-
firms the relevance of N-methylation of indolinones in kinase

2962
N. Höttecke et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2958–2963
Figure 3. Kinase screening of 43 kinases from compounds 1 (BSc3930), 12 (BSc3944), 13 (BSc3890), 15 (BSc3927). Kinase inhibition is indicated by positive values. Negative
values indicate kinase activation.
inhibition. This N–H bond is essential for the inhibition of most ki-
nases by indolinones.
The inhibitory activity of 1, 12, 13, 15 on CK1d cannot be corre-
lated to the inhibition of
ure 4 for the Ab38 level reduction at a concentration of 10
These data agree with three possible explanations: (1) IC261
c-secretase activity which is shown in Fig-
lM.
does not directly affect
uct is responsible for
c
-secretase activity but a degradation prod-
c-secretase inhibition, (2) IC261 is coinci-
dently a weak
CK1d or CK1
responsible for the cellular activity. To investigate a direct effect
on -secretase activity, IC261 was tested in a cell-free assay using
c
-secretase inhibitor, which independently targets
Figure 5. Cell-free
685,45826 GSI as control.
c-secretase assay in the presence of IC261, Sunitinib, or L-
e
activity. (3) Another, yet unidentified kinase is
c
The cellular activity of IC261 and its activity in cell-free assays
is inconsistent with an exclusively CK1d mediated effect on Ab
lipid-reconstituted purified c-secretase and purified APP C100-His6
as substrate for Ab generation,25 that is, under conditions, where
secretion via
isoforms, for example, CK1
c
-secretase inhibition but an influence of other CK1
or by b-secretase could not be ex-
no additional metabolism is present (Fig. 5).
e
No effect on the Ab production by c-secretase was observed at
cluded. Another potential mechanism, a Notch mediated effect,
50 lM, the highest effective concentration used in cultured cells by
was excluded by Flajolet et al.6 The cell-free assay is neither sub-
the study of Flajolet et al.6 IC261 did also not exert a modulatory
ject to CK1d or CK1
is suggested for the interference of Ab secretion by IC261: (a) cel-
lular -secretase inhibition may occur at high concentration of
IC261 or through a metabolic activation of IC261. It is not clear,
however, whether the interference of IC261 with -secretase activ-
ity at high concentration (in the cell-free assay at 250 M, i.e., five
times above the concentration used by Flajolet et al.6 in the cellular
assay) does reflect enzyme inhibition. The observed reduction in
e dependent regulation, thus a pleiotropic effect
effect on the production of Ab38, Ab40 and Ab42 species in the
cell-free assay at this concentration.
c
-Secretase activity was also
M IC261, however, -secretase inhibition
M. This may be due to the resemblance of
c
not affected at 150
was observed at 250
l
l
c
c
IC261 to known c-secretase inhibitors derived from NSAIDs, which
l
are characterized by IC50ies around 100–300 lM, for example,
2.2,27,28 Consistent with previous results,25,29 presence of the highly
c-
potent GSI L-685,45826 (structure see Supplementary data) at
secretase activity could also be due to a damage of the lipid envi-
ronment by this particular compound in the in vitro assays; (b) up-
stream modulation of Ab metabolism by an unidentified mode of
action, which does not necessarily involve CK1d at low concentra-
tions, which is also confirmed by Flajolet et al. investigation.6 Sun-
itinib, a promiscuous kinase inhibitor, was selected for structural
similarity to IC261 and submitted to the assay panel. It reduces
Ab-secretion in the cellular assay. However, Sunitinib did not dis-
play an effect on Ab generation in the cell-free assay suggesting
that an unidentified kinase may be involved in Ab-secretion in
the cellular assay.
0.5
l
M completely blocked
c
-secretase activity in this assay.
100
80
60
40
20
BSc3930
BSc3944
BSc3890
BSc3927
Even a subtle variation of the IC261 substitution pattern re-
sulted in complete loss of CK1 inhibition but still exerts Ab lower-
ing capability (e.g., 13).
0
0
50
100
δ
CK1 inhibition [%]
In conclusion, our IC261 SAR data are inconsistent with CK1-
dependent inhibition of
are not suitable as lead structure for
c
-secretase. The most potent compounds
Figure 4. Correlation plot of CK1d activity inhibition and Ab38 level reduction at a
concentration of 10 M.
c-secretase modulation
l

N. Höttecke et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2958–2963
2963
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in vivo, as long as they display significant inhibition of Flt3. Such
off-target activity will impair further development.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.110.
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