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OCR for page 24
Colloquium
Identification of benzothiazoles as potential
polyglutamine aggregation inhibitors of
Huntington's disease by using an
automated filter retardation assay
Volker Heiser*t, Sabine Engemann*t, Wolfgang Brocker*t, llona Dunkel*, Annett Boeddrich*t, Stephanie Waelter*$,
Edd i Nord hoff*t, Rudi Lu rz*, Nancy Sch ugardt*$, Susan ne Rauten berg *t, Christian Herhaus§, Gerhard Barn ickel§,
Henning Bottcher§, Hans Lehrach*, and Erich E. Wanker*$ll
*Max-Planck-lnstitut fur Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany; and §Merck KGaA, Frankfurter Strasse 250, D-64271
Darmstadt, Germany
Preventing the formation of insoluble polyglutamine containing
protein aggregates in neurons may represent an attractive thera-
peutic strategy to ameliorate Huntington's disease (HD). There-
fore, the ability to screen for small molecules that suppress the
self-assembly of huntingtin would have potential clinical and
significant research applications. We have developed an auto-
mated filter retardation assay for the rapid identification of chem-
ical compounds that prevent HD exon 1 protein aggregation in
vitro. Using this method, a total of 25 benzathiazole derivatives
that inhibit huntingtin fibrillogenesis in a dose-dependent manner
were discovered from a library of ~184,000 small molecules. The
results obtained by the filter assay were confirmed by immuno-
blotting, electron microscopy, and mass spectrometry. Further-
more, cell culture studies revealed that 2-amino-4,7-dimethyl-
benzathiazol-6-ol, a chemical compound similar to riluzole,
significantly inhibits HD exon 1 aggregation in vivo. These findings
may provide the basis for a new therapeutic approach to prevent
the accumulation of insoluble protein aggregates in Huntington's
disease and related glutamine repeat disorders.
Huntington's disease (HD) is a progressive neurodegenera-
tive disorder with no effective treatment (1~. The disease is
caused by an elongated CAG trinucleotide repeat expansion
located within exon 1 of the IT-15 gene encoding huntingtin, an
~350-kDa protein of unknown function. The CAG repeat is
translated into a polyglutamine (polyQ) stretch. In HD patients,
huntingtin is expressed with 38-180 glutamine residues, whereas
in healthy individuals the protein is synthesized with 8-37
glutamine residues (2, 3~. Thus, the disease develops when a
critical length of about 37 glutamine residues (pathological
threshold) is exceeded, whereas a polyQ tract of fewer than 37
glutamine residues is tolerated by neuronal cells (4~.
The accumulation of ubiquitinated polyQ-containing protein
aggregates in neuronal inclusions is a pathological hallmark of
HD and related glutamine repeat disorders (5~. Whether the
formation of huntingtin aggregates in brain is the cause, or
merely the consequence, of disease, however, is still unclear (6~.
Within the last few years, aggregation of polyQ-containing
proteins has been reproduced in various in vitro and in vivo model
systems (7-11~. Evidence has been presented that the process of
aggregate formation is causally linked with disease progression.
Recently, Yamamoto et al. (12) have shown that blockage of HD
exon 1 expression in symptomatic transgenic mice results in
disappearance of insoluble protein aggregates, as well as motor
dysfunction, suggesting that protein aggregation in vivo is asso-
ciated with disease progression.
16400-16406 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4
The formation of insoluble polyQ-containing protein aggre-
gates in vitro and in cell culture model systems has been inhibited
by specific antibodies (13), peptides (14), heat shock proteins
(15, 16), and chemical compounds (17~. Moreover, antibodies
(18), ~-sheet breaker peptides (19), and chemical compounds
(20-24) that prevent protein aggregation in various models of
Alzheimer's and prion diseases have been described. However,
the effect of these and other potential therapeutic molecules on
the progression of neurodegenerative disorders in humans needs
to be evaluated.
In this study, we have developed an automated filter retarda-
tion assay for the identification of HD exon 1 aggregation
inhibitors. In contrast to conventional methods, our assay is
eminently suited for high-throughput detection of chemical
compounds that block huntingtin fibrillogenesis because it allows
the parallel, nonradioactive screening of up to 384 potential
aggregation inhibitors on a single filter membrane. Here, we
report the rapid screening and characterization of a previously
uncharacterized class of polyglutamine aggregation inhibitors
from large chemical compound libraries by using this approach.
Materials and Methods
Chemical Compounds, Enzymes, and Instrumentation. A library con-
taining 184,880 chemical compounds was provided by Merck. All
compounds were dissolved in 100% DMSO at a cone. of 3 mM.
Riluzole was purchased from BioTrend (Cologne, Germany).
