Discovery of Small Molecules that Induce the Degradation of Huntingtin

Article abstract of DOI:10.1002/anie.201706529

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt), and removal of toxic mHtt is expected to be an effective therapeutic approach. We designed two small hybrid molecules (1 and 2) by linking a ligand for ubiquitin ligase (cellular inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt aggregates, anticipating that these compounds would recruit cIAP1 to mHtt and induce selective degradation by the ubiquitin-proteasome system. The synthesized compounds reduced mHtt levels in HD patient fibroblasts and appear to be promising candidates for the development of a treatment for HD.

Full text of DOI:10.1002/anie.201706529

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Accepted Article
Title: Discovery of Small Molecules that Induce Degradation of
Huntingtin
Authors: Shusuke Tomoshige, Sayaka Nomura, Kenji Ohgane, Yuichi
Hashimoto, and Minoru Ishikawa
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201706529
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http://dx.doi.org/10.1002/ange.201706529

Angewandte Chemie International Edition
10.1002/anie.201706529
COMMUNICATION
Discovery of Small Molecules that Induce Degradation of
Huntingtin
[
a][b]
[a][b]
[a]
[a]
Shusuke Tomoshige,
Sayaka Nomura,
Kenji Ohgane, Yuichi Hashimoto, and Minoru
[
a]
Ishikawa *
a
Abstract: Huntington’s disease (HD) is an autosomal dominant
neurodegenerative disorder caused by aggregation of mutant
huntingtin (mHtt), and removal of toxic mHtt is expected to be an
effective therapeutic approach. We designed two small hybrid
molecules 1 and 2, by linking a ligand for ubiquitin ligase (cellular
inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt
aggregates, anticipating that these compounds would recruit cIAP1
to mHtt and induce selective degradation via the ubiquitin-
proteasome system. The synthesized compounds reduced mHtt
levels in HD patients’ fibroblasts, and appear to be promising
candidates for treatment of HD.
Ligand for
target protein
Ligand for
ubiquitin ligase
(E3)
Small molecule degradation inducers
Ub
Ub
Ub
target
proteinꢀ
degradation
proteasome
E3ꢀ
Ub
b
Neurodegenerative disorders, such as Alzheimer’s disease
Probe for
aggregates
Ligand for
cIAP1
(
AD), Parkinson’s disease (PD) and polyglutamine (polyQ)
Small molecule degradation inducers
targeting protein aggregates
diseases, are caused by aggregates of misfolded proteins,
which can be stained by fluorescent small molecules such as
thioflavin T (ThT) that specifically bind to the aggregates. Among
the nine polyQ diseases, Huntington’s disease (HD) is an
autosomal dominant neurodegenerative disorder caused by
expansion of the CAG repeat sequence in HTT exon 1 gene to
mHtt aggregates
Ub
Ub
Ub
Proteasome
>
35 repeats; the resulting gene encodes aggregation-prone
Ub
mutant huntingtin (mHtt) with an extended N-terminal polyQ
cIAP1ꢀ
Degradation
[
1]
tract. Due to its beta-sheet-rich structure, mHtt accumulates in
neuronal cells as intracellular aggregates. These aggregates,
including soluble oligomer intermediates, cause neuronal cell
death, resulting in various symptoms such as motor dysfunction,
O
OH
c
[
2]
S
N
H
N
that progress and lead to death within 15-20 years. Several
groups have reported various approaches to target the toxic
mHtt aggregates, including small molecules that inhibit or
O
N
H
N
Ph
2
H
O
NH2
1
[
3]
accelerate aggregation of mHtt
or induce a molecular
[
4]
chaperone that suppresses the aggregation, expression of 46-
[
5]
O
OH
mer peptides that induce autophagy in order to degrade mHtt,
H
N
O
[
6,7]
S
and gene silencing of mHtt.
