A chemical chaperone-based drug candidate is effective in a mouse model of amyotrophic lateral sclerosis (ALS)

Article abstract of DOI:10.1002/cmdc.201500045

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg^-1 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.

Full text of DOI:10.1002/cmdc.201500045

DOI: 10.1002/cmdc.201500045
Full Papers
A Chemical Chaperone-Based Drug Candidate is Effective
in a Mouse Model of Amyotrophic Lateral Sclerosis (ALS)
[
a]
[a]
[b]
[b]
[c]
Tamar Getter, Ilana Zaks, Yael Barhum, Tali Ben-Zur, Sebastian Bçselt,
[d]
[a]
[e]
[a]
[a]
Simpson Gregoire, Olga Viskind, Tom Shani, Hugo Gottlieb, Omer Green,
Moran Shubely, Hanoch Senderowitz, Adrian Israelson, Inchan Kwon,
Susanne Petri, Daniel Offen, and Arie Gruzman*
[a]
[a]
[e]
[d, f]
[c]
[b]
[a]
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative
disease characterized by the selective death of motor neurons
and skeletal muscle atrophy. The majority of ALS cases are ac-
quired spontaneously, with inherited disease accounting for
only 10% of all cases. Recent studies provide compelling evi-
dence that aggregates of misfolded proteins underlie both
types of ALS. Small molecules such as artificial chaperones can
prevent or even reverse the aggregation of proteins associated
with various human diseases. However, their very high active
concentration (micromolar range) severely limits their utility as
drugs. We synthesized several ester and amide derivatives of
chemical chaperones. The lead compound 14, 3-((5-((4,6-dime-
thylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethyl-
propan-1-amine oxide shows, in the micromolar concentration
range, both neuronal and astrocyte protective effects in vitro;
À1
at daily doses of 10 mgkg 14 improved the neurological
functions and delayed body weight loss in ALS mice. Members
of this new chemical chaperone derivative class are strong can-
didates for the development of new drugs for ALS patients.
Introduction
Amyotrophic lateral sclerosis (ALS) is the most common motor
neuron disease in humans, affecting people at any time during
adulthood, although the average age of onset begins in the
supporting the idea that perhaps ALS is a multifactorial disease
with both environmental and genetic components. Fewer than
10% of ALS cases have a familial history (familial ALS, fALS)
[
1]
[2]
mid-fifties. Patients usually die within 3–5 years of symptoms
and are linked to certain mutations in various genes. One of
[
1]
onset, usually due to respiratory failure. The only available
FDA-approved drug, riluzole, offers only a marginal delay in
the most common mutations found in fALS is a mutation in
[2]
the gene for superoxide dismutase 1 (SOD1). More than 120
[
1]
[2]
disease progression. About 90% of ALS patients do not have
any genetic abnormalities (sporadic form of the disease, sALS),
mutations in SOD1 are linked to the disease. Interestingly,
SOD1 encodes an enzyme that plays an essential role in
human physiology as a scavenger of superoxide radicals. How-
ever, not all of the ALS-linked SOD1 mutants are physiological-
+
[
a] T. Getter, I. Zaks, O. Viskind, Dr. H. Gottlieb, O. Green, M. Shubely,
[3]
ly inactive. Moreover, SOD1 knockout mice do not develop
Prof. H. Senderowitz, Dr. A. Gruzman
Department of Chemistry, Faculty of Exact Sciences
Bar-Ilan University, Ramat Gan, 529002 (Israel)
E-mail: gruzmaa@biu.ac.il
ALS, while the expression of mutant SOD1 causes the dis-
[3]
ease. The large number of ALS-causing SOD1 mutations sug-
gests that almost any alteration in the structure of SOD1 will
be cytotoxic, leading specifically to ALS and not to other dis-
eases. All other fALS-related mutations are not ALS specific.
Mutations in those genes are associated with other neurologi-
cal disorders as well, in particular with frontotemporal demen-
[
b] Y. Barhum, Dr. T. Ben-Zur, Prof. D. Offen
Felsenstien Medical Research Center, Sackler Faculty of Medicine
Tel Aviv University, Petah-Tikva, 49100 (Israel)
[
c] Dr. S. Bçselt, Prof. S. Petri
Department of Neurology, Hannover Medical School
Hannover, 30625 (Germany)
[4]
tia. SOD1 is the only gene, that when mutated in almost all
[
d] Dr. S. Gregoire, Prof. I. Kwon
possible positions, leads to ALS. The disease pathology and
symptoms in SOD1-linked fALS patients closely resemble those
of sALS patients. In light of the linkage of the SOD1 gene to
fALS, it was hypothesized that there may be a common molec-
Department of Chemical Engineering, University of Virginia
P.O. Box 400071, Charlottesville, VA 22904-4741 (USA)
[
e] T. Shani, Dr. A. Israelson
Department of Physiology & Cell Biology, Faculty of Health Sciences
Ben-Gurion University of the Negev, Beer-Sheva, 84105 (Israel)
[5]
ular basis for both forms of the disease. There are many re-
WT
[
f] Prof. I. Kwon
ports that oxidized/misfolded wild-type SOD1 ( SOD1) indu-
School of Materials Science & Engineering
Gwangju Institute of Science and Technology (GIST)
Gwangju, 500-712 (Republic of Korea)
[6]
ces the selective death of motor neurons. Accordingly, several
studies have investigated the possible role of structural
changes in mutated SOD1 that cause toxic effects in motor
+
[
] Present address: Prolor Biotech, Nes-Ziona (Israel)
[7]
neurons. Indeed, several common features of nearly all SOD1
mutants associated with ALS have been found, in particular,
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500045.
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[
8]
the increased formation of aggregates. It has also been
shown that different factors, among them oxidative stress, lead
to alterations in appropriate folding of SOD1 in motor neurons
2
+
2+
following the loss of Cu and Zn ions, accelerating aggrega-
[
9]
tion of the protein. Indirect evidence supports the idea that
WT
toxic forms of oxidized/misfolded SOD1 are secreted from
2 2
Scheme 1. Reagents and conditions: a) 3% aq. H O , 72 h, RT.
glia cells into the extracellular space, leading to motor neuron
[
10]
death.
Artificial chaperones including polyols, trimethylamino N-
oxide (TMAO), tauroursodeoxycholic acid, taurine, 4-phenylbu-
tyric acid, and various amino acid derivatives have been shown
to reverse the mislocalization and aggregation of proteins as-
using the Boekelheide rearrangement. The ester was hydro-
lyzed in the presence of sodium methoxide in methanol to
obtain the corresponding primary alcohols 7 and 8. Glutaric
anhydride was used to form the cleavable linker between the
lipophilic pyridine-based moiety and the chemical chaperones.
After esterification of 7 and 8, the free carboxylic acid was in-
troduced to obtain 9 and 10. Compound 9 was conjugated in
the presence of the coupling reagent PyBOP to the chemical
[
11]
sociated with different human diseases. The mechanisms by
which chemical chaperones function are not fully understood.
The major limitation in using chemical chaperones as drugs is
their very high active concentration, in the millimolar range.
For example, the active concentrations of TMAO needed for
chaperone-like effects in cell cultures is in the range of
chaperone,
3-hydroxy-N,N-dimethylpropan-1-amine
oxide
[
12]
1
.0 mm. Tauroursodeoxycholic acid shows an effect on ag-
gregation of misfolded proteins at a concentration range of
.2–1 mm in vitro and at a more acceptable dose in vivo,
500 mg per day p.o. (the compound is used therapeutically
(Scheme 2) to obtain the ester derivative 11. The identical reac-
tion between 9 and 3-amino-N,N-dimethylpropan-1-amine
oxide resulted in the amide derivative 12. Finally, 9 was conju-
gated in a similar way with taurine to obtain the correspond-
ing amide 13. Subsequently, 10 was converted using the same
synthetic approach into the ester derivative of 3-hydroxy-N,N-
dimethylproban-1-amine oxide (14) or into the amide deriva-
tive of 3-amino-N,N-dimethylproban-1-amine oxide (15). In ad-
dition, three imidazole-based compounds of TMAO and 4-phe-
nylbutyric acid (methyl ether) were synthetized. Briefly, com-
mercially available 1-(3-aminopropyl)imidazole was conjugated
with glutaric anhydride, leading to the formation of an ester
derivative of 3-hydroxy-N,N-dimethylpropan-1-amine oxide to
obtain 17. The 1-(3-aminopropyl)imidazole was also directly
conjugated with carboxylic acid derivative of TMAO: 3-car-
boxy-N,N-dimethylproban-1-amine oxide to obtain the corre-
sponding amide 18. The latter compound was also synthesized
via a direct conjugation between the 1-(3-aminopropyl)imida-
zole and 4-(4-methoxyphenyl)butyric acid. The synthesis of the
corresponding amide 19 was successfully accomplished in
a similar manner.
0
~
[
13]
for the treatment of cholestatic liver diseases). 4-Phenylbuty-
ric acid is active in vitro at concentrations of 0.5–5 mm and in
[
14]
vivo at daily doses of 0.5–20 g. In the search for small mole-
cules that can prevent the formation of toxic aggregates at
concentrations lower than those required by available com-
pounds, we decided to prepare more lipophilic, yet still water-
soluble derivatives of known chemical chaperones. In this way
we planned to decrease their active concentrations and allow
them to serve as ALS drug candidates.
Results and Discussion
TMAO and 4-methoxyphenylbutyric acid have poor membrane
[
15,16]
permeability.
