Potent aminocyclitol glucocerebrosidase inhibitors are subnanomolar pharmacological chaperones for treating gaucher disease

Article abstract of DOI:10.1021/jm300342q

Amino-myo-inositol derivatives have been found to be potent inhibitors of glucocerebrosidase (GCase), the β-glucosidase enzyme deficient in Gaucher disease (GD). When tested using lymphoblasts derived from patients with GD homozygous for N370S or L444P mu

Full text of DOI:10.1021/jm300342q

Article
pubs.acs.org/jmc
Potent Aminocyclitol Glucocerebrosidase Inhibitors are
Subnanomolar Pharmacological Chaperones for Treating Gaucher
Disease
Ana Trapero,† Patricia Gonzalez-Bulnes,† Terry D. Butters,‡ and Amadeu Llebaria*,†
́
†Departament de Química Biomed
Barcelona, Spain
̀
ica, Institut de Química Avanca̧ da de Catalunya (IQAC−CSIC), Jordi Girona 18-26, 08034
‡Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United
Kingdom
S
* Supporting Information
ABSTRACT: Amino-myo-inositol derivatives have been
found to be potent inhibitors of glucocerebrosidase (GCase),
the β-glucosidase enzyme deficient in Gaucher disease (GD).
When tested using lymphoblasts derived from patients with
GD homozygous for N370S or L444P mutations, the
compounds enhanced GCase activity at very low concen-
trations. The most potent inhibitor, (1R,2S,3R,4S,5S,6R)-5-
(nonylamino)-6-(nonyloxy)cyclohexane-1,2,3,4-tetraol had a
Ki of 1 nM using isolated enzyme and an IC50 of 4.3 nM when
assayed in human fibroblast cell culture. This aminocyclitol
produced maximum increases of GCase activities of 90% in
N370S lymphoblasts at 1 nM and 40% in L444P at 0.01 nM
following a three-day incubation. In addition to inhibitory
potency, this compound has the permeability, subcellular distribution, and cell metabolism characteristics that are important for
use as a pharmacological chaperone. It is a remarkable finding that picomolar concentrations of aminocyclitols are sufficient to
enhance activity in the L444P variant, which produces a severe neuronopathic form of GD without clinical treatment.
therapeutic approach, now referred to as pharmacological
chaperone therapy (PCT), has emerged as a promising
alternative. PCT is based on the use of competitive GCase
inhibitors that are capable of enhancing residual hydrolytic
activity at subinhibitory concentrations.9 This counterintuitive
approach may be rationalized by the fact that although the
defective enzyme is predisposed to misfolding and/or
instability, it is still catalytically active. Recent evidence showing
that accumulation of substrate in GD results from low levels of
functional GCase protein instead from a low intrinsic catalytic
GCase activity10 would reinforce the feasibility of the PCT
approach. Reversible competitive inhibitors alter or stabilize the
defective and misfolded GCase, avoiding its premature
degradation in the endoplasmic reticulum (ER) by the quality
control mechanism of the cell and allowing its normal
trafficking to the lysosome. Thus, PCT is highly promising
for the treatment of GD; this strategy indeed combines the
benefits of the small-molecule approach, including oral
bioavailability and the potential to cross the blood−brain
barrier, with the specificity of an enzyme-directed approach.
INTRODUCTION
■
Glycosphingolipid lysosomal storage disorders, also named
glycosphingolipidoses, are a group of inherited diseases caused
by defects in the lysosomal degradation of glycosphingolipids.1
Among them, Gaucher disease (GD) is the most prevalent, and
it is caused by a deficiency in the activity of β‑glucocere-
brosidase (GCase, acid β-glucosidase, EC 3.2.1.45) encoded by
GBA1 gene, which catalyzes cleavage of the β‑glycosidic bond
of glucosylceramide to release glucose and ceramide.2
A
deficiency in enzyme activity results in the accumulation of
undegraded substrate in the lysosomes of macrophages, causing
severe symptoms. Clinically, GD is classified into three major
types based on the absence (type 1) or the presence and
severity (types 2 and 3) of central nervous system
involvement.3 The two most prevalent missense mutant
forms of GCase reported in patients with GD are N370S and
L444P.4 Patients homozygous or heterozygous for N370S
GCase typically present with a non-neuronopathic form of GD,
whereas those homozygous for L444P GCase usually display a
more severe neuronopathic form.