Doxycycline and elastase were obtained from Sigma-Aldrich,
and trypsin (modified version) was purchased from La Roche
(Mannheim, Germany). The Cy3-labeled donkey anti-rabbit IgG
was obtained from Jackson ImmunoResearch. Parallel liquid
handling in 96-and 384-well format plates was performed with
Tomtec (Orange, CT) Quadra 96SV and Quadra 384S robots.
This paper results from the Arthur M. Sackier Colioquium of the Nationai AcacJemy of
Sciences, "Self-Perpetuating Structurai States in Biology, Disease, and Genetics," heicJ
March 22-24, 2002, at the Nationai AcacJemy of Sciences in Washington, DC.
Abbreviations: HD, Huntington's cJisease; polyQ, polygiutamine; MAEDi-TOF, matrix-
assistecl laser cJesorption ionization-time-of-flight; GST-HD51, GSE-HD exon ~ fusion pro-
tein with 51 giutamines; HDQ51, Fiag-taggecJ HD exon ~ protein with 51 giutamines.
"Present acJciress: Scienion AG, Voimerstrasse 7a, D-12489 Beriin, Germany.
"Present acJciress: Max-Deibruck-Centrum fur Molekuiare MecJizin, Robert-Rossie-Strasse
10, D-13122 Berlin, Germany.
To whom reprint requests shouicJ be sent at the present acJUress: Max-Deibruck-Centrum
fur Molekuiare Medizin, Robert-Rossie-Strasse 10, D-13122 Berlin, Germany. E-maii:
ewanker~mcJc-beri in.cJe.
www. pnas.org/cg i/cloi/ ~ 0. ~ 073/pnas. ~ 82426599
OCR for page 25
Construction of Plasmids and Protein Purification. Standard proto-
cols for DNA manipulations were followed. Escherichia cold
SURE (stop unwanted rearrangement events; Stratagene) was
used as host strain for plasmid construction and protein expres-
sion. Plasmids pCAG51 and pCAG512\P are described elsewhere
(7, 25~. E. cold SURE carrying the plasmids pCAG51 or
pCAG51AP was used for the expression of GST-HD51 (GST-
HD exon 1 fusion protein with 51 glutamines) and GST-
HD512\P fusion proteins, respectively. Recombinant proteins
were purified under native conditions by affinity chromatogra-
phy on glutathione agarose as described (25), and aliquots were
subjected to matrix-assisted laser desorption ionization-time-of-
flight (MALDI-TOF) MS for sequence confirmation. Recom-
binant proteins were stored at a cone. of 25 ,uM at -80°C.
High-Throughput Screening Procedure. In each well of six hundred
384-well microtiter plates, 15 Al of a 20 ,uM solution of the
chemical compound to be tested (in 150 mM NaCl, 20 mM
Tris HCl (pH 8.0), 2 mM CaCl2, and 6.7% DMSO) was mixed
with 15 ,ul of predigested GST-HD51 fusion protein (1.25 ,uM)
by using a pipetting robot. Total removal of the GST tag from
the fusion protein GST-HD51 was achieved by elastase treat-
ment (3 min at 37°C) before the addition of chemical com-
pounds. Samples were incubated for 16 h at 37°C to allow
aggregate formation. Reactions were stopped by addition of 30
,ul of 4% SDS/100 mM DTT, followed by heating at 98°C for 10
min. Aliquots corresponding to 250 ng of GST-HD51 protein
were filtered through a cellulose acetate membrane (0.2 ,um,
Schleicher & Schuell) by using a 384-well vacuum dot blot
apparatus. Captured aggregates were detected by incubation
with HD1 antibody (diluted 1:5,000), followed by incubation
with alkaline phosphatase-conjugated anti-rabbit secondary an-
tibody and the fluorescent substrate AttoPhos. Signals corre-
sponding to SDS-insoluble aggregates were quantified by using
AIDA 2.0 image analysis software (Raytest, Straubenhardt,
Germany).
SDS/PAGE and Western Blotting. Proteins present in whole cell
extracts or aggregation reactions were denatured, separated by
SDS/PAGE (10 or 12%), and transferred to nitrocellulose.
Membranes were blocked with 3% nonfat dry milk in Tris-
buffered saline (TBS) containing 0.05% Tween 20 and incubated
with the HD1 antibody (7~. Secondary antibody was peroxidase-
conjugated anti-rabbit IgG (Roche). Immunoreactive protein
was detected by using enhanced chemiluminescence reagent
(ECL, Amersham Pharmacia).
