Potent inhibitors of Huntingtin protein aggregation in a cell-based assay

Article abstract of DOI:10.1016/j.bmcl.2009.01.087

A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activ

Full text of DOI:10.1016/j.bmcl.2009.01.087

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1715–1717
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent inhibitors of Huntingtin protein aggregation in a cell-based assay
Alison Rinderspacher a, Maria Laura Cremona b, Yidong Liu a, Shi-Xian Deng a, Yuli Xie a, Gangli Gong a,
b
b
b
c
d
c
´
Nathalie Aulner , Udo Többen , Katherine Myers , Caty Chung , Monique Andersen , Dušica Vidovic ,
Stephan Schürer c, Lars Brandén b, Ai Yamamoto d, Donald W. Landry a,
*
a Division of Experimental Therapeutics, Department of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032, USA
b Genome Center, Columbia University, 1150 St. Nicholas Ave., New York, NY 10032, USA
c Scientific Computing, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458, USA
d Department of Neurology, Columbia University, 630 West 168th Street, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a
high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small
Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 December 2008
Revised 24 January 2009
Accepted 27 January 2009
Available online 30 January 2009
Keywords:
Huntington’s disease
Huntingtin protein
Polyglutamine aggregates
Inhibitors of huntingtin protein aggregation
High-throughput screen
Quinazoline
Huntington’s disease (HD), a member of the trinucleotide-re-
peat neurological disorders, is caused by a heritable, polygluta-
mine-expansion mutation in the NH3-terminus of the Huntingtin
protein (Htt).1–3 The intracellular accumulation of insoluble aggre-
gates of mutant Htt is a hallmark of HD, but the precise mechanism
of toxicity remains a matter of conjecture.4 In mouse models of HD,
expression of the NH3-terminal portion of the mutant Htt protein
recapitulates key features of the disease with mice displaying a
phenotype of progressive neurological impairment. Neurons in
these models5,6 show an intrinsic ability to clear polyQ aggregates,
and accompanying this clearance, a reversal of the HD-like neuro-
logic symptoms is observed. Macroautophagy, the lysosome-med-
iated degradation of cytosolic proteins, is implicated as the cellular
pathway responsible for aggregate clearance.7–10 Using a unique
two-tiered functional genetic screen, we previously found that
macroautophagy-mediated clearance of accumulated mutant pro-
tein can be increased through IRS-2 stimulation in a cell-based
model of HD.7 In order to search for small-molecule activators of
Htt clearance, we used this model in a high-throughput fluorescent
cell-based assay format to monitor the aggregation and clearance
of a tet-regulatable,11 conditionally expressed mutant protein con-
sisting of the first 17 NH3-terminal amino acids of Htt followed by
a polyQ stretch of 103 residues fused to a monomeric cyan fluores-
cent protein (mCFP).7 These stable transfectants do not manifest
the acute polyQ-length-dependent cell death observed after tran-
sient transfection7 and thus provided an excellent platform for
the identification of modulators of protein degradation which act
over a period of days.
The degree of aggregation in this assay is measured by the pres-
ence and magnitude of fluorescent intracellular inclusions. We
screened a 10K chemical-compound library, provided by the
NIH-sponsored MLSCN, for diminution of aggregation of mutant
protein after incubation for 72 h. We identified six hits: five tetra-
cycline derivatives, likely suppressing tet-regulatable gene expres-
sion, and quinazoline 1a (Fig. 1), with an IC50 of approximately
* Corresponding author. Tel.: +1 212 305 5838; fax: +1 212 305 3475.
E-mail address: dwl1@columbia.edu (D.W. Landry).
Figure 1. Structure analysis of our lead compound.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.087