Because good blood–brain barrier (BBB) per-
meation is mandatory for any compound in the treatment of
ALS, the conjugation of these chemical chaperones to mem-
brane-permeating moieties such as aromatic amines (methyl-
[
15–17]
pyridines and imidazole) was attempted.
We used a methy-
The resulting compounds were screened in the ALS in vitro
hSOD1G93A
[21]
lated derivative of 4-phenylbutyric acid (a known chemical
chaperone) to avoid the effect of the negatively charged
phenol moiety on membrane penetration, hypothesizing that
within the cell, the methyl ether will be transformed back to
the phenolic form by the well-known metabolic oxidative O-
model,
NSC-34 motor neuron-like cells. This model
was established based on experimental data obtained from
human SOD1/G93A transgenic mice that become paralyzed
[22]
due to motor neuron loss and die at the age of 5–6 months.
ALS-related pathophysiological properties including increased
reactive oxygen species formation, abnormal mitochondrial
membrane potential, increased vulnerability to toxins/stress
conditions, and even the formation of insoluble protein aggre-
gates that contain mutated SOD1 were detected in these cells,
[
18–20]
dealkylation reaction.
Amide and ester bonds were chosen
for the formation of linkers between chemical chaperones and
aromatic amine moieties.
Precursor compounds S-1–S-3 were synthesized as outlined
in Scheme 1. The tertiary amines in three starting molecules
were oxidized with a 3% hydrogen peroxide solution. Alto-
gether, eight different derivatives of TMAO, taurine, and 4-phe-
nylbutyric acid were synthesized as shown in Scheme 2. Com-
mercially available 2,3,5-trimethylpyridine (1) and 2,4,6-trime-
thylpyridine (2) were oxidized by hydrogen peroxide into the
corresponding N-oxides 3 and 4 followed by conversion of the
methyl group at the second position into acetyl esters 5 and 6,
[23]
making them a suitable model for in vitro ALS studies. The
potential cytoprotective effect of the tested compounds on
the viability of these cells following 24 h pre-incubation with
the ER stress inducer tunicamycin was studied. Figure 1A
shows that only compound 14 was able to prevent the cyto-
hWTSOD1
toxic effect of tunicamycin in both
NSC-34 and
hSOD1G93A
NSC-34 motor neuron-like cells. Importantly, compound
14 did not demonstrate a significant protective cellular effect
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Scheme 2. Reagents and conditions: a) 3% aq. H
2
O
2
, AcOH, 6 h, 808C; b) acetic anhydride, 4 h, reflux; c) MeONa, MeOH, 4 h, RT; d) glutaric anhydride, dry THF,
30 min, RT; e) 3-hydroxy-N,N-dimethylpropan-1-amine oxide (S-1)/3-amino-N,N-dimethylpropan-1-amine oxide (S-2)/taurine/3-carboxy-N,N-dimethylpropan-1-
amine oxide (S-3)/4-(4-methoxyphenyl)butyric acid, PyBOP, Et N, DMF, 10 h, RT.
3
in rat L6 myotubes (pretreated with tunicamycin) which do not
express human SOD1 (data not shown).
was that only a relatively long incubation (24–48 h) with 14
had a cytoprotective effect. Both shorter (5–12 h) and longer
incubation periods (72 h) were not beneficial for cell viability.
These data suggest that intracellular accumulation of 14 is
needed for maximizing its biological effect, but degradation
probably occurs during long incubations of the compound,
thus lowering its activity.
We note that even small structural changes may affect the
biochemical parameters (e.g., cell permeation, intracellular dis-
position, stability, binding to misfolded proteins) of the synthe-
sized compounds. For example, the ester bond derivative 14
was found to be active, whereas compound 15, in which this
bond is replaced by the more stable amide bond, was found
to be inactive. Compound 14 was found to be stable under
physiological conditions (378C, pH 7.4) for 24 h (Supporting In-
formation Figure S1) Furthermore, its active concentration was
found to be 20-fold lower (50 mm versus 1 mm) than that of
the chaperone alone (TMAO). Taken together, these data sup-
port the hypothesis that the cytoprotective effect was caused
by compound 14 and not by its potential hydrolysis product
We did not succeed in isolating sufficient amounts of the
hSOD1G93A
motor neurons from the
transgenic mice to test the
effect of 14 in this primary cell culture. Thus, the neuroprotec-
tive effect of 14 was validated in primary astrocytes, which
hSOD1G93A
were isolated from spinal cords of the
transgenic mice
as described in the Experimental Section below. It has been
shown that primary astrocytes can be used as a model system
to investigate ALS molecular mechanisms and to evaluate po-
[24]
(
TMAO), which was demonstrated to be cytoprotective by
tential therapies for ALS.
Similar to the activity in
cells, compound 14 protected
[
12]
hSOD1G93A
itself.
NSC-34 motor neuron-like
hSOD1G93A
Based on these results, only compound 14 was further in-
primary mouse astro-
vestigated. The dose–response and time course effect of this
cytes from ER stress which was induced by tunicamycin when
hSOD1G93A
compound were tested in
NSC-34 motor neuron-like
the cells were incubated with the compound for 48 h (Fig-
hSOD1G93A
cells as shown respectively in Figure 1B,C. At concentrations
below 25 mm, 14 did not show any significant effect on cell via-
bility. The maximal effect was obtained at a concentration of
ure 1D). In contrast to what was observed in the
NSC-
34 motor neuron cell model, the compound was unable to
demonstrate a cytoprotective effect after 24 h incubation. This
result suggests that the cytoprotective intracellular concentra-
150 mm. An interesting result obtained from this experiment
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hWTSOD1
hSOD1G93A
Figure 1. In vitro biological evaluation of compound 14. A) Preliminary screening of synthesized compounds.
and
NSC-34 motor neuron-like
cells (grey and black columns, respectively) were grown as described in the Experimental Section. Cells were initially exposed to 0.75 mm tunicamycin for 12 h
and then exposed to trimethylamino N-oxide (TMAO) for 24 h (T, 1 mm), p-phenylbutyric acid (PB, 500 mm) or to 50 mm of the test compound for 24 h. Follow-
hSOD1G93A
ing exposure, a standard MTT analysis was conducted. B) Dose–response analysis of the effect of 14.
NSC-34 motor neuron-like cells were grown and
treated with tunicamycin as described for panel A. Cells were exposed to increasing concentrations of 14 and TMAO as indicated. After 24 h, MTT analysis
was conducted as described in the Experimental Section. C) Time course analysis of the effect of 14. Cells were treated as described in panel A. Compound
1
4 (50 mm) was added to cultures at indicated time points (&). MTT analysis was conducted as described in the Experimental Section for each indicated time
hSOD1G93A
for the treated and non-treated cells (~). D) Dose–response effect of 14 in primary
tained as described in the Experimental Section. The cultures were pre-incubated with naked chaperone (3-hydroxy-N,N-dimethylproban-1-amine oxide,
00 mm, NC), 10 (linker with dimethylpyridine moiety, 200 mm, LP) and increasing concentrations of 14 for 48 h. Afterward, the cells were exposed to tunica-
mice astrocytes. The primary cultures of mice astrocytes were ob-
2
mycin (1 mm) for an additional 24 h. MTT analysis was conducted as described in the Experimental Section. Significant difference between untreated control
cells and tunicamycin-treated cells is indicated by #, and between tunicamycin-treated control and cells exposed to test compound by *; p ꢀ0.05 meanÆSE
(
n=6).
tion of 14 is not identical in different cell types or that the rate
of accumulation is cell dependent.
like cells under ER stress conditions. Figure 2A,B show that 14
dose-dependently (50–150 mm) reduces the appearance of mis-
folded mutated SDS-resistant high-molecular-weight SOD1 rel-
ative to control cells, most likely due to its chaperoning activi-
ty. TMAO also reduced the level of misfolded SOD1, but at
a much higher concentration (1 mm).
Precipitation of aggregated protein into inclusion bodies is
a common cytopathological hallmark of the majority of neuro-
[
25]
degenerative diseases.
Mutated SOD1 can aggregate into
[5]
high-molecular-weight insoluble protein complexes. Such
SDS-resistant species are detectable in western blots of SOD1
in lysates obtained from transfected cells, spinal cord extracts
from transgenic mice expressing mutant SOD1, and in autopsy
samples of ALS patients. We therefore studied the effect of 14
In addition, the effect of 14 on the level of two well-known
ER stress markers—binding immunoglobulin protein (BiP) and
[26]
C/EBP homologous protein (CHOP) —was investigated as
shown in Figure 2A,B. Misfolded and aggregated proteins
cause ER stress, which lead to increased intracellular levels of
both proteins. The levels of both markers were significantly
on the formation of SDS-resistant high-molecular-weight SOD1
hSOD1G93A
(the aggregate-like species) in
NSC-34 motor neuron-
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hSOD1G93A
Figure 2. In vitro biological evaluation of compound 14. A) Western blot analysis.