MS and Microscopic Analysis. The conditions for proteolytic diges-
tion of GST-HD51AP with trypsin have been described (17~. Mass
maps of digested proteins were recorded on a Bruker Scout MTP
Reflex III MALDI mass spectrometer (Bruker Daltonik, Ger-
many) using the matrix or-cyano-4-hydroxycinamic acid. For elec-
tron microscopic analysis, the trypsin-digested GST-HD51 fusion
protein was adjusted to a final cone. of 50 ,ug/ml in 40 mM Tris HCl
(pH 8.0) and 150 mM NaCl. Samples were negatively stained
with l~o uranyl acetate and viewed in a Philips CM100 electron
microscope (Philips Electron Optics, Eindhoven, The Nether-
lands). Electron micrographs of perinuclear inclusion bodies in 293
Tet-Off cells were generated as described (11~.
Culturing of 293 Tet-Off Cells. Cells were grown in ~-glutamine-free
minimum essential medium with Earle's salts (GIBCO/BRL)
supplemented with 10% FBS, 2 mM ~-glutamine, 100 units/ml
penicillin, 100 ,ug/ml streptomycin, 100 ,ug/ml G418, 150 ,ug/ml
hygromycin B. and 10 ng/ml doxycycline in poly-~-lysine-coated
cell culture flasks. HDQ51 (Flag-tagged HD exon 1 protein with
51 glutamines) expression was induced by thoroughly washing
the cells with PBS and adding fresh medium lacking doxycycline.
Heiser et a/.
After incubation for 4 h in the absence of doxycycline, chemical
compounds were added to the medium at the indicated concen-
trations. The compound-containing medium was changed daily,
and, after 72-76 h of incubation, cells were harvested and protein
extracts were prepared as described (26~. Aliquots correspond-
ing to 10-25 ,ug of protein were used for the cellulose acetate
filter retardation assay (25~. Protein concentration was deter-
mined by using the Bradford protein assay.
Indirect Immunofluorescence Microscopy. 293 Tet-Off cells were
grown in Leighton tubes (Costar) in the presence or absence of
PGL-135 (cone. 25 or 50 ,uM). Expression of HDQ51 was
induced for 3 days by removal of doxycycline from the culture
medium. Chemical compounds were added 4 h after incubation
of cells in doxycycline-free medium. Indirect immunofluores-
cence microscopy was performed after 3 days as described (114.
Results
Inhibitor Screening. To identify polyQ aggregation inhibitors from
large chemical libraries, a high-throughput in vitro screening
assay was developed. The principle of this automated filter
retardation assay is shown schematically in Fig. 1A. The assay is
based on the finding that polyQ-containing protein aggregates
are resistant to SDS and selectively retained on a cellulose
acetate filter, whereas SDS-soluble protein under the same
conditions is not. The aggregates retained on the filter mem-
brane are then detected and quantified by immunoblot analysis
using specific antibodies (25~.
GST-HD51 was incubated for 3 min at 37°C with elastase,
resulting in the complete removal of the GST tag from the fusion
protein and the initiation of the aggregation process (data not
shown). Then, immediately, chemical compounds were added to
give a final cone. of 10 ,u M by using a pipetting robot. Incubation
was continued for 16 h at 37°C to permit aggregate formation.
Proteins were reduced and denatured by boiling in 2% SDS/50
mM DTT and filtered through a cellulose acetate membrane by
using a 384-well dot blot apparatus. After washing of the
membrane with 0.2~o SDS, the amount of insoluble polyQ-
containing protein aggregates retained on the surface was
quantified by immunoblotting and subsequent image analysis.
Compounds that reduced the signal intensity by more than 15~o
relative to the signal intensity of noninhibited HD51 aggregation
reactions were identified as hits (Fig. 1B, F8~. As positive
controls, elastase-digested GST-HD51 protein treated with the
known polyglutamine aggregation inhibitors Congo red and
thioflavine S (17) (control I), as well as undigested GST-HD51
protein (control II), which does not aggregate (7), were applied
to the filter membrane. Samples treated with the solvent DMSO
and untreated GST-HD51 elastase cleavace oroducts were used
as negative controls (Fig. 1B).