Despite these efforts, no effective
N
3H
Ph
N
O
NH2
clinical treatment is yet available, because the modes of action
N
N
N
[
8]
of the chemical aggregation modulators remain unclear and
biological approaches have several limitations, including poor
metabolic stability, limited selectivity and low permeability of
2
O
[
9]
reagents.
We believe, therefore, that novel approaches
S
H
N
O
N
H
N
H
Ph
employing small molecules are required for effective treatment of
2
N
O
HD.
3
Figure 1. Small molecule-mediated protein-degradation technology and the
newly designed small hybrid molecules. (a) Proposed mechanisms.
Degradation inducers induce complex formation between target protein and
ubiquitin ligase (E3), followed by ubiquitination (Ub) of target protein, and
proteasomal degradation. (b) The concept of this study. Small hybrid
molecules consisting of a probe for aggregates linked to a ligand of cIAP1 are
expected to induce degradation of mHtt aggregates. (c) Structures of 1-3.
[a]
Dr. S. Tomoshige, Ms. S. Nomura, Dr. K. Ohgane, Prof. Y.
Hashimoto, and Dr. M. Ishikawa
Institute of Molecular and Cellular Biosciences, The University of
Tokyo
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
E-mail: m-ishikawa@iam.u-tokyo,ac.jp
[b]
These authors contributed equally to this work.
The concept of hybrid molecules with a dual mode of action is
the focus of the attention in small molecule drug discovery
Supporting information for this article is given via a link at the end
of the document.
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Angewandte Chemie International Edition
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COMMUNICATION
[
10]
programs. In recent years, a strategy has been developed for
degradation of target proteins based on the use of small hybrid
molecules composed of ligand for the target protein and a ligand
for ubiquitin ligase (E3); these hybrid molecules recruit E3 to the
target protein, leading to selective degradation via the ubiquitin-
of wtHtt as well as that of mHtt after transcription without
cytotoxicity (Figure S4). Considering reports that (1) purified
[
19]
wtHtt containing 32Q formed small aggregates in vitro, (2) the
[
20]
smallest polyQ peptide able to bind to ThT (6) is 6Q peptide,
and (3) ThT can bind to soluble oligomers of amyloid beta as
[
11,12]
[17]
proteasome system (UPS)
(Figure 1a). Based on our work
well,
we assume that wtHtt also aggregate slightly or form
[
12]
in this area,
we hypothesized that a small hybrid molecule
soluble oligomers and 1 and 2 may also interact with small
aggregates or soluble oligomers of wtHtt to induce their
degradation.
designed to bring mHtt and E3 into close proximity would induce
degradation of mHtt in living cells. Since no small-molecular
ligand for Htt has yet been discovered, we focused on the
aggregation-prone nature of mHtt and planned to utilize small-
molecule probes for targeting the protein aggregates (Figure
[
21]
In vitro competitive binding assay
by using aggregates of
[
22]
62Q peptides
indicates that 1 and 2 bind to turbid 62Q
aggregates (Figure S5). Next, we explored whether 1 and 2
induce non-physiological complex formation [aggregate-
compound-cIAP1] by means of two ELISA assays using
immobilized model insoluble aggregates of 62Q and GST-fused
1
b). For this purpose, we focused on BTA (4) and PDB (5)
[
13,14]
(
Figure S1), because of reports on brain penetration
and a
[
15,16]
linker introduction.
It is not clear whether these probes can
[
12a]
specifically bind to soluble oligomers as well as insoluble polyQ
aggregates, although Thioflavin T (ThT) (6, Figure S1) can bind
baculovirus IAP repeat 3 (GST-BIR3),
the bestatin analog-
binding site of cIAP1, and immobilized GST-BIR3 and soluble
GST-62Q. Small molecules induced interaction between cIAP1
and both insoluble 62Q aggregates and soluble GST-62Q that
was blocked in the presence of BTA or MeBS (Figures 3, S6 and
S7). It is known that GST-62Q has an amyloid-like
[
17]
to polyQ aggregates and soluble oligomers of amyloid beta.