NSC-34 motor neuron-like cells were treated as described in Fig-
ure 1A. Subsequently, cell lysates were produced as described in the Supporting Information. Standard SDS-PAGE (10%) was conducted. Western blots were
obtained by using commercially available antibodies as described in the Supporting Information. Experiments were run n=4 times, and representative gels
are shown. B) Western blot bands density calculations. The density of the signal of SDS-resistance high-molecular-weight SOD1 species (&) was normalized
according to the amount of normal size monomer SOD1 band. The signal of BiP (&) and CHOP (&) were normalized according to the density of a-tubulin;
*
p ꢀ0.05, meanÆSE (n=4). C) Effect of compound 14 on the level of misfolded mutant SOD1. Recombinant proteins were incubated for 20 min with increas-
ing concentrations of compound 14. After incubation, the immunoprecipitation (IP) procedure was performed, and it was followed by PAGE–western blot
hG93A
hG85R
analysis as described in the Experimental Section. D) IP band density calculations. The densities of the signals of
malized according to the density of the unbound SOD1 band. The control (not treated by 14) band density was taken as 100% for each SOD1 mutant. The
naked” chaperone, trimethylamino N-oxide (TMAO), 3-hydroxy-N,N-dimethylproban-1-amine oxide, and 10 (linker with dimethylpyridine moiety), were not
SOD1 (&) and
SOD1 (&) were nor-
“
active at 200 mm (data not shown). *p ꢀ0.05, meanÆSE (n=3).
lower in cells treated with 14 at 10 mm than in untreated cells.
Increasing concentrations of 14 did not significantly decrease
any of the tested ER stress markers. Importantly, the cell-pro-
tective and anti-aggregation effects of the compound likely
follow different mechanisms of action, and higher concentra-
tions of 14 are probably needed for neuroprotection (see
above). Interestingly, TMAO treatment, which was used as
a positive control, decreased the level of BiP and failed to de-
crease the amount of CHOP.
ALS in humans which is accompanied by the accumulation of
[27]
the protein in astrocytes and motor neurons. Using an im-
munoprecipitation assay with an antibody that specifically rec-
ognizes misfolded SOD1 only, compound 14 (at 200 mm) was
found to decrease the levels of both SOD1 mutants by 35–
40% (Figure 2C,D).
The potential anti-aggregation effect of compound 14 was
hWTSOD1-EGFP
hSOD1A4V-EGFP
further tested in transfected
and
HEK293T
cells. This mutation is common in many fALS patients and is
[28]
Furthermore, the ability of compound 14 to directly reduce
the amount of misfolded human recombinant mutated SOD1
was investigated. Two mutated human SOD1 recombinant pro-
teins were chosen for this analysis: G93A and G85R. The G85R
mutation in SOD1 is associated with a rapid progression of
associated with a clinically severe disease manifestation. As
shown in Figure 3, 14 (200 mm) decreased the expression levels
hA4V
hWT
and aggregation of
SOD1 as well as
SOD1 in these cells.
hA4V
The reduced expression level of
SOD1-EGFP as well as
hWT
SOD1-EGFP suggests enhanced degradation rates for both
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ing the disease. In line with this expectation, a small-molecule
proteasome activator was recently reported by Trippier et al.,
5-(((2,4-dichloro-6-methylphenyl)thio)methyl)-1,2-dihydro-3H-
pyrazol-3-one, which demonstrated biological activity in ALS
[30]
mice.
Using a commercially available proteasome activity
assay kit, we performed the experiment and determined that
compound 14 is not a proteasome activator even at supra-
pharmacological concentrations (Supporting Information Fig-
ure S2).
Despite its relatively high active concentration in vitro, com-
pound 14 was tested in vivo. For the in vivo evaluation we
hSOD1G93A
used
transgenic mice, which are commonly used as an
[31]
ALS model. This model of ALS mice is also widely used for
[31]
ALS drug screening. Compound 14 or a saline control were
injected daily (10 mgkg i.p., soluble in saline) from day 40
À1
(
from birth) to day 125 of the experiment. Female and male
mice were treated separately. As shown in Figure 4A,B, chronic
treatment with 14 slowed the progression of body weight loss
hSOD1G93A
in
transgenic mice. The loss of skeletal muscle mass (re-
sulting in total body weight loss) is one of the hallmarks of the
[1]
disease. Thus, the ability of the compound to slow this pro-
cess may translate into a significant effect in ALS patients.
Treatment with 14 also delayed the onset and slowed the pro-
gression of the loss of neurological functions (extension reflex
of both hind paws) in both groups of ALS mice, starting from
day 90 of treatment (Figure 4C,D). The effect was especially
dramatic in females. From day 120, the control group showed
a significant paralysis of the lower legs relative to almost
normal lower leg movement ability in treated female mice (see
the movie file in the Supporting Information). Following with-
drawal of the treatment on day 130 (due to ethical considera-
tions for prevention of misery to the animals), no statistically
significant differences in life span were observed in the treated
hWT
Figure 3. Anti-aggregation effect of 14 in transfected SOD1-EGFP and
hA4V
SOD1-EGFP HEK293T cells. A) The fluorescence microscopic images of the
transfected HEK293T cells expressing EGFP fusion of SOD1 variants. The
images of transfected HEK293T cells expressing EGFP fusion of SOD1 and
th
mice, and both groups died by around the 155 day (Figure 5).
hWT
Indeed, in many studies using this specific model, the effect of
the studied compounds was evident in the motor symptoms
hA4V
SOD1 in the absence or the presence of 200 mm of 14 were taken at two
days post-transfection (white arrows: intracellular SOD1 mutant aggregates;
scale bar: 50 mm). B) The calculated data of the cellular fluorescence. The
mean cellular fluorescence of the transfected HEK293T cells expressing EGFP
[31]
and was not reflected in the general survival rate.
Many of the symptoms arising during the course of the dis-
ease are untreatable so far, and all efforts should be made to
improve the quality of life, helping to maintain the patient’s
hWT
hA4V
fusion of SOD1 and
SOD1 in the absence (&) or presence (&) of 14.
Values represent the mean cellular fluorescence with error bars denoting
standard deviations (n=3). To determine whether the addition of 14 affects
the mean cellular fluorescence, two-sided Student’s t-tests were applied to
the data (*p <0.05). The “naked” chaperone, trimethylamino N-oxide
[32]
autonomy for as long as possible. Thus, the results of the
present study support the use of 14 as a novel drug candidate
for the delay of symptom progression, prevention of deteriora-
tion of motor functions, and the improvement in the quality of
life of ALS patients, even though the compound was not
shown to prolong survival rate. Moreover, the mechanism of
action of compound 14 might also be useful in the develop-
ment of universal drugs for the treatment of disorders that are
related to general protein misfolding and aggregation.
(
TMAO), and lipophilic domain with the linker (10) did not significantly
affect the cellular fluorescence.
SOD1 variants. Because the addition of 14 did not significantly
decrease the HEK293T cell growth rate, inhibition of protein
synthesis may not be the cause of the reduced expression
level. Therefore, the apparent reduction of intracellular aggre-
hA4V
gates of
SOD1 in the presence of 14 is likely due to the
Conclusions
[29]
promoted degradation of the entire human SOD1 species.
To further probe this point, we tested the ability of compound
4 to activate the proteasome. Given the large number of mis-
folded/aggregated proteins implicated in ALS, the activation of
one of the most important disposal mechanisms of the cell
Herein we report a novel compound, 3-((5-((4,6-dimethylpyri-
din-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-
amine oxide (14), which demonstrated biological effect in ALS
mice. The molecule was designed as a lipophilic derivative of
a known chemical chaperone (TMAO). The compound showed
1
(even in a nonspecific manner) is a viable approach to combat-
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hSOD1G93A
Figure 4. In vivo evaluation of compound 14. Chronic treatment with 14 prevent loss of the body weight and neurological function in
transgenic
À1
mice. Mice were daily treated i.p. with 10 mgkg of 14 (&) beginning from day 40 and continuing until day 130; the untreated control group (*) is shown
for comparison. Body weight was determined every other day until day 125 in A) male and B) female mice. The effect of 14 on neurological function was
measured from day 90. Mice were tested every other day by measuring reflex of both hind paws in C) males and D) females. Significant difference between
treated and control mice is indicated by *; ANOVA, p <0.001 meanÆSD (n=15).
prevention of body mass loss and an improvement in neuro-
Experimental Section
logical functions. The in vitro study revealed several character-
Materials
istics of its mechanism of action. Compound 14 significantly
decreased the formation of misfolded mutated SOD1 (G93A
and G85R, recombinant proteins). Evidence presented in this
study indicate that this compound is able to prevent ER-stress-
induced apoptosis of NSC-34 motor neuron-like cells and pri-
mary mouse astrocytes (both types of cells were transfected
with mutated SOD1 G93A, which leads to fALS in humans). In
addition, 14 decreased the levels of known ER stress markers
All chemical reagents, solvents, and acids were purchased from
Sigma–Aldrich, Acros Organic, Alfa Aesar, Bio-Lab Ltd., Merck, or
IU-CHEM Ltd., and were all used as received. Anhydrous THF was
obtained by distillation from a boiled blue colored mix containing
sodium (1% w/v) and benzophenone (0.2% w/v). Column chroma-
tography was performed on silica gel 60 (230–400 mesh; Merck).