--~- r-
High-throughput screening of an ~184,000-chemical com-
pound library by using the automated filter retardation assay
resulted in the identification of about 300 chemical compounds
that inhibited HD51 protein aggregation in a dose-dependent
manner. Among these, we identified 25 benzothiazole derivatives
by cluster analysis. Benzothiazole derivatives have been shown
oreviouslv to be effective in treating neurodegenerative dis-
orders such as amyotrophic lateral sclerosis (27~. For example,
riluzole (2-amino-6-trifluoromethoxybenzothiazole), a potent
antagonist of glutamate release, has been reported to slow down
disease progression in amyotrophic lateral sclerosis patients (28,
29~. First clinical data on riluzole treatment of HD patients
indicate a positive effect on hyperkinesia (30), and recently
riluzole was reported to prolong the lifespan of a mouse model
of HD (31~. However, its mode of action is largely unknown. The
identification of benzothiazoles as polyglutamine aggregation
inhibitors is highly relevant because they are very promising
candidates for future drug development. The structures of the
PNAS | December 10, 2002 | vol. 99 | supp~. 4 | 16401
OCR for page 26
A
protease fusion protein
· ~
+
cleavage reaction
___ ~
_ __
___
0~o
o
-
formation of aggregates
_ _ _ ~ ~
image analysis and
hit identification
Bcompound library
(384 well format)
~3,
filter retardation assay
(384 well format)
li 11 11
or ~ I
it
mmunodetection of
aggregates
control ~ control I'
DMSO
Thio-S ~:
A
B
C
F
G
H
Congo Red | | - Protease
DMSO ~ + Protease
1/2 3 4 5 6 7 8 9 10 11\12
.~-~-4
Low Is .
~ .e e. ee.~e ~ ~ ,
.> ~~:~e
·
~~-eee.~*~ee.~e
~ ~—~.~. : ~
:~t ~~.~.
le.~
.. - ~~. ~ .~1~.
1~. .. .~1
it: - ~~e ~ o.~.
· Ieee.~e ~ ~ .~.
—.~ee ·~.
61~e .~1
.1~4 .~-1~.
~ r
I 1I
FX inactive ~ - inactive
inactive ~ ~ ~ active
III IV
Fig. 1. Development of an automated filter retardation assay for high-
throughput drug screening. (A) Flow chart of the automated filter retardation
assay. (B) Effect of chemical compounds on HD exon 1 aggregation as moni-
tored by the filter assay. GST-HD51 fusion protein at a cone. of 1.25 ,uM was
predigested for 3 min at 37°C with elastase to remove totally the GSTtag from
the fusion protein and to initiate aggregation of the HDQ51 protein. Imme-
diately afterthis step, various chemical compounds were added (final cone. 10
,uM) and samples were incubated for an additional 16 h at 37°C. Then,
aggregation reactions were filtered through a cellulose acetate membrane by
using a 384-well dot blot apparatus. Captured aggregates were detected by
immunoblotting using the HD1 antibody. On each filter membrane, 320
different chemical compounds were tested (squares A2-H11). Squares A1-H1
and A12-H12 were used for control samples. In square F8, an inhibitory
compound was identified. ThioS, thioflavine S.
16402 1 www.pnas.org/cgi/doi/10.1073/pnas.182426599
eight most effective compounds in the in vitro screen having ICso
values in the range of 1-11 ,uM are shown in Table 1.
The effect of increasing concentrations of the compounds
PGL-001, PGL-005, and PGL-034 on HD51 aggregation as
monitored by the filter retardation assay is shown in Fig.2A and
B. PGL-001, PGL-005, and PGL-034 inhibited HD51 aggrega-
tion with IC50 values of 1.2, 8.2, and 2.2 ,uM, respectively,
whereas the solvent DMSO alone was ineffective. The inhibitory
effect of the benzothiazole derivatives PGL-001 and PGL-034 on
HD51 aggregation in vitro was also confirmed by electron
microscopy (Fig. 3A). After an 18-h incubation of trypsin-
digested GST-HD51 fusion protein (final cone. 100 ng/,ul)
without added chemical compound, numerous clusters of high-
molecular-weight fibrils with a ribbon-like morphology (diame-
ter, 20-40 rim) were detected. In strong contrast, treatment of
the trypsin-digested GST-HD51 protein with PGL-001 or PGL-
034 (each at 20 AM) resulted in the appearance of only a few
fibrils with a diameter of 6-8 nm. In agreement with previous
observations (7), electron microscopy of undigested GST-HD51
protein (negative control) predominantly showed spherical par-
ticles with a diameter of 6-7 nm.
Recently, we have shown that formation of insoluble HD exon
1 protein aggregates in vitro is a nucleation-dependent process
(32) and that addition of chemical compounds such as Congo red
or thioflavine S to the aggregation reaction significantly delays
the assembly of monomeric HD exon 1 protein into high-
molecular-weight fibrillar structures (17~. To test whether the
benzothiazole derivatives PGL-001 and PGL-034 have a similar
effect on HD51 protein aggregation, a time course experiment
was performed in which the decrease of soluble HD51 protein
in the presence or absence of chemical compounds was moni-
tored by SDS/PAGE and immunoblotting (Fig. 3B). After 24 h
of incubation of trypsin-digested GST-HD51 protein, SDS-
soluble HD51 protein was detected only in the presence of
PGL-001 and PGL-034, indicating that these compounds have
delayed the assembly of monomeric HD51 into insoluble protein
aggregates. The soluble HD51 protein that resisted aggregate
formation was readily accessible to proteolytic degradation.