As a cIAP1-recognizing moiety, we decided to conjugate bestatin
7, Figure S1) through an amide bond, because bestatin methyl
(
amide BE04 (8, Figure S1) is more selective for cIAP1 over
peptidases as compared with bestatin methyl ester MeBS (9,
Figure S1), and bestatin amide derivatives do not induce auto-
ubiquitination of cIAP1 at low concentration, unlike bestatin ester
[
3]
conformation,
and this result indicates that both small
molecules can induce interaction even between soluble
oligomers of mHtt and cIAP1.
[
18]
derivatives.
Hence, we designed and synthesized 1 and 2
consisting of cIAP1 ligand BE04 conjugated to BTA or PDB,
respectively (Figure 1c and Schemes S1 and S2).
a1.4"
1.2"
b
1"
.9"
.8"
.7"
We examined the effects of 1 and 2 on mHtt levels in two
fibroblasts derived from two patients with HD (HD fb). At the
concentration of 10 µM, 1 and 2 reduced the mHtt level in both
HD fb (Figures 2 and S2a), which suggests that 1 and 2 work
effectively as down-regulators of mHtt. Compound 1 reduced the
mHtt level in a dose dependent manner (Figure S2b)
0
0
0
1"
0
.6"
0.8"
0.5"
0.6"
0.4"
0.2"
0
.4"
.3"
1
0 µM 1 (h)
-
24
48
10 µM 2 (h)
-
24
48
0
IB:polyQ
0.2"
IB:polyQ
0.1"
0
"
0"
IB:Htt
IB:Htt
1
9
4
(µM) - 3 10 30 10 10 10 10 10 2 (µM) - 3 10 30 10 10
(µM) -
(µM) -
-
-
-
-
-
-
3 10 -
-
-
9 (µM) -
-
-
- 3 10
IB:tubulin
IB:tubulin
-
- 10 30100
Figure 2. Compounds 1 and 2 reduced the Htt level in HD fb (GM04281).
Western blot analyses stained with both anti-polyQ antibody and anti-Htt
antibody were performed.
Figure 3. Demonstration of (a) 1-induced and (b) 2-induced interaction
between immobilized 62Q aggregates and GST-BIR3 by ELISA. Experiments
were performed in triplicate. Gray bars indicate means of each data points,
and black cross marks indicate data points.
Although these fibroblast cells express both mHtt and wild
type Htt (wtHtt), we could hardly detect wtHtt with anti-Htt
antibody after treatment with either 1 or 2 (Figures 2 and S2).
Therefore, we investigated the activity of the compounds by
using fibroblasts derived from a normal subject (normal fb)
expressing only wtHtt. First, we confirmed that 1 and 2 reduced
the wtHtt level in normal fb (Figure S3a). Next, to confirm that 1
and 2 do not affect transcription of the HTT gene itself, we
analyzed the mRNA abundance of Htt in HD fb. qPCR analysis
showed that 1 and 2 did not affect mRNA abundance (Figure
S3b). Taken together, these results indicate that the transcription
of HTT gene is not targeted by 1 and 2, but they reduce the level
The hydroxy and amino groups of BE04 are crucial for binding
[
18]
to cIAP1
amino groups showed no degradation-inducing activity towards
and a derivative not containing the hydroxy and
[
12b]
the target protein.
Thus, we designed and synthesized 3
(Figure 1) as a negative control. Neither BTA alone, BE04 alone,
the combination of them, nor 3 reduced the mHtt level in HD fb
(Figure S8a). Next, the proteasome dependence of the
degradation was investigated. Pretreating HD fb with
proteasome inhibitors MG132 and bortezomib abrogated the 1-
mediated down-regulation of mHtt (Figure S8b). All these results
support our conclusion that 1 and
2 bind to both mHtt
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Angewandte Chemie International Edition
10.1002/anie.201706529
COMMUNICATION
aggregates and cIAP1, and thereby induce proteasomal
degradation of mHtt.
probe for protein aggregates, and we showed that both
compounds induced degradation of mHtt in living cells. In
particular, they were effective against mHtt in cells derived from
HD patients and mHtt with a much longer polyQ repeat
sequence (145Q). Analysis of the mode of action revealed that 1
and 2 induce complex formation between Htt aggregates and
cIAP1, and this leads to proteasomal degradation of mHtt.