Analytical and preparative HPLC (Young Lin Instruments, Anyang,
Korea) were performed on LUNA C18 preparative (10 mm, 100ꢁ
(CHOP and BiP) in NSC-34 motor neuron-like cells. Finally, this
3
0 mm) or analytical (5 mm, 250ꢁ4.6 mm) columns, both from Phe-
novel derivative of TMAO decreased the formation of misfold-
ed mutant SOD1 aggregates in NSC-34 motor neuron-like cells
nomenex Inc. (Torrance, CA, USA). HPLC purification was carried
out with an increasing linear gradient of CH CN in H O. Purity of
3
2
(
G93A) and in HEK293T cells transfected by another related to
the synthesized compounds was confirmed by HPLC analysis (Sup-
porting Information Table S1). Analytical TLC was carried out on
pre-coated silica gel 60 F254 (Merck) sheets using UV absorption
and iodine physical adsorption for visualization. Mass spectra were
recorded on a Finnigan Model 400 instrument using a QToF micro-
spectrometer (Micromass, Milford, MA, USA), using electrospray
ionization (ESI) in the positive ion mode. Data were processed
using mass LynX ver. 4.1 calculation and de-convolution software
ALS in humans SOD1 mutant (A4V). Future detailed characteri-
zation of the mechanism of action of compound 14 in vivo is
needed. Also, the compound may not be specifically selective
for ALS-induced protein misfolding. Therefore, we plan to test
the compound in other types of neurodegenerative disease
models, such as mouse models of Alzheimer’s, Parkinson’s, and
Huntington’s disease.
(
(
Waters Corp., Milford, MA, USA). High-resolution mass spectra
HRMS) were obtained using an LTQ Orbitrap XL (Thermo Scientific,
Waltham, MA, USA). Melting points were measured with a Fisher–
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with a synthetic peptide corresponding to residues surrounding
Gly584 of the human protein, which recognizes the mouse protein
as well. These two antibodies and anti-a-tubulin 1 were purchased
from Cell Signaling (Danvers, MA, USA). B8H10 antibody was sup-
plied by MediMabs, (Montreal, QC, Canada). The proteasome activi-
ty assay kit was purchased from Abcam (Cambridge, UK).
Chemistry
General procedure for the preparation of S-1 and S-2: To a stirred
solution of commercially available 3-(dimethylamino)propan-1-ol/3-
(
dimethylamino)propan-1-amine (2.62 mmol), 3% aq. H O (5 mL)
2 2
was added dropwise, and the solution was stirred for 72 h at room
temperature. The water was removed by lyophilization, and the re-
sulting crystalline mass was dissolved in 20 mL MeOH and filtered.
MeOH was evaporated under reduced pressure to obtain an N-
oxide derivative.
3
-Hydroxy-N,N-dimethylpropan-1-amine oxide (S-1): The title
compound was obtained starting from 3-(dimethylamino)propan-
1
1
2
1
-ol (colorless syrup, 93%): H NMR (300 MHz, D O): d=2.06 (m,
2
H), 3.45 (s, 6H), 3.43 (m, 2H), 3.7 (t, J=6 Hz, 2H), 4.79 ppm (s,
H); C NMR (75.49 MHz, D O): d=25.78, 57.15, 58.54, 67.74 ppm;
MS (ES, 30 eV) m/z (%): 120 (100) [M+H] , 142 (5) [M+Na] .
13
2
+
+
3
-Amino-N,N-dimethylpropan-1-amine oxide (S-2): The title com-
pound was obtained starting from 3-(dimethylamino)propan-1-
1
amine (yellow oil, 91%): H NMR (600 MHz, D O): d=1.79 (m, 2H),
2
2
1
3
.56 (t, J=7.8 Hz, 2H), 3.00 (s, 6H), 3.17 ppm (m, 2H); C NMR
Figure 5. Survival curves following the treatment with compound 14 (&);
(
150.9 MHz, D O): d=25.2, 37.65, 57.19, 67.93 ppm; MS (ES, 30 eV)
the untreated control group (*) is shown for comparison. A) Effect of 14 on
2
+
hSOD1G93A
m/z (%): 119 (100) [M+H] .
the survival rate in male
vival rate in female
transgenic mice. B) Effect of 14 on the sur-
hSOD1G93A
transgenic mice. The experiment was conducted
General procedure for the preparation of S-3: To a stirred solu-
tion of commercially available 4-(dimethylamino)butyric acid hy-
drochloride (29.8 mmol), a solution of NaOH (10% w/v) was added
to reach pH 14. Subsequently, 3% aq. H O (200 mL) were added
as described in the Experimental Section. Significant difference between
treated and untreated mice is indicated by *; p ꢀ0.05 meanÆSD (n=15).
2
2
dropwise, and the reaction mix was stirred for 72 h at room tem-
perature. The reaction was quenched by the addition of 1m HCl to
reach pH 3 and extracted with EtOAc (3ꢁ50 mL). The combined or-
Johns melting point apparatus (Waltham, MA, USA). Hank’s bal-
anced salt solution (HBSS) was purchased from Life Technologies
ganic phases were dried over Mg SO , and the solvent was re-
(
Carlsbad, CA, USA). b-Mercaptoethanol, PMSF, sodium orthovana-
2
4
moved under reduced pressure to obtain an N-oxide derivative.
date, sodium-b-glycerophosphate, sodium dodecyl sulfate (SDS),
and sodium pyrophosphate decahydrate were purchased from Alfa
Aesar (Karlsruhe, Germany). EGTA was from AppliChem (Darmstadt,
Germany). Dulbecco’s modified Eagle’s medium (DMEM), heat-inac-
tivated and normal fetal calf serum (FCS), l-glutamine, penicillin/
streptomycin, nystatin, neomycine-G418 and trypsin were pur-
chased from Biological Industries (Beth-Haemek, Israel). Bromphe-
nol blue, N,N,N,N-tetramethylethylenediamine, ammonium persul-
fate, fat-free milk, acrylamide solution for gel preparation, and pro-
tein molecular weight markers were purchased from Bio-Rad (Her-
cules, CA, USA). BSA, methylthiazolyl blue (MT) was from Chem-
Impex International (Wood Dale, IL, USA). Bradford reagent, DMSO,
DNAse, DTT, EGTA, glycerol, IGEPAL, NaCl, PBS tablets, protease in-
hibitor cocktail for mammalian cells, para-phenylbutyric acid, PBS
tablets, Tris·HCl, tunicamycin, trimethylamino N-oxide (TMAO),
Triton X-100, and NaF were purchased from Sigma–Aldrich Chemi-
cals (Rehovot, Israel). Hygromycin B was purchased from Molecula
3
-Carboxy-N,N-dimethylpropan-1-amine oxide (S-3): The title
compound was obtained starting from 4-(dimethylamino)butyric
acid hydrochloride (white oil, 90%): H NMR (400 MHz, D O): d=
1
2
2
2
1
.08 (m, 2H), 2.32 (t, J=7.08 Hz, 2H), 3.37 (s,6H), 3.51 ppm (m,
13
H); C NMR (100.6 MHz, D O): d=19.17, 48.60, 55.94, 68.53,
2
+
79.65 ppm; MS (ES, 30 eV) m/z (%): 148 (100) [M+H] , 170 (16)
+
[M+Na] .
General procedure for the preparation of 3 and 4: To a stirred so-
lution of commercially available 2,3,5-collidine/2,4,6-collidine
(76.8 mmol), AcOH (85 mL) and 30% aq. H O2 solution (33 mL)
2
were added. The mixture was heated at 808C for 6 h. The reaction
progress was followed by TLC. After cooling, the mixture was
poured into H O (50 mL) and neutralized by the addition of a satu-
rated NaHCO3 solution. The aqueous layer was extracted with
CH Cl (3ꢁ200 mL); the combined organic phases were dried over
2
2
2
Mg SO , and the solvent was removed under reduced pressure.
2
4
(
Shaftesbury, UK). The following antibodies were used: rabbit poly-
[33]
clonal anti-SOD1 antiserum (Santa Cruz Biotechnology, TX, USA);
CHOP, monoclonal antibody which was produced based on a syn-
thetic peptide corresponding to residues surrounding Leu159 of
the human CHOP protein which also recognizes the mouse pro-
tein. Anti-Bip rabbit mAb was produced by immunizing rabbits
2,3,5-Trimethylpyridine 1-oxide (3):
obtained starting from 2,3,5-collidine (yellow oil, 57%): H NMR
(400 MHz, CDCl ): d=2.32 (s, 3H), 2.38 (s, 3H), 2.53 (s, 3H), 7.16 (s,
H), 8.21 (s, H), AcOH 2.1 ppm (s, 6H); C NMR (100.6 MHz, CDCl3):
The title compound was
1
3
13
d=13.02, 17.31, 18.93, 131.41, 134.34, 136.81, 145.45, AcOH 20.72,
&
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+
1
73.46 ppm; MS (ES, 30 eV) m/z (%): 138 (100) [M+H] , 160 (18)
sure. The resulting syrup was purified by silica gel column (n-
hexane/EtOAc 50:50).
+
[M+Na] .
[
36]
5
-((3,5-Dimethylpyridin-2-yl)methoxy)-5-oxopentanoic acid (9):
2
,4,6-Trimethylpyridine 1-oxide (4): The title compound was ob-
1
The title compound was obtained starting from 7 (white solid,
7%, mp: 878C): H NMR (300 MHz, D O): d=1.89 (quint, J=7.5 Hz,
H), 2.27 (t, J=7.5 Hz, 2H), 2.44 (s, 3H), 2.45 (s, 3H), 2.54 (t, J=
.5 Hz, 2H), 5.39 (s, 2H), 8.06 (s, 1H), 8.33 ppm (s, 1H); C NMR
tained starting from 2,4,6-collidine (yellow oil, 61%): H NMR
400 MHz, CDCl ): d=2.09 (s, 3H), 2.29 (s, 6H), 6.76 ppm (s, 2H);
1
4
2
7
(
2
3
1
3
C NMR (100.6 MHz, CDCl3): d=17.18, 19.21, 124.00, 135.81,
1
3
+
1
47.21 ppm; MS (EI, 30 eV) m/z (%): 137 (82) [M] .