Thus, incubation for 30 min with proteinase K resulted in
complete degradation of HD51 protein, whereas the high-
molecular-weight polyQ-containing protein aggregates were
largely resistant to proteinase K treatment (Fig. 3B). The results
obtained by SDS/PAGE and immunoblotting were confirmed by
MALDI-TOF MS analyses using a GST-HD exon 1 fusion
protein, GST-HD517\P, that lacks the proline-rich region C-
terminal to the polyQ tract (25~. As shown in Fig. 3C, after
incubation for 24 h, soluble HD512\P protein was detected only
in samples that contained the compound PGL-001. In control
samples lacking an added chemical compound, HD512\P assem-
bled quantitatively into high-molecular-weight aggregates that
are not accessible to MALDI-TOF MS analysis (17~. Similar
results were obtained with PGL-005 and PGL-034 (data not
shown), indicating that benzothiazoles efficiently slow down the
assembly of HD exon 1 protein into pro/ease-resistant high-
molecular-weight aggregates in vitro.
Inhibition of HD Exon 1 Protein Aggregation in a Cell Culture Model
System of HD. To determine whether benzothiazoles are also
capable of inhibiting HD exon 1 protein aggregation in viva, a
cell-based 96-well microtiter plate huntingtin aggregation assay
was developed. In this assay, the tetracycline (tet)-regulated
expression system (33) was used for the synthesis of HDQ51 in
293 Tet-Off cells (11~. Cultivation of cells for 48-72 h in the
absence of doxycycline induces the expression of HDQ51, re-
sulting in the formation of large perinuclear inclusion bodies that
mainly consist of fibrillar HDQ51 protein (11~. The HDQ51
fibrils formed in 293 Tet-Off cells have a diameter of ~10 nm
(data not shown).
Heiser et a/.
OCR for page 27
Table 1. Benzothiazoles inhibit HD exon 1 aggregation in vitro
PG L-01 6
PG L-31 7
PG L-005
PG L-072
PG L-077
PG L-001
HAN
NH2
o
CN>¢ ~
NH2
art/
>¢N
AH
HO Br
~ O
IC50 values shown are the average of four independent determinations (+SE).
Induced 293 Tet-Off cells expressing HDQ51 were grown for
72 h in the presence or absence of benzothiazole derivatives.
Then, the cells were lysed, and the protein concentration in the
A AM 0.2 0.4 0.8 1.5 3.1 6.2 12.5 25 50 100
r T .
PGL-034 . ~ . ~ . ~ ~ ·~ 0 ~ , ~ ~,.~ HI . ~
PG L-005 . ~ . ~ . ~ . ~ . ~ .. O . ~ ..
— ~ }~.~ ~ Hi . ~,
PGL-001 . ~ *. ~ . ~ . ~^ . . ~ . .
~ . _._ _ ~~ ~ ~._ :
. . ., · .. · . ~ . O . · . · . · . · . · . ·
;. in. ~ _~ ~~ _ ~ ~ .. _ ~
fool
.~
a) 7s
Cal
._
.= 50
.>
~= 25
o
0 1
solvent
PGL-005
PGL-034
· PGL-001
10 100 1000
concentration (pM)
Fig. 2. In vitro inhibition of HD exon 1 aggregation by benzothiazoles. (A)
Effect of the indicated chemical compounds on HD exon 1 aggregation as
monitored bythe filter retardation assay. (B) Quantification of the dot blot results
shown in A. The signal intensity from the sample without added chemical com-
pound (solvent) was arbitrarily set as 100. For structure of the chemical com-
pounds, see Table 1.
Heiser et al.