Current therapeutic strategies5 for GD involve either enzyme
replacement therapy,6 or pharmacological GCase substrate
reduction,7 which are of limited efficacy for disease variants
affecting the central nervous system.8 In the past decade, a new
Received: March 12, 2012
Published: April 19, 2012
© 2012 American Chemical Society
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This gives the opportunity also to treat types of lysosomal
storage diseases involving the central nervous system.
In recent years, a number of different classes of non-
carbohydrate11 and carbohydrate-like compounds, including
diaminocyclitols,12 polyhydroxylated bicyclic isoureas and
guanidines,13 isofagomine,14−16 iminoxylitols (α-C9-DIX and
2-O-hexyl-DIX),17,18 polyhydroxylated lactams,19 bicyclic nojiri-
mycin and galactonojirimycin analogues (NOI-NJ and 6S-NOI-
GNJ),20,21 and deoxynojirimycin derivatives (NN-DNJ)22,23
have been reported as GCase inhibitors. Therefore, these
compounds have potential as pharmacological chaperones for
the treatment of GD and induce enhancement of the GCase
activity in mutant cell lines (Figure 1).
Figure 2. Chemical structures of aminocyclitol derivatives evaluated in
this work.
hydroxyl group. In addition, the X-ray crystal structure of
GCase indicates the existence of an annulus of hydrophobic
residues around the entrance to the glucose binding site27 that
generate two hydrophobic pockets in the GCase active center.28
We reasoned that aminocyclitol recognition could be increased
by the presence of two alkyl groups, which would mimic the
two lipophilic chains present in glucosylceramide, the natural
substrate of GCase. Therefore, the hydroxyl at C2 position of
compounds 1 and 3 was replaced with a methyl or a nonyl
ether groups to give amino-scyllo-inositols 4−5 and amino-myo-
inositols 6−7, respectively. Finally, we obtained the O-nonyl
primary amine myo-8 and the N,N-dinonyl substituted amino-
myo-inositol 9 designed to have a wider coverage of the alkyl
group effects on GCase inhibition.
The biological activity of these new aminocyclitol derivatives
was determined using recombinant GCase, and the selectivity
inhibitory profile of compounds examined for other glyco-
sidases and glucosylceramide synthase (GCS) from cell
homogenates. We have also studied the selectivity of the
compounds for the lysosomal β-glucosidase enzyme (GCase)
vs the nonlysosomal β-glucosidase enzyme encoded by the
GBA2 gene in mouse tissue homogenates. After studying the
toxicity of compounds, the inhibitory effect of aminocyclitol
derivatives was examined in wild-type human fibroblasts. Next,
the effect of these compounds on the stabilization of
recombinant GCase activity after thermal denaturation was
determined as a measure for potential pharmacological
chaperone activity. Furthermore, these compounds effects as
pharmacological chaperones for N370S (non-neuronopathic
form) or L444P (neuronopathic form) GCase mutants and
hence their potential as therapeutics for treating GD were also
investigated.
Figure 1. Chemical structures of pharmacological chaperones for
GCase.
In this context, aminocyclitols also have potential applica-
bility as pharmacological chaperones for GCase.24−26 In
particular, N-nonyl substituted amino-scyllo-inositol 1 (Figure
2) is a competitive inhibitor of recombinant GCase and was
able to stabilize the enzyme against thermal denaturation24 but
had little effect in fibroblasts obtained from Gaucher patients in
enhancing enzyme activity.25 A program on optimization of this
family was undertaken to develop new aminocyclitol derivatives
with improved pharmacological chaperone profiles for the
treatment of GD. We have recently reported several potent
GCase inhibitors having a bicyclic structure13 or a diaminocy-
clohexane scaffold12 with promising cellular activities in
Gaucher cells. Interestingly, a strong effect of the stereo-
chemistry of the cyclohexane nitrogen and hydroxyl sub-
stituents on the biological activity was evident in these families.