(
100.6 MHz, [D ]acetone): d=17.50, 17.61, 20.80, 33.05, 33.31,
6
General procedure for the preparation of 5 and 6: The com-
pounds were synthesized using the Boekelheide rearrangement.
65.57, 132.66, 133.75, 139.48, 147.12, 151.18, 172.86, 174.32 ppm;
MS (ES, 30 eV) m/z (%): 252 (100) [M+H] .
[34]
+
Solution of 3/4 (19 mmol) was added to acetic anhydride (40 mL)
and was heated at reflux for 4 h. After cooling, excess acetic anhy-
dride was evaporated under reduced pressure (high-vacuum oil
pump) to obtain black crude. The resulting syrup was purified by
silica gel column (petroleum ether/EtOAc/MeOH 10:30:60).
5
-((4,6-Dimethylpyridin-2-yl)methoxy)-5-oxopentanoic acid (10):
The title compound was obtained starting from 8 (white solid,
1
4
9%, mp: 648C): H NMR (400 MHz, [D ]DMSO): d=1.77 (quint, J=
6
7
.40 Hz, 2H), 2.26 (t, J=7.40 Hz, 2H), 2.27 (s, 3H), 2.40 (s, 3H), 2.44
(
t, J=7.40 Hz, 2H), 5.05 (s, 2H), 7.00 (s, 1H), 7.01 ppm (s, 1H);
1
3
(
3,5-Dimethylpyridin-2-yl)methyl acetate (5): The title compound
C NMR (100.6 MHz, [D ]DMSO): d=20.06, 20.37, 23.66, 32.65,
6
1
was obtained starting from 3 (yellow oil, 69.5%): H NMR (400 MHz,
32.89, 65.99, 119.18, 122.97, 147.78, 154.79, 157.23, 172.32,
174.06 ppm; MS (ES, 30 eV) m/z (%): 252 (100) [M+H] , 174 (3)
[M+Na] .
+
CDCl ): d=1.99 (s, 3H), 2.18, 2.22 (s, 6H), 5.08 (s, 2H), 7.20 (s, H),
3
1
3
+
8
5
3
.16 ppm (s, H); C NMR (100.6 MHz, CDCl ): d=17.23, 20.14,
3
6.02, 131.22, 132.37, 138.21, 146.70, 149.91, 169.95 ppm; MS (ES,
0 eV) m/z (%): 180 (100) [M+H] , 202 (16) [M+Na] .
5
-((3-(1H-Imidazol-1-yl)propyl)amino)-5-oxopentanoic acid (16):
+
+
The title compound was obtained starting from S-10 (white solid,
1
6
4%, mp: 958C): H NMR (300 MHz, D O): d=1.72 (quint, J=
2
(
4,6-Dimethylpyridin-2-yl)methyl acetate (6): The title compound
1
7.75 Hz, 2H), 2.01 (quint, J=6.63 Hz, 2H), 2.12 (m, 4H), 3.13 (t, J=
.63 Hz, 2H), 4.04 (t, J=6.62 Hz, 2H), 7.26 (s, 1H), 7.34 (s, 1H),
was obtained starting from 4 (yellow oil, 71%): H NMR (300 MHz,
6
CDCl ): d=2.10 (s, 3H), 2.29 (s, 3H), 2.48 (s, 3H), 5.14 (s, 2H), 6.91
3
13
13
8.36 ppm (s, 1H); C NMR (75.49 MHz, D O): d=22.58, 29.21, 35.63,
2
(
s, H), 6.98 (s, H), AcOH 2.03 (s, 3H), 10.21 ppm (s, 1H); C NMR
3
6.27, 36.88, 46.42, 121.52, 122.29, 135.79, 170.51, 176.89 ppm; MS
(
CDCl , 75.49 MHz): d=20.87, 20.99, 23.04, 65.95, 120.56, 124.17,
3
+
+
(ES, 30 eV) m/z (%): 240 (100) [M+H] , 262 (3) [M+Na] .
1
3
49.52, 154.22, 157.63, 170.87, AcOH 20.99, 176.52 ppm; MS (ES,
0 eV) m/z (%): 180 (100) [M+H] , 202 (47) [M+Na] .
+
+
General procedure for the preparation of (11–15, 17–19): To
a suspension of acid derivative (1.6 mmol) in DMF (3 mL), Et N
3
General procedure for the preparation of 7 and 8: The com-
(1 mL) and PyBOP (1.8 mmol) were added. The mixture was stirred
pounds were obtained using a modification of the procedure re-
[35]
at room temperature for 30 min. Then, R-OH/R-NH (1.6 mmol) dis-
2
ported by Ellervik and Magnusson. Compound 5/6 (18 mmol)
solved in DMF (2 mL) was added to the reaction. The reaction mix-
ture was stirred for 18 h. After that, DMF was evaporated under
high pressure to obtain a yellow oil. The product was purified by
HPLC.
was dissolved in MeOH (170 mL), and 1m NaOCH in MeOH (3 mL)
3
was added. The mixture was stirred for 3 h at room temperature.
Excess MeOH was evaporated under reduced pressure. The reac-
tion was quenched by the addition of H O (50 mL) and extracted
2
with EtOAc (3ꢁ50 mL). The combined organic phase was washed
with brine and dried over Mg SO , and the solvent was removed
under reduced pressure. The resulting syrup was purified by
a silica gel column (petroleum ether/EtOAc/MeOH 10:30:60).
3-(5-((3,5-Dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)-N,N-
dimethylpropan-1-amine oxide (11): The title compound was ob-
tained starting from 9 (yellow oil, 39%): H NMR (400 MHz,
2
4
1
[D ]acetone): d=1.90 (quint, J=7.40 Hz, 2H), 2.24 (s, 3H), 2.25
6
(
quint, J=6.62 Hz, 2H), 2.32 (s, 3H), 2.39 (t, J=6.46 Hz, 2H), 2.43 (t,
(
3,5-Dimethylpyridin-2-yl)methanol (7): The title compound was
J=7.10 Hz, 2H), 3.22 (s, 6H), 3.45 (m, 2H), 4.19 (t, J=6.26 Hz, 2H),
5
[
6
1
3
1
obtained starting from 5 (yellow oil, 47.3%): H NMR (400 MHz,
CDCl ): d=2.11 (s, 3H), 2.22 (s, 3H), 4.57 (s, 2H), 7.20 (s, H),
8
6
13
.17 (s, 2H), 7.40 (s, 1H), 8.20 ppm (s, 1H); C NMR (100.6 MHz,
3
D ]acetone): d=17.92, 18.01, 21.16, 24.10, 33.67, 58.68, 62.90,
6
1
3
.12 ppm (s, H); C NMR (100.6 MHz, CDCl ): d=16.32, 17.77,
3
6.48, 68.18, 133.88, 139.41, 147.88, 151.81, 162.77, 173.16,
1.42, 128.97, 131.45, 138.43, 145.28, 153.43 ppm; MS (ES, 30 eV)
+
73.33 ppm; ESI-HRMS (m/z) [M+H]
calcd for C H N O :
18 28 2 5
+
+
m/z (%): 138 (100) [M+H] , 160 (4) [M+Na] .
53.20710, found: 353.20746; MS (ES, 30 eV) m/z (%): 353 (46) [M+
+
H] .
(4,6-Dimethylpyridin-2-yl)methanol (8): The title compound was
1
obtained starting from 6 (yellow solid, 49%, mp: 438C): H NMR
300 MHz, CDCl ): d=2.30 (s, 3H), 2.50 (s, 3H), 4.66 (s, 2H), 6.84 (s,
3
-(5-((4,6-Dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)-N,N-
(
3
dimethylpropan-1-amine oxide (14): The title compound was ob-
tained starting from 10 (yellow oil, 33%): H NMR (400 MHz,
1
3
1
H), 6.87 ppm (s, H); C NMR (CDCl , 75.49 MHz): d=21.00, 24.02,
3
6
4.00, 118.56, 122.95, 148.28, 157.17, 158.35 ppm; MS (ES, 30 eV)
[
3
3
D ]acetone): d=1.90 (quint, J=7.14 Hz, 2H), 2.25 (m, 2H), 2.31 (s,
H), 2.42 (s, 3H), 2.44 (t, J=7.33 Hz, 2H), 2.51 (t, J=7.44 Hz, 2H),
6
+
m/z (%): 138 (100) [M+H] .
.16 (s, 6H), 3.38 (m, 2H), 4.21 (t, J=6.32 Hz, 2H), 5.11 (s, 2H), 6.99
General procedure for the preparation of 9, 10, and 16: Com-
pound 7/8/S-10 (14 mmol) was dissolved in dry THF (15 mL). Sub-
sequently, glutaric anhydride (14 mmol) was dissolved in dry THF
13
(s, 1H), 7.04 ppm (s, 1H); C NMR (100.6 MHz, [D ]acetone): d=
6
2
1
3
0.96, 23.97, 33.60, 58.71, 62.81, 67.16, 68.10, 119.93, 123.67,
48.79, 156.24, 158.52, 173.03, 173.18 ppm; MS (ES, 30 eV) m/z (%):
(
2 mL) and was added dropwise to the reaction solution while stir-
+
+
+
53 (100) [M+H] , 375 (4) [M+Na] ; ESI-HRMS (m/z) [M+H]
ring at room temperature for 1 h. The reaction was quenched by
the addition of H O (50 mL) and extracted with EtOAc (3ꢁ150 mL).
calcd for C H N O : 353.20710, found: 353.20758.