HTS no. Structure Name IC50, AM
PG L-374 H2N - \=NH2 Benzothiazole-2,5,6-triamine 3 5 + 1.2
N NH2
PG L-0 34 H Nl~SJi`NH2 [ 6, 6 ] B i be n zoth i a zo l y l -2, 2 -d i a m i n e 2.2 + 0. 2
H2N - \ ~ (CS/~NH2 6-(2-Amino-benzothiazol-6-ylsulfanyl)-benzothiazol-2-ylamine 7.5 + 2.0
2-[3-(6-Am ino-benzothiazol-2-yl)-phenyl]-benzothiazol-6-ylamine 2.5 + 1 .6
3-Amino-N-[4-(6-methyl-benzothiazol-2-yl)-phenyl]-benzamide 8.2 + 0.5
3-Methoxy-N-[4-(6-methyl-benzothiazol-2-yl)-phenyl]-benzamide 9.3 + 7.2
4-[(4-BenzOthiazol-2-yl-phenylimino)-methyl]-2,6-dibromo-benzene-1,3-diol 1 1.0 + 4.5
N-(6-Phenylcarbamoyl-benzothiazol-2-yl)-terephthalamic acid methyl ester 1.2 + 0.5
cell extracts was determined. Because the protein concentration
largely correlates with the cell density, a reduction of protein
concentration by the addition of a given chemical compound
indicates that the compound is toxic for 293 Tet-Off cells. For the
detection of SDS-insoluble protein aggregates, protein extracts
were filtered through a cellulose acetate membrane, and the
amount of captured aggregates was quantified by immunoblot-
ting and image analysis.
We tested more than 100 benzothiazole derivatives, including
those identified by the high-throughput in vitro screen (Table 1),
as well as several structural analogues obtained by computer
analysis. Unexpectedly, we found that the most potent com-
pounds in the cell-free assays- such as PGL-001, PGL-005,
PGL-016, and PGL-034 (Figs. 2 and 3; Table 1) were toxic for
293 Tet-Off cells (data not shown). Therefore, their activity on
HDQ51 aggregation cannot be evaluated in this cell model
system of HD. A similar result was also obtained, when the
FDA-approved compound riluzole was tested in the cell-based
assay (Fig. 4A). Increasing concentrations of riluzole markedly
reduced the protein concentration, suggesting that the effect of
this compound on HDQ51 aggregation is due to toxicity, but not
due to an inhibition of this process (Fig. 4 B and C). In strong
contrast, the benzothiazole derivatives PGL-135,PGL-137, and
PGL-201 identified by computer analysis because of structural
homology to the compounds shown in Table 1 were nontoxic for
293 Tet-Off cells (Fig. 4~. We found that the compounds
PGL-135 and PGL-137, but not PGL-201, inhibited HDQ51
aggregation in the cell-based assay. As shown in Fig. 4B,
treatment of 293 Tet-Off cells with increasing concentrations of
PGL-135 inhibited HDQ51 aggregation in a dose-dependent
manner (ICso value ~40 EM). In comparison, PGL-137 sup-
pressed HD exon 1 aggregation with an IC50 value of ~100 ,uM
PNAS | December 10, 2002 | vol. 99 | supple. 4 | 16403
OCR for page 28
DMSO PGL-034 PGL-001
+1 + 1'-+-- +1 +
1 + ~ I -T~+~
100
50
o
50
I* COntrOI
1''''1''''1''''1'lllIlllll'lllIll'' 0
~ lo, ~
O O O m/Z
O O O
O O O
O O O
A
COm OUnd
P
Tr~DSIn
,.
Proteinase K
Aggregates
HD51
monomer
HD5 1/\P
.
. ~ PG L-001
IlililililIiIIIIIIllIlill
Cal
0 0 0 m/z
O O O
O O O
O O O
Fig. 3. Analysis of HD exon 1 aggregation in vitro by using secondary assays.
(A) Electron micrographs of HD51 fibrils formed in the presence or absence of
the indicated benzothiazoles. Trypsin-digested GST-HD51 protein at 1.5 ,uM
was incubated for 16 h at 37°C either without chemical compound (post
control) or with PGL-001 and PG L-034 (final cone. 20 ,uM). Representative
examples of fibrillar structures are shown. (B) Western blot analysis of trypsin-
digested GST-HD51 fusion protein. Aggregation reactions were performed for
16 h in the presence or absence of the indicated chemical compounds. Where
indicated (+), aliquots were taken and incubated for additional 30 min with
10 ng/~l proteinase K. Samples corresponding to 200 ng of fusion protein
were analyzed by SDS/PAGE and immunoblotting using the HD1 antibody. (C)
Effect of the compound PG L-001 on HD exon 1 aggregation as monitored by
MALDI-TOF MS. GST-HD51AP at 2.5 ,uM was incubated for 18 h at 37°C with
trypsin in the absence (control) or presence of PG L-001 (final cone. 20 ,uM).
Analysis by MALDI-TOF MS revealed that, only in the presence of PGL-001,
monomeric HD51 ~\P peptide (monoisotopic mass of 8,074.74 Da) is detectable;
without added compound, the peptide has assembled into insoluble
high-molecular-weight protein aggregates that cannot be detected by
MALDI-TOF MS.
(data not shown). Together, these results indicate that the
benzothiazole derivatives PGL-135 and PGL-137 are nontoxic
for 293 Tet-Off cells and inhibit HDQ51 aggregation in vivo.