These studies have concluded that the compounds with better
affinity for the active site of the enzyme are those having myo-
configuration, a type of stereochemistry not previously
considered in the aminocyclitols having a single nitrogen
substitution such as 1, where a scyllo scaffold was always
retained.24,26 These precedents stimulated the study of the
stereochemical effects on the activity of this family of
compounds. Accordingly, we synthesized several amino-myo-
inositols (Figure 2) by inverting the configurations of two
cyclitol stereogenic centers in scyllo-1 at either C1 (compound
2), bearing the amino group, or C2 (compound 3), bearing a
CHEMISTRY
■
The final enantiopure aminocyclitols 3−9 and achiral 2 were
synthesized from azido alcohols 10−16 according to the
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a
Scheme 1. Synthesis of Aminocyclitol Derivatives 2−9
a
Reagents and conditions: (a) NaH, C9H19I or CH3I, DMF, 0 °C, 71−93%; (b) LiAlH4, THF, rt, 72−95%; (c) C8H17CHO, NaBH3CN, AcOH,
MeOH, rt, 75−90%; (d) Pd/C, THF, HCl, H2 (2 atm), rt, 86−94%.
sequence described in Scheme 1. The starting materials, azido
alcohols 10 and 11, were obtained by the regio- and
diastereoselective opening of racemic tetra-O-benzyl conduritol
B epoxide29 and enantiopure tetra-O-benzyl conduritol B
epoxide,30 respectively, and azido alcohol 14 was synthesized
from intermediate 11 according to our previously reported
route.30
Amino-myo-inositols 2 and 3 were synthesized by reductive
amination of nonanal with amino alcohols 1729 and 20,30
respectively, to give the corresponding protected N-nonyl
aminocyclitols, which were transformed into the final
compounds after O-benzyl hydrogenolysis (Scheme 1). A
similar strategy was followed for the preparation of the N,N-
dinonyl aminocyclitol 9 from amino alcohol 20, but in this case,
an excess of aldehyde (2.5 equiv) and NaBH3CN (5 equiv)
were used in the reductive amination step.
The N,O‑dialkyl aminocyclitols 4−7 were obtained by
O‑alkylation of azido alcohols 11 and 14, followed by azide
reduction, reductive amination, and O-benzyl deprotection.
Finally, simultaneous removal of the O-benzyl groups and
reduction of the azido group in intermediate 16 by catalytic
hydrogenation over Pd/C catalyst under acidic conditions
afforded the O-nonyl primary amine 8 (Scheme 1) .
at pH 5.2. The inhibitory activities of all compounds against
imiglucerase are summarized in Table 1.
The N-nonyl aminocyclitols 1 and 2 exhibited Ki values of
1.924 and 17.9 μM, respectively. Surprisingly, N-nonyl amino-
cyclitol 3 (myo-configuration) was found to be a 60-fold better
inhibitor than 1 (scyllo-configuration) and 562-fold better than
2 (myo-configuration), the compound epimeric with 1 at the
nitrogen substituted cyclohexane carbon. Therefore, the
Table 1. Inhibitory Activity of Aminocyclitols 1−9 against
Imiglucerase
IC50 (μM)
a
compd
pH 7.0
pH 5.2
Ki (μM)
b
b
1
0.78
24.9
3.9
1.9
2
52.6
0.08
4.4
17.9
3
0.025
0.67
0.032
1.58
4
5
0.17
0.67
0.13
0.004
0.45
1.72
0.28
6
0.019
0.005
0.12
0.053
0.001
0.13
7
8
9
0.53
0.82
c
c
BIOLOGICAL RESULTS AND DISCUSSION
NN-DNJ
0.30
0.66
0.30
■
a
Imiglucerase Inhibition. Compounds 2−9 were evaluated
as inhibitors of recombinant GCase (imiglucerase, Cerezyme)
The inhibition was competitive in all cases (determined at pH 5.2).