18
28
2
5
2
The combined organic phase was washed with brine and dried
over Mg SO , and the solvent was removed under reduced pres-
3-(5-((3,5-Dimethylpyridin-2-yl)methoxy)-5-oxopentanamido)-
N,N-dimethylpropan-1-amine oxide (12): The title compound was
2
4
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1
obtained starting from 9 (yellow oil, 46%): H NMR (400 MHz, D O):
Biology
2
d=1.87 (quint, J=7.03 Hz, 2H), 2.02 (m, 2H), 2.25 (t, J=7.45 Hz,
Animals
2
3
H), 2.48 (t, J=7.45 Hz, 2H), 3.30 (s, 6H), 3.24 (t, J=6.78 Hz, 2H),
.46 (m, 2H), 5.30 (s, 2H), 7.91 (s, 1H), 8.25 ppm (s, 1H); C NMR
13
G93A
Mice overexpressing SOD1
ratories (Bar Harbor, ME, USA). The
model was developed and characterized as a model for ALS by
Gurney et al. The animals were housed in standard conditions:
constant temperature (22Æ18C), humidity (relative, 40%), and
a 12 h light/dark cycle and were allowed free access to food and
water. Male mice with hemizygous background were bred with
were purchased from Jackson Labo-
(
100.6 MHz, D O): d=17.98, 18.58, 21.93, 24.34, 34.10, 36.13, 37.66,
G93A
2
SOD1 transgenic mouse
5
1
3
8.00, 63.66, 68.93, 136.58, 138.02, 142.77, 146.75, 147.82, 176.23,
+
77.42 ppm; ESI-HRMS (m/z) [M+H]
calcd for C H N O :
[22]
18
29
3
4
52.22308, found: 352.22372; MS (ES, 30 eV) m/z (%): 352 (100)
+
[M+H] .
3
-(5-((4,6-Dimethylpyridin-2-yl)methoxy)-5-oxopentanamido)-
control C57Bl females so that each litter would generate hemizy-
N,N-dimethylpropan-1-amine oxide (15): The title compound was
G93A
1
gous
SOD1 transgenic mice and littermate controls. Newborn
obtained starting from 10 (yellow oil, 42%): H NMR (400 MHz,
mice were genotyped by PCR analysis using the following primers.
IL2 primers: CTA GGC CAC AGA ATT GAA AGA TCT and GTA GGT
GGA AAT TCT AGC ATC ATC C and the
D O): d=1.91 (quint, J=7.31 Hz, 2H), 2.02 (m, 2H), 2.29 (t, J=
2
7
.45 Hz, 2H), 2.31 (s, 3H), 2.44 (s, 3H), 2.49 (t, J=7.45 Hz, 2H), 3.18
hG93A
SOD1 primers: CAT CAG
(
s, 6H), 3.26 (t, J=6.61 Hz, 2H), 3.34 (m, 2H), 5.09 (s, 2H), 7.09 (s,
13
CCC TAA TCC ATC TGA and CGC GAC TAA CAA TCA AAG TGA. Ge-
nomic DNA was extracted from tail biopsies using the D-Tail DNA
extraction kit (Syntezza Bioscience, Jerusalem, Israel). The experi-
ments were performed in accordance with local and international
regulations, every effort was made to reduce the number of ani-
mals used and to minimize their suffering. Primary astrocyte cul-
tures were obtained from littermates which were positive for the
transgene. The Tel Aviv University is an Association for Assessment
and Accreditation of Laboratory Animal Care (AAALAC) internation-
ally accredited institution.
1
2
1
H), 7.12 ppm (s, 1H); C NMR (100.6 MHz, D O): d=19.65, 20.00,
2
1.49, 22.49, 32.31, 35.82, 56.79, 65.46, 67.32, 120.44, 124.00,
51.06, 152.24, 156.84, 174.51, 175.24 ppm; ESI-HRMS (m/z) [M+
+
H] calcd for C H N O : 352.22308, found: 352.22314; MS (ES,
3
1
8
29
3
4
+
0 eV) m/z (%): 352 (38) [M+H] .
2
-(5-((3,5-Dimethylpyridin-2-yl)methoxy)-5-oxopentanamido)e-
thanesulfonic acid (13): The title compound was obtained starting
1
from 9 (yellow oil, 54%): H NMR (300 MHz, D O): d=1.81 (quint,
2
J=7.11 Hz, 2H), 2.19 (t, J=7.11 Hz, 2H), 2.26 (s, 3H), 2.28 (s, 3H),
2
2
6
1
1
.44 (t, J=7.11 Hz, 2H), 2.95 (t, J=6.67 Hz, 2H), 3.44 (t, J=6.67 Hz,
H), 5.20 (s, 2H), 7.70 (s, 1H), 8.15 (s, 1H), Et N (1.19, t, J=7.33 Hz,
3
13
H), (3.11 ppm, q, J=7.33 Hz, 4H); C NMR (75.49 MHz, D O): d=
2
6.89, 17.35, 20.63, 34.87, 35.28, 49.88, 63.47, 134.44, 135.97,
Cell cultures
43.30, 143.62, 147.63, 147.63, 175.86, Et N (8.48, 46.92 ppm); ESI-
3
G93A
+
Human
SOD1 transfected motor neuron-like cells (NSC-34):
HRMS (m/z) [M+H] calcd for C H N O S: 359.12713, found:
15
22
2
3
hG93ASOD1
hWTSOD1
NSC-34 and
NSC-34 cells were kindly provided by
3
59.12753; MS (ESÀ, 30 eV) m/z (%): 357 (37) [MÀH].
-(5-((3-(1H-Imidazol-1-yl)propyl)amino)-5-oxopentanamido)-
Prof. Nava Zisapel (Tel Aviv University, Israel). Cells were grown in
DMEM (22.5 mm glucose) supplemented with 15% heat inactivated
3
À1
N,N-dimethylpropan-1-amine oxide (17): The title compound was
obtained starting from 16 (yellow oil, 74%): H NMR (400 MHz,
D O): d=1.77 (quint, J=7.54 Hz, 2H), 1.96 (m, 4H), 2.16 (m, 4H),
FCS, 1 mm glutamine, and antibiotics (100 mgmL
penicillin,
1
À1
À1
À1
100 mgmL
streptomycin, 200 mgmL
hygromicine and
2
700 mgmL neomycin-G418) at 378C in a 5% CO humidified at-
2
3
7
2
1
.07 (m, 2H), 3.23 (m, 4H), 4.00 (t, J=6.79 Hz, 2H), 6.95 (s, 1H),
mosphere.
13
.09 (s, 1H), 7.61 ppm (s, 1H); C NMR (100.6 MHz, D O): d=21.12,
2
hWTSOD1
hSOD1A4V
HEK293T and
tained at 378C and 5% CO
tific) supplemented with fetal bovine serum (FBS; 10% v/v), strep-
HEK293T cells: HEK293T cells were main-
2.52, 28.82, 29.69, 34.34, 35.87, 35.99, 43.96, 56.90, 67.36, 119.54,
+
2
in DMEM/High Glucose (Thermo Scien-
27.15, 137.35, 175.18, 175.36 ppm; ESI-HRMS (m/z) [M+H] calcd
for C H N O : 340.23432, found: 340.23477; MS (ES, 30 eV) m/z
1
6
29
5
3
À1
À1
+
+
tomycin sulfate (100 mgmL ), and penicillin (100 UmL ).
(
%): 340 (53) [M+H] , 362 (4) [M+Na] .
4
1
-((3-(1H-Imidazol-1-yl)propyl)amino)-N,N-dimethyl-4-oxobutan-
-amine oxide (18): The title compound was obtained starting
1
from S-10 (yellow oil, 68%): H NMR (300 MHz, D O): d=1.99 (m,
Mice astrocyte primary cultures
2
2
H), 2.24 (m, 4H), 3.03 (m, 2H), 3.19 (s, 6H), 3.32 (m, 2H), 4.33 (t,
Astrocyte primary cultures were prepared from the cortex of new-
born mice as previously described by Apricꢂ et al., with minor
changes. The cortex tissue was dissected from newborn mice
J=7.15 Hz, 2H), 7.42 (s, 1H), 7.50 (s, 1H), 8.66 ppm (s, 1H);
1
3
C NMR (75.49 MHz, D O): d=20.23, 27.75, 34.07, 36.68, 46.20,
2
[37]
5
7.16, 69.89, 121.32, 121.74, 135.32, 181.48 ppm; ESI-HRMS (m/z)
+
(postnatal days 1–3) and the meninges were removed. Spinal cords
were isolated from newborn mice as described in the isolation pro-
tocol. Purified tissues were mechanically dissociated in cold PBS
and digested by incubation with trypsin in PBS (1:5 v/v) for 10 min
at 378C. The reaction was stopped by the addition of complete
[M+H] calcd for C H N O : 255.18155, found: 255.18199; MS
1
2
22
4
2
+
+
(
ES, 30 eV) m/z (%): 255 (100) [M+H] , 277 (13) [M+Na] .