The effect of PGL-135 on the formation of HDQ51 aggregates
16404 1 www.pnas.org/cgi/doi/10.1073/pnas.182426599
in 293 Tet-Off cells was also examined by indirect immunoflu-
orescence microscopy (Fig. 5A). Treatment of cells with PGL-
135 significantly reduced the formation of large perinuclear
inclusion bodies in a dose-dependent manner. Cultivation of the
cells with 25 and 50 ,uM PGL-135 resulted in a 30 and 50%
reduction of the average number of inclusion bodies per cell,
respectively, consistent with the results obtained by the filter
retardation assay (Fig. 4B).
Discussion
In this study, we have developed cell-free and cell-based assays
for the identification of chemical compounds that prevent the
formation of SDS-insoluble HD exon 1 aggregates. Using a
high-throughput in vitro filter retardation assay, we have iden-
tified about 300 chemical compounds in a library of ~184,000
small molecules that suppress the accumulation of polyQ-
containing huntingtin aggregates in a dose-dependent manner.
Among these compounds, five larger groups with 5-35 structur-
ally related small molecules were found. In this study, we have
concentrated on the analysis of one group of small molecules
containing 25 different benzothiazole derivatives.
Benzothiazoles are highly interesting molecules for drug de-
velopment, because they already have been shown to be useful
for treating cerebrovascular and neurodegenerative disorders.
For example, riluzole extends the survival of neurons in amyo-
trophic lateral sclerosis patients (28, 29~. It also has been tested
for therapy of HD patients, where treatment with riluzole has
positive effects on choreatic hyperkinesia (30~. Moreover, it has
been reported that riluzole leads to an extended lifespan in a
mouse model of HD (31~. Immunohistochemistry revealed
profound changes in ubiquitination of HD-characteristic neuro-
nal intranuclear inclusions, but no significant size reduction of
hunting/in-positive inclusions was observed, indicating that this
molecule does not directly interfere with huntingtin aggregation
(31~.
Our data show that benzothiazole derivatives are potent
inhibitors of HD exon 1 aggregation in vitro and in cell culture
model systems of HD. However, the mechanism of action of
these molecules on a molecular level is unclear. We have shown
previously that polyQ-containing HD exon 1 aggregates are
formed by a nucleation-dependent polymerization (32~. Thus,
aggregation does not proceed immediately but after a lag phase
during which monomers slowly assemble into unstable oligo-
meric structures (nuclei). Once these structures have formed, the
addition of further monomers becomes thermodynamically fa-
vorable, resulting in the rapid accumulation of large fibrils (4~.
Currently, the structure of the huntingtin oligomers and fibrils is
unknown; however, it is likely that both structures consist of
cross ,B-sheets (4, 32~.
We propose that benzothiazoles slow down HD exon 1 ag-
gregation because they bind to polyQ-containing [3-sheet struc-
tures. Similar to Congo red, they may interfere with nucleus
formation and/or fibril growth. However, detailed in vitro
drug-protein binding studies will be necessary to address these
questions in more detail. Currently, it is not known whether
benzothiazole derivatives such as PGL-001 preferentially inter-
act with monomers, oligomers, or fibrillar structures. We have
found that PGL-001 inhibits the aggregation of HDQ51 protein
(cone. 0.625 ,uM) with an ICso of 1.2 ,uM, suggesting that a
protein-inhibitor stoichiometry of 1:2 is necessary and sufficient
to inhibit HD exon 1 aggregation in vitro.
The activity of benzothiazoles to prevent huntingtin aggrega-
tion was also assessed in a cell culture model of HD. We found
that, at cone. of 10-150 ,uM, the most potent compounds in the
cell-free assays such as PGL-001 or PGL-034 as well as the
FDA-approved drug riluzole (Fig. 4), are toxic for 293 Tet-Off
cells. Thus, the impact of these compounds on HD exon 1
aggregation in vivo cannot be determined. In comparison, the
Heiser et a/.
OCR for page 29
A
HO`¢ it ~ - NH2
PGL-135 PGL-1 37
HO`¢:CS~ <
2-amino- 4,7-dimethyl- 2-dimethylamino-
benzothiazol-6-ol benzothiazol-6-ol
PGL-201 F Riluzole
IF
Fed
/ - NH2
6-methyl-benzothiazol-
2-ylamine
B 120 1
-
° 100
8
o
-
~ 60
SO
5? 40
~ 20
C 120 j
2 100 1
o
o
. _
Q
=
8
io
o
80
O ;
80
Sol
40 -
20 ~
1:C ,,~NH2
2-amino-6-trifluoro-
methoxybenzothiazole
~~~
PGL-201 ~ ~;
PGL-135
Riluzole
_ , .