See ref 24. See ref 17.
b
c
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configurations of amino groups and hydroxyl groups (encircled
in Figure 2) have an important effect on imiglucerase
inhibition. However, the N,N-disubstituted aminocyclitol 9
was significantly less active than the N-alkyl substituted
aminocyclitol 3, an observation that is in agreement with the
results previously reported24 in a related family of amino-
cyclitols where the N,N-dialkylated compounds were also found
to be much less active than N-monoalkylated derivatives.
The Ki values of the O-methyl aminocyclitols 4 and 6 were
similar to, or slightly higher than, the hydroxylated amino-
cyclitols 1 and 3, respectively. In contrast, extension of the
length of the O-alkyl chain improved the potency considerably
(compounds 5 and 7). The most potent inhibitor found in this
work, the N,O-dinonyl aminocyclitol 7, displayed a remarkable
Ki value of 1 nM, which is 32-fold more potent than N-nonyl
aminocyclitol 3 and 53-fold than O-methyl aminocyclitol 6.
However, the free amine 8 was much less active, indicating that
the presence of N-alkyl chain in the inhibitor is favorable for the
inhibition of imiglucerase.
Active-site binding GCase inhibitors are pharmacological
chaperone candidates, and expected to interact with cellular
GCase in the ER, assisting folding and trafficking at neutral pH.
Consequently, aminocyclitols 2−9 were also evaluated as
inhibitors of imiglucerase at pH 7.0 to determine the effect of
pH on its activity (see Table 1). For comparative purposes, our
previously reported aminocyclitol 124 and NN-DNJ were also
tested under the same experimental conditions. Interestingly,
aminocyclitols 1−9 inhibit recombinant GCase at pH 7.0 and
5.2 with comparable potencies, indicative of a similar activity on
the enzyme at both ER and lysosome pH environments.
Inhibition of GCase in Wild-Type Human Fibroblasts.
The cellular GCase inhibition23 of aminocyclitols 1−9 and NN-
DNJ was also studied in wild-type human fibroblasts after 24 h
of incubation at 50 μM. Because at this concentration inhibitors
5, 7, and 9 displayed signs of cytotoxicity, these aminocyclitols
were tested at 5 μM, well below the CC50 determined for these
compounds (see Table S2, Supporting Information). The
GCase inhibition in wild-type human fibroblasts at above
concentrations is shown in Figure 4A. A good correlation
between the Ki values against imiglucerase and GCase
inhibition in cell culture was observed, except for the N,N-
disubstituted aminocyclitol 9, which showed a noticeable
inhibition in cellular assays, higher than could be expected
from data obtained in isolated enzyme inhibition experiments.
The iminosugar NN-DNJ and aminocyclitol derivatives 1, 2,
and 4 showed between 23 and 56% of inhibition at 50 μM,
whereas aminocyclitols 3, 5−8, and 9 displayed more than 39%
of GCase inhibition at 5 μM (see Figures 3B, 4A, and Figure
S8, Supporting Information). The high potency of compounds
3, 5−8, and 9 required analysis at lower concentrations,
showing that these are extremely potent cellular GCase
inhibitors at nanomolar to low micromolar concentrations,
with aminocyclitol 7 being the most potent inhibitor of cellular
GCase with a IC50 value of 4.3 nM.
All tested compounds were found competitive inhibitors of
imiglucerase, as illustrated in Figure 3A for 7 (see also Figures
S1−S7, Supporting Information). It is worth mentioning the
low Ki values found for amino-myo-inositols 3, 6−7, and 8 that
were in the 1−130 nM range. The reasons for the increase in
potency in compounds 3−9 compared with 1 are presently
unclear and will be addressed in future structural studies.