N-(3-(1H-Imidazol-1-yl)propyl)-4-(4-methoxyphenyl)butanamide
19): The title compound was obtained starting from 4-(4-methoxy-
(
1
À1
phenyl)butyric acid (white solid, 54%, mp: 588C): H NMR
DMEM, supplemented with 10% FBS, 100 mgmL streptomycin,
À1
À1
(
(
400 MHz, D O): d=1.84 (quint, J=7.59 Hz, 2H), 2.01 (m, 2H), 2.18
m, 2H), 2.55 (t, J=7.59 Hz, 2H), 3.11 (t, J=6.04 Hz, 2H), 3.77 (s,
100 UmL penicillin, 12.5 UmL nystatin, 2 mm l-glutamine and
2
À1
50 mgmL DNAse. At this point, the tissue was mechanically disso-
3
8
H), 4.17 (t, J=6.04 Hz, 2H), 6.91 (d, J=8.62 Hz, 2H), 7.18 (d, J=
.62 Hz, 2H), 7.40 (s, 1H), 7.44 (s, 1H), 8.59 ppm (s, 1H); C NMR
ciated once more to ensure full dissociation into single cells. The
cells were washed with complete medium once and centrifuged at
1100 rpm for 7 min. The supernatant was removed and the pellet
was re-suspended in complete medium. The cells were plated at
13
(
100.6 MHz, D O): d=28.25, 30.37, 34.98, 36.61, 37.40, 48.20, 56.92,
2
115.53, 121.83, 123.11, 131.26, 136.00, 136.22, 158.58, 178.28 ppm;
+
4
À2
ESI-HRMS (m/z) [M+H] calcd for C H N O : 302.18630, found:
a density of 10 cellscm . Cell cultures were grown in complete
17
23
3
2
3
02.18665.
medium and maintained at 378C and 5% CO2.
&
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The cells were allowed to proliferate until confluence was ach-
ieved; at this point microglia cells were eliminated by shaking at
cate unless otherwise mentioned. For transfected HEK293T cells
treated with 14, compound 14 (200 mm) was added to cell culture
medium three times during the transfection, when the medium
was replaced.
2
50 rpm for 18 h on a horizontal orbital shaker followed by remov-
al of the microglia-containing medium. After a few hours the cells
were trypsinized and re-plated. Astrocyte cultures from this pas-
sage (P1) were used for all studies. Cells were seeded in 96-well
plates at a concentration of 10000 cells per well and allowed to
attach. All experiments were done in serum-free medium. Cell via-
bility was measured by Alamar blue 10% (AbD Serotec, Kidlington,
UK). The results were read at l 590 nm using a Fluostar device.
[29]
Fluorescence microscopy was performed as previously reported.
Two days post-transfection, the transfected HEK293T cells were ex-
amined by fluorescence microscopy using a VistaVision inverted
fluorescence microscope (VWR, Radnor, PA, USA). Images were cap-
tured using a DC-2C digital camera. The fluorescence excitation
wavelength range was between 420 and 485 nm and the emission
wavelength was 515 nm. Flow cytometric analysis of cellular fluo-
[29]
rescence was performed as previously reported. Two days post-
transfection, the transfected HEK293T cells were trypsinized,
washed twice with 1ꢁ PBS and re-suspended in 500 mL 1ꢁ PBS.
The fluorescence intensities of the HEK293T cells expressing SOD1
variant-EGFP fusion protein were measured using a C6 flow cytom-
eter (BD Biosciences, San Jose, CA, USA). The excitation wavelength
was 488 nm and the fluorescence emission was detected at
585 nm. Only GFP-positive cells were used to calculate the mean
cellular fluorescence. Each sample was prepared in triplicate, and
cellular fluorescence indicates mean cellular fluorescence unless
otherwise noted.
MTT cell viability measurement
This assay measures the reduction of a tetrazolium component in
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide]
into an insoluble formazan product by the mitochondria of viable
À1
cells. Cells were incubated with MTT (2 mgmL ) in growth
medium for 30 min at 37C8. The medium was then aspirated, and
DMSO was added to solubilize the colored crystals and absorbance
at 570 nm was measured in an ELISA reader. The amount of color
produced is directly proportional to the number of viable cells.
Western blot protocols
Immunoprecipitation
Western blot of SOD1 and the SDS resistance mutant human SOD1
dimer (32–35 kDa): Cells were treated as described for the MTT
preparation. After that, whole-cell lysates were prepared in the fol-
lowing manner: the lysis buffer contained 50 mm Tris·HCl, pH 7.5,
hWT
hG93A
hG85R
Recombinant SOD1,
SOD1, and
SOD1 were expressed in
sf9 cells and purified using hydrophobic interaction chromatogra-
phy (HIC) and ion-exchange chromatography (IEX), as described
[38]
1
1
mm EDTA, 1 mm EGTA, 1 mm Na VO , 150 mm NaCl, 50 mm NaF,
0 mm sodium glycerophosphate, 5 mm sodium pyrophosphate,
previously. Proteins (4 mg) were solubilized in immunoprecipita-
tion (IP) buffer [50 mm Tris·HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA,
0.5% Nonidet P-40 plus protease inhibitors] and incubated over-
night with B8H10 (MediMabs, Montreal, QC, Canada) antibodies
previously cross-linked to Dynabeads protein G (Invitogen, Wal-
tham, MA, USA) with dimethyl pimelimidate (Pierce, Waltham, MA,
USA) according to the manufacturer’s instructions. The beads were
magnetically isolated and washed three times with IP buffer. Sam-
ples were eluted with boiling in 2ꢁ sample buffer. For immuno-
blotting, proteins were separated by SDS-PAGE, transferred to ni-
trocellulose membranes and probed with goat anti-SOD1 (C-17;
SCB) antibodies.
3
4
and 1 mm PMSF, supplemented with 0.1% (v/v) NP-40, 0.1% (v/v)
b-mercaptoethanol, and protease inhibitor cocktail (1:100 dilution).
The cells were washed with ice-cold PBS, and 1 mL lysis buffer was
then added and incubated at 48C for 40 min. The resulting cell ly-
sates were centrifuged at 8700 g for 30 min at 48C, and the result-
ing supernatant fractions were separated and kept at À208C until
use. Protein content in the supernatant was determined according
to the Bradford assay, using a BSA standard dissolved in the same
buffer. Aliquots (5–60 mg of protein) were mixed with the sample
buffer [62.5 mm Tris·HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol,
5
0 mm DTT, and 0.01% (w/v) bromophenol blue], heated at 958C
for 5 min. The proteins were separated on 10% SDS-PAGE and
western blot analyses were performed using rabbit polyclonal anti-
SOD1 antiserum (Santa Cruz) followed by an HRP-conjugated anti-
rabbit IgG secondary antibody before visualization with ECL plus
solution using the Image Quant program (GE Biosciences, Pitts-
burgh, PA, USA); 25 mg total protein was loaded in each lane.
Proteasome activation
The test was conducted using a proteasome activity assay kit ac-
cording to the manufacturer’s instructions (Abcam, Cambridge,
UK). Lysates of the INS-1E cells were used as a source of the pro-
[39]
teasome.
hG93A
Western blot of ER stress markers in
SOD1 transfected motor
neuron-like cells (NSC-34): A cell lysis and standard western blot–
PAGE analysis were conducted. The CHOP and BiP antibodies were
used according to the manufacturer’s instructions. A total of 25 mg
protein was loaded in each lane. The total protein concentration in
the samples was determined by the Bradford method.
Measurement of mice neurological functions
As disease progresses, SOD1 mice gradually begin to lose their
ability to extend their legs. From day 90 the mice were evaluated
weekly for their hind-limb reflex by a blinded observation. Each
mice was given the following score: 0 for full extension of both
hind limbs, 1 for extension of the legs of no more than 50% of the
capacity, and 2 for no extension in both hind limbs.
SOD1-EGFP transfection, fluorescence microscopic analysis,
flow cytomeric analysis
Transfection of HEK293T cells was performed as previously report-
Statistical analysis
[
29]
ed. Cells were seeded on six-well plates one day prior to trans-
fection. Once the cells grew to 80–90% confluence, they were
transfected with 3.5 mg of the appropriate plasmid via the calcium
phosphate precipitation method. All samples were treated in tripli-
In vitro results are given as mean ÆSEM. Statistical significance
(p<0.05) was calculated among experimental groups using the
two-tailed Student’s t-test. The QuickCalcs online service (Graph-
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11
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Pad Software: www.graphpad.com/quickcalcs/ttest1.cfm was used
for statistical evaluations. Statistical significance for the in vivo
study was determined by ANOVA (Supporting Information
Table S2). The Kaplan–Mayer test was used to determine the statis-
tical significance for the survival test. Significance was considered
if p<0.05.
Osta, A. Ricobaraza, J. Oyarzabal, R. Franco, Curr. Med. Chem. 2011, 18,
5545–5553.
[
[
[
15] S. A. Hitchcock, L. D. Pennington, J. Med. Chem. 2006, 49, 7559–7583.
16] L. Di, H. Rong, B. Feng, J. Med. Chem. 2013, 56, 2–12.
17] a) S. Okuno, K. Sakurada, H. Ohta, H. Ikegaya, Y. Kazui, T. Akutsu, T. Taka-
tori, K. Iwadate, Toxicol. Appl. Pharmacol. 2008, 227, 8–15; b) E. Pop,
W. M. Wu, N. Bodor, J. Med. Chem. 1989, 32, 1789–1795.
[18] T. S. Dowers, J. P. Jones, Drug Metab. Dispos. 2006, 34, 1288–1290.