10 100
Compound (,uM)
~~_~:
PGL-201 ~
PGL-135 ~ 4,,~f:
Riluzole
v 1
Compound (,uM)
D ~ ~1
6 12 25 50 75 4.5 9 18 24 36 ,uM
___ ~
Fig. 4. Inhibition of HD exon 1 aggregation in 293 Tet-Off cells. (A) Structure of
chemical compounds counteracting HDQ51 aggregation in vivo. (B) Quantifica-
tion of filter retardation assay results. Cells were incubated for 72 h in the
presence of various concentrations of the indicated chemical compounds. Protein
extracts were prepared and filtered through a cellulose acetate membrane;
captured SDS-insoluble protein aggregates were detected with the HD1 anti-
body. The dots corresponding to the control reactions without added chemical
compound (not shown) were arbitrarily set as 100. (C) Relative protein concen-
trations of the cell extracts analyzed in B. (D) Western blot of cell extracts
preparedfrom293Tet-Offcellsaftertreatmentwith increasing concentrationsof
the chemical compound PGL-135 and the solvent DMSO. The arrow indicates the
HDQ51 monomer.
Heiser et a/.
A
B 20
B_`
- o
of
-
~n 15
o
._
c.) 10
a_
a_
._
5
-
a)
Cal
Or
T
it
.
control 25 ,uM 50 AM
(DMSO) PGL-135 PGL-135
Fig. 5. Immunofluorescence microscopy analysis. (A) 293 Tet-Off cells ex-
pressing Flag-tagged HD exon 1 protein with 51 glutamines (HDQ51) were
cultivated for 72 h in the presence or absence of PGL-135. Formation of
inclusion bodies with aggregated HDQ51 protein was followed by indirect
immunofluorescence microscopy using the anti-Flag antibody. Inclusion bod-
ies are indicated as red dots by the arrow. Nuclei were counterstained with
Hoechst. A total of about 5,000 PGL-135-treated and untreated cells were
examined. (B) Quantification of cells with inclusion bodies.
structurally related benzothiazole derivatives PGL-135 and
PGL-137, identified by computer analysis, were nontoxic in the
cell-based assay under the same conditions. We suggest that the
compounds PGL-135 and PGL-137, similar to the most active
compounds in the cell-free assay, directly interact with mutant
HD exon 1 protein and thereby slow down the formation of
insoluble protein aggregates in viva. However, at this stage, we
cannot exclude an indirect mode of action in the cell culture
model of HD. For example, misfolding and aggregation of
polyQ-containing HD exon 1 protein could be prevented by
stimulation of a heat shock response or activation of the ubiq-
uitin/proteasome system (11, 15, 16~. Experiments to address
these questions are needed.
Our studies show that the automated filter retardation assay
described above is suitable for high-throughput screening of
PNAS | December to, 2002 | vow. 99 | suppl. 4 | 16405
OCR for page 30
chemical compounds that prevent aggregate formation. Using
this cell-free assay, very large compound libraries can be
screened because the system is fast, robust, and relatively cheap.
Furthermore, the amount needed of each substance for the
aggregation reaction is small compared with other in vitro
aggregation drug screening assays (344. Another strength of this
assay is its ability to detect not only compounds that inhibit fibril
growth (35), but also compounds that prevent nucleation.
A basic problem with drug screenings is that they tend to yield
different results in cell-free, as opposed to cell-based, assays.
Many compounds that show beneficial activities in the cell-free
experiments are inactive or toxic in cell culture. We, e.g., found
that benzothiazole derivatives that showed the greatest inhibi-
tory effect in vitro were toxic in the cell assays. This finding
means that the first step in screening can be to some extent
misleading and that only the combination of cell-free and
cell-based assays will result in the successful identification of
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16406 1 www.pnas.org/cgi/doi/10.1 073/pnas.182426599
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in the patient's neurons.
We have described a previously uncharacterized class of
chemical compounds that prevent the formation of HD exon 1
aggregates in vitro and are biologically active in cell cultures.
Now, the inhibitory activity of these molecules will be demon-
strated in transgenic mouse models of HD and ultimately in
humans.
We thank K. Genser, A. Droge, S. Schnogl, and E. Scherzinger for critical
reading of the manuscript. This work has been supported by grants from
the Deutsche Forschungsgemeinschaft (WA1151/1-2 and WA1151/2-
1), the Huntington's Disease Society of America, the Human Frontier
Science Program Organization, and the Bundesministerium fur Bildung,
Wissenschaft, Forschung und Technologie (BioFuture Project 0311853~.
24
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Heiser et a/.
Representative terms from entire chapter:
retardation assay