Several groups have reported impairment of UPS in
neurodegenerative disorders, whereas there is increasing
evidence that UPS is still operative in neurodegenerative
The longer the polyQ repeat structure in mHtt, the earlier
symptoms appear in patients and the more severe they
[
23]
become,
considered to be more difficult. Hence, we investigated whether
could induce degradation of mHtt with a much longer polyQ
repeat, using the enhanced green fluorescent protein-tagged
and clinical treatment of HD with longer polyQ is
1
[
24]
exon 1 of Htt containing 145Q (145QHtt-EGFP). Compound 1
reduced the 145QHtt-EGFP level in Hela cells transiently
expressing 145QHtt-EGFP, but not the EGFP level in Hela cells
transiently expressing EGFP (Figure 4a). These hybrid
molecules did not decrease the levels of EGFP and tubulin and
did not affect cell viability (Figure S4), suggesting high selectivity
for Htt over unrelated proteins. In accordance with the results in
Figure S3a, the level of 23QHtt-EGFP (this repeat number does
not cause HD) was also reduced in Hela cells transiently
expressing 23QHtt-EGFP (Figure S9a). In contrast, neither BTA
alone, BE04 alone, the combination of them, nor 3 reduced the
[
25]
disorders. We showed that hijacking E3 ligase induced by the
small molecule would be effective therapeutic approach for
neurodegenerative diseases. We also showed the novel
methodology
for
hybrid
small
molecule-mediated
posttranslational knockdown of target proteins for which small-
molecular ligands for aggregation-prone proteins have not been
identified. The design concept of targeting the aggregated state
of proteins differs from previous approaches of posttranslational
knockdown using specific ligands to target proteins. We believe
that this methodology would be applicable to target other
aggregation-prone proteins, besides Htt, that cause
neurodegenerative diseases including AD, PD and other polyQ
diseases.
1
45QHtt-EGFP level (Figure S9b). Moreover, to exclude the
possibility of accelerated formation of insoluble aggregates of
mHtt, fluorescence-microscopy analysis was also performed.
Treatment with 1 significantly reduced the number of aggregates
of 145QHtt-EGFP (Figures 4b and S10). These results suggest
that 1 and 2 can be effective in reducing mHtt with much longer
polyQ repeats, and does not enhance the aggregation.
Acknowledgements
1
45QHtt-EGFP
The work described in this article was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of
Education, Culture, Sports, Science and Technology, Japan, and
the Japan Society for Promotion of Science. This work was also
supported financially by Platform for Drug Discovery, Informatics,
and Structural Life Science. We are grateful to Prof. Mikihiko
Naito for helpful discussions. The following cells/DNA samples
were obtained from the NIGMS Human Genetic Cell Repository
at the Coriell Institute for Medical Research: GM04281,
GM01187, GM07492 and CH00019.
a
EGFP
-
-
-
+
1
2
(10 µM)
(10 µM)
+
-
1
(µM)
-
1
3
10
IB : polyQ
IB:tubulin
IB : GFP
IB : tubulin
b
1
1
1
1
60"
40"
20"
00"
*
Keywords: Drug design, Huntington’s disease, Medicinal
chemistry, Neurodegenerative disorders, Small molecule-protein
degrader
8
6
4
2
0"
0"
0"
0"
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Small molecules for huntington’s
disease: Two chemical classes of
small molecules induced degradation
of pathogenic mutant huntingtin in
living cells.
Shusuke Tomoshige Sayaka Nomura,
Kenji Ohgane, Yuichi Hashimoto, and
Minoru Ishikawa*
Page No. – Page No.
Discovery of Small Molecules that
Induce Degradation of Huntingtin
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