[19] B. B. Brodie, J. Axelrod, J. Pharmacol. Exp. Ther. 1949, 97, 58–67.
[20] H. Nagahori, Y. Tomigahara, N. Isobe, H. Kaneko, J. Agric. Food Chem.
Acknowledgements
2
009, 57, 10845–10851.
21] H. D. Durham, S. Dahrouge, N. R. Cashman, Neurotoxicology 1993, 14,
87–395.
22] M. E. Gurney, H. Pu, A. Y. Chiu, M. C. Dal Canto, C. Y. Polchow, D. D.
Alexander, J. Caliendo, A. Hentati, Y. W. Kwon, H. X. Deng, W. Chen, P.
Zhai, R. L. Sufit, T. Siddique, Science 1994, 264, 1772–1775.
[
[
This study was supported by the Bar-Ilan University (Israel)
through a new faculty grant (to A.G.) and by the Israeli Ministry
of Industry, Trade and Labor through the KAMIN program (2012–
3
2
015 to D.O. and A.G.). I.K. acknowledges support from the
Korean Government through the National Research Foundation
NRF) of Korea (2014R1A2A1A11050322). A.I. received a grant
[
23] a) C. Gomes, C. Escrevente, J. Costa, Neurosci. Lett. 2010, 475, 145–149;
b) B. Daniel, O. Green, O. Viskind, A. Gruzman, Amyotroph. Lateral Scler.
Frontotemporal Degener. 2013, 14, 434–443; c) A. Neymotin, N. Y. Calin-
gasan, E. Wille, N. Naseri, S. Petri, M. Damiano, K. T. Liby, R. Risingsong,
M. Sporn, M. F. Beal, M. Kiaei, Free Radical Biol. Med. 2011, 51, 88–86.
24] A. M. Haidet-Phillips, M. E. Hester, C. J. Miranda, K. Meyer, L. Braun, A.
Frakes, S. W. Song, S. Likhite, M. J. Murtha, K. D. Foust, M. Rao, A. Eagle,
A. Kammesheidt, A. Christensen, J. R. Mendell, A. H. M. Burghes, B. K.
Kaspar, Nat. Biotechnol. 2011, 29, 824–828.
(
from the Israeli Science Foundation (ISF 124/14). T.G. is the recipi-
ent of a Lev Zion Fellowship. The authors thank Dr. M. Kanovsky
for editing this article.
[
Keywords: amyotrophic
lateral
sclerosis
·
chemical
[
[
[
25] M. D. Kawaja, Curr. Opin. Mol. Ther. 2005, 7, 565–568.
chaperones · drug development · in vivo studies · superoxide
dismutase 1
26] R. Sano, J. C. Reed, Biochim. Biophys. Acta 2013, 1833, 3460–3470.
27] L. I. Bruijn, M. W. Becher, M. K. Lee, K. L. Anderson, N. A. Jenkins, N. G.
Copeland, S. S. Sisodia, J. D. Rothstein, D. R. Borchelt, D. L. Price, D. W.
Cleveland, Neuron 1997, 18, 327–338.
[
[
[
[
[
1] S. Vucic, J. D. Rothstein, M. C. Kiernan, Trends Neurosci. 2014, 37, 433–
442.
[
28] D. R. Rosen, A. C. Bowling, D. Patterson, T. B. Usdin, P. Sapp, E. Mezey, D.
McKenna-Yasek, J. P. O’Regan, Z. Rahmani, R. J. Ferrante, M. J. Brown-
stein, N. W. Kowall, M. F. Beal, H. R. Horvitz, R. H. Brown, Jr., Hum. Mol.
Genet. 1994, 3, 981–987.
2] Y. Sheng, M. Chattopadhyay, J. Whitelegge, J. S. Valentine, Curr. Top.
Med. Chem. 2012, 12, 2560–2572.
3] Z. Xu, Amyotroph. Lateral Scler. Other Mot. Neuron Disord. 2000, 1, 225–
2
34.
4] Y. Iguchi, M. Katsuno, K. Ikenaka, S. Ishigaki, G. Sobue, J. Neurol. 2013,
60, 2917–2927.
[
29] S. Gregoire, S. Zhang, J. Costanzo, K. Wilson, E. J. Fernandez, I. Kwon,
Biotechnol. Bioeng. 2014, 111, 462–474.
2
[
30] P. C. Trippier, K. T. Zhao, S. G. Fox, I. T. Schiefer, R. Benmohamed, J.
Moran, D. R. Kirsch, R. I. Morimoto, R. B. Silverman, ACS Chem. Neurosci.
5] a) A. Gruzman, W. L. Wood, E. Alpert, M. D. Prasad, R. G. Miller, J. D.
Rothstein, R. Bowser, R. Hamilton, T. D. Wood, D. W. Cleveland, V. R. Lin-
gappa, J. Liu, Proc. Natl. Acad. Sci. USA 2007, 104, 12524–12529;
b) D. A. Bosco, G. Morfini, N. M. Karabacak, Y. Song, F. Gros-Louis, P. Pasi-
nelli, H. Goolsby, B. A. Fontaine, N. Lemay, D. McKenna-Yasek, M. P.
Frosch, J. N. Agar, J.-P. Julien, S. T. Brady, R. H. Brown, Jr., Nat. Neurosci.
2
014, 5, 823–829.
[
31] S. Perrin, Nature 2014, 507, 423–425.
[
32] a) A. M. Vincent, S. A. Sakowski, A. Schuyler, E. L. Feldman, Drug Discov-
ery Today 2008, 13, 67–72; b) L. C. Wijesekera, P. N. Leigh, Orphanet. J.
Rare Dis. 2009, 4, 3.
2
010, 13, 1396–1403.
6] E. Kabashi, P. N. Valdmanis, P. Dion, G. A. Rouleau, Ann. Neurol. 2007, 62,
53–559.
[
33] G. D. Couch, P. J. Burke, R. J. Knox, C. J. Moody, Tetrahedron 2008, 64,
[
2816–2823.
5
[
[
[
[
34] V. Boekelheide, W. J. Linn, J. Am. Chem. Soc. 1954, 76, 1286–1291.
35] U. Ellervik, G. Magnusson, J. Org. Chem. 1998, 63, 9314–9322.
36] P. Imming, X. G. Yang, Arch. Pharm. 1994, 327, 747–750.
[7] A. Tiwari, L. J. Hayward, Neurodegener. Dis. 2005, 2, 115–127.
[8] P. J. Hart, Curr. Opin. Chem. Biol. 2006, 10, 131–138.
[9] J. D. Rothstein, Ann. Neurol. 2009, 65, S3–S9.
37] K. Apricꢂ, P. M. Beart, D. Crawford, R. D. O’Shea, J. Neurosci. Res. 2004,
[
10] M. Basso, S. Pozzi, M. Tortarolo, F. Fiordaliso, C. Bisighini, L. Pasetto, G.
Spaltro, D. Lidonnici, F. Gensano, E. Battaglia, C. Bendotti, V. Bonetto, J.
Biol. Chem. 2013, 288, 15699–15711.
7
5, 751–759.
[
38] L. J. Hayward, J. A. Rodriguez, J. W. Kim, A. Tiwari, J. J. Goto, D. E. Cabelli,
J. S. Valentine, R. H. Brown, Jr., J. Biol. Chem. 2002, 277, 15923–15931.
39] L. Pasternak, E. Meltzer-Mats, G. Babai-Shani, G. Cohen, O. Viskind, J.
Eckel, E. Cerasi, S. Sasson, A. Gruzman, Chem. Commun. 2014, 50,
[
[
[
[
11] R. S. Rajan, K. Tsumoto, M. Tokunaga, H. Tokunaga, Y. Kita, T. Arakawa,
Curr. Med. Chem. 2011, 18, 1–15.
12] S. S. Cho, G. Reddy, J. E. Straub, D. Thirumalai, J. Phys. Chem. B. 2011,
[
11222–11225.
1
15, 13401–13407.
13] S. Vang, K. Longley, C. J. Steer, W. C. Low, Glob. Adv. Health Med. 2014, 3,
8–69.
5
14] a) A. F. Collins, H. A. Pearson, P. Giardina, K. T. McDonagh, S. W. Brusilow,
Received: January 29, 2015
G. J. Dover, Blood 1995, 85, 43–49; b) M. Cuadrado-Tejedor, A. Garcꢃa-
Published online on && &&, 0000
&
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FULL PAPERS
Low concentration efficacy: Com-
pound 14 demonstrated biological ef-
fects in ALS mice, showing the preven-
tion of body mass loss and improve-
ments in neurological function. In vitro
studies revealed that this lead com-
pound significantly decreases the for-
mation of misfolded mutated super-
oxide dismutase 1 (SOD1) and prevents
ER-stress-induced apoptosis. In addition,
T. Getter, I. Zaks, Y. Barhum, T. Ben-Zur,
S. Bçselt, S. Gregoire, O. Viskind, T. Shani,
H. Gottlieb, O. Green, M. Shubely,
H. Senderowitz, A. Israelson, I. Kwon,
S. Petri, D. Offen, A. Gruzman*
&& – &&
A Chemical Chaperone-Based Drug
Candidate is Effective in a Mouse
Model of Amyotrophic Lateral
Sclerosis (ALS)
14 decreased the levels of known ER
stress markers and decreased the forma-
tion of misfolded mutant SOD1 aggre-
gates.
ChemMedChem 0000, 00, 0 – 0
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13
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