1716
A. Rinderspacher et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1715–1717
Table 1
Table 2
Modifications of regions A and C: IC50 values of our piperazine analogs 1–4
Modifications of regions B and C: IC50 values of our secondary amine analogs 5–11
Compound
X
R
IC50, lM
Compound
X
R
IC50, lM
5a
5b
5c
5d
6a
6b
6c
6d
7
8
9
10
11
Br
Cl
F
Phenol
Phenol
Phenol
Phenol
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Phenoxyphenyl
4-Phenoxyphenyl
4-(Cyclopentyloxy)phenyl
4-(Benzyloxy)phenyl
Ethyl (4-phenoxy)acetate
1.11
0.71
6.38
>15.8
Inactive
3.44
1a
1b
1c
1d
2a
2b
2c
2d
3a
4a
4b
4c
4d
Br
Cl
F
H
Br
Cl
F
H
Br
Br
Cl
F
Ethyl
Ethyl
Ethyl
Ethyl
Methyl
Methyl
Methyl
Methyl
2.21
3.63
5.26
>10
1.31
>10
>10
>10
2.74
H
Br
Cl
F
Inactive
10.5
H
Cl
NH2
Cl
Cl
Cl
Inactive
Inactive
Inactive
Inactive
4.48
H
4-(1,3-Benzodioxol-5-ylmethyl)
4-(1,3-Benzodioxol-5-ylmethyl)
4-(1,3-Benzodioxol-5-ylmethyl)
4-(1,3-Benzodioxol-5-ylmethyl)
Inactive
1.07
>10
H
>10
Table 3
Quinazoline derivatives 12–14 of Sarkar et al.: IC50 values in our assay
Scheme 1. General procedure for the synthesis of our quinazoline compounds.
Compound
R
IC50, lM
12
13
14
Propyl
Allyl
Benzyl
8.13
5.36
6.13
4
lM. The structure of 1a was confirmed by synthesis and dose-
dependent activity was demonstrated with an IC50 of 2.2 M (Table
l
1). A structure–activity relationship (SAR) study was conducted to
identify more potent derivatives. Three regions of the quinazoline
were selected for modification: A, the distal piperazine N-alkyl
group; B, the piperazine ring itself; and C, the 6-bromo group on
the aromatic core. We envisioned that analogs would be easily syn-
thesized from common 4-chloro-6-halo-quinazoline intermediates
(Scheme 1).12 As this work proceeded, Sarkar et al., seeking
enhancers of autophagy, reported several bromo-substituted quin-
azolines13 and we synthesized three of their compounds, 12–14,
for comparison (Table 3).
Substitution at the N-ethyl group of quinazoline 1a with either
smaller or bulkier groups did not alter activity (Table 1). In con-
trast, substitution at the 6-position with fluorine or hydrogen
was not well tolerated compared to substitution with bromine or
chlorine. This pattern for substitution at the 6-position was also
observed in the series based on replacement of the piperazine ring
with 40-substituted phenylethylene amine. That is, the activity of
the 6-fluoro- and dehalogenated derivatives was generally lower
(Table 2). In general, the secondary amines of Table 2 were supe-
rior to the corresponding piperazines of Table 1. Quinazoline itself
is inactive in our assay and any substitutions of the quinazoline
core with other cores resulted in loss of activity. Substitution of
the amino group with an alkoxy group also resulted in loss of activ-
ity. The most potent inhibitor of accumulation of aggregated mu-
Sarkar compounds 12–14 were reported to have EC50 values of
approximately 50 M in a yeast assay. While no rank order was re-
l
ported for this assay, the potency of the compounds in PC12 cells
was 12 > 13 > 14 and in COS-7 cells was 14 > 13 > 12.13 In contrast,
we found low micromolar potency in our system with a rank order
of 12 > 14 > 13 (Table 3). It remains to be formally demonstrated
that our assay and that of Sarkar et al. are directed to the same
target.
The decrease in the number of aggregates per cell observed with
our compound may be due to the clearance of inclusions because
the number of inclusions after the administration of compounds
such as 5b is less than the number before treatment. Nonetheless,
other factors may be at play, such as enhanced cell division leading
to cytoplasmic dilution, and thus an apparent decrease in the num-
ber of inclusions, or enhanced cell death leading to a preferential
loss of aggregate-containing cells. To eliminate these possibilities,
we examined cell numbers and cell death after administration of
compound 5b. As shown in Figure 2, despite the clearance of inclu-
sions, neither signs of cytotoxicity nor differences in the total num-
ber of cells were observed.
In summary, we prepared and identified four potent inhibitors
of Huntingtin protein aggregation, 5b, 4b, 5a, and 2a. Our library
of analogs provides a starting point for mechanistic studies to
determine the target of action.
tant Htt fragment was compound 5b with an IC50 of 0.71 lM.
Alkylation of the 40-hydroxyl group of compound 5b was not toler-
ated, cf compounds 7–10.

A. Rinderspacher et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1715–1717
1717
Figure 2. Compound 5b leads to a dose-dependent decrease in the number of aggregates per cell in the absence of increased proliferation or cell death. Cells were seeded in a
96-well format and treated with 2 g/mL doxycycline (Dox), 0.1% DMSO (No Dox) as control, or compound 5b. (A) Percent (%) cells with aggregates. (i) The overall effect of 5b
l
on % cells with aggregates with respect to Dox and No Dox. ANOVA reveals a statistically significant effect of ‘Treatment’ on ‘% Cells with aggregates’ (F(2,37) = 14.486,
p < 0.001. *p < 0.05 in Fisher Post hoc analysis when compared to No Dox). (ii) 5b exerts a dose-dependent decrease of aggregates. (B) % Dead cells. There is no effect on cell
death of 5b as compared to Dox or No Dox (i) or across dose (ii). (F(2,37) = 0.346; p = 0.7095). (C) Number of cells per well. There is no effect on the total cell number of 5b as
compared to Dox or No Dox (i) or across dose (ii). (F(2,37) = 2.225; p = 0.1224).
9. Iwata, A.; Christianson, J. C.; Bucci, M.; Ellerby, L. M.; Nukina, N.; Forno, L. S.;
Acknowledgments
Kopito, R. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13135.
10. Filimonenko, M.; Stuffers, S.; Raiborg, C.; Yamamoto, A.; Malerød, L.; Fisher, E.
The authors would like to acknowledge Teresa Wojtasiewicz
for technical support and would like to thank the NIH’s Molecular
Libraries Initiative for funding our research.
M. C.; Isaacs, A.; Brech, A.; Stenmark, H.; Simonsen, A. J. Cell Biol. 2007, 179, 485.
11. Gossen, M.; Bujard, H. Annu. Rev. Genet. 2002, 36, 153.
12. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury 300 T NMR
spectrometer. High-resolution mass spectrometry was done by Dr. Yasuhiro
Itagaki at Columbia University. 4,6-Dichloroquinazoline (I, X = Cl) was stirred
at room temperature in
a 4:1 mixture of ethanol (4.0 mL) and pyridine
References and notes
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