Effects of Compounds on the Activity of other
Glycosidases and GCS. To test the selectivity profile, the
new aminocyclitol derivatives 2−9 were tested as inhibitors
against a series of commercial glycosidases (see Table S1,
Supporting Information), including α-glucosidase (baker’s yeast
and rice), β-glucosidase (crude almond), β-galactosidase
(bovine liver), and α-galactosidase (green coffee beans). All
compounds were inactive when tested at 100 μM for
α‑galactosidase, almond β-glucosidase, and α-glucosidases.
Moreover, only aminocyclitols 2, 7, and 8 displayed weak
inhibition of β-galactosidase (between 74 and 84% inhibition at
100 μM).
The selectivity of compounds 1−9 for GCase was also
evaluated toward other lysosomal glycosidases in wild-type
human fibroblasts. After 24 h of incubation, less than 16%
inhibition of the lysosomal α-glucosidase, α-galactosidase, and
β-galactosidase enzymes by compounds 5 and 9 was observed
at 5 μM, whereas the rest of compounds showed no appreciable
inhibition (see Figure 4A for inhibitory data of all compounds
toward all enzymes mentioned). In stark contrast, the
iminosugar NN-DNJ showed no anomer specificity and
behaved as a strong inhibitor of lysosomal α-glucosidase and
β-glucosidase, in agreement with literature reports.21,31
Finally, only aminocyclitol derivatives 5, 7, and 9 showed
moderate inhibition (between 56 and 98%) on GCS from cell
homogenates at 250 μM, but were inactive when tested at 50
μM (see Table S1, Supporting Information). This result is in
agreement with the correlation between lipophilicity and the
Figure 3. Aminocyclitol 7 is a potent inhibitor of GCase.
(A) Lineweaver−Burk plot for the inhibition of imiglucerase by 7
(empty square, 0 nM; triangle, 1.56 nM; solid square, 3.13 nM; circle,
4.68 nM) at pH 5.2 (Ki = 1.03 nM). Regression lines rise from data
obtained in two different experiments with duplicates. (B) GCase
inhibition of aminocyclitol 7 in wild-type human fibroblasts after 24 h
incubation time at the indicated inhibitor concentrations. Experiments
were performed in triplicate, and the mean SD is shown.
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Figure 4. (A) Inhibition of lysosomal human β-glucosidase (GCase), α-glucosidase (α-Glu), α-galactosidase (α-Gal), and β-galactosidase (β-Gal) in
intact wild-type human fibroblasts after 24 h of incubation at nontoxic concentrations: 5 μM for 5, 7, and 9 and 50 μM for the rest of the
compounds. Experiments were performed in triplicate, and bars represent the mean SD. (B) Inhibition of GCase (black bar) and GBA2 (gray bar)
in mouse tissue homogenates. Compounds were assayed at 50 and 5 μM (see also Figure S9, Supporting Information) and 3 mM substrate. Each bar
represents the mean SD of two independent experiments with duplicates.
GCS inhibitory potency that was also observed in other series
of compounds.32
compounds as pharmacological chaperones.23,24 In this context,
the effect of aminocyclitols on the stabilization of recombinant
GCase activity after thermal denaturation at 48 °C was
determined in the presence and in the absence of increasing
concentrations of compounds at different incubation times (see
Figures S10 and S11, Supporting Information) as a measure for
potential pharmacological chaperone activity. For the matter of
comparison, NN-DNJ23 and aminocyclitol 124 were also tested
under the same experimental conditions. The stabilization ratio
values found for each compound at 50 μM (for 1−2, 4, and
NN-DNJ) or 1 μM (for 3, 5−8, and 9) and 60 min incubation
time are summarized in Table 2. The N-nonyl aminocyclitols
1−2 at 50 μM and N,N-disubstituted aminocyclitol 9 at 1 μM
exhibited stabilization ratios between 1.2 and 2.3 after 1 h of
incubation time at 48 °C, clearly less potent than compounds 3,
5−7, and 8, which showed stabilization ratios between 2 and 29
at only 1 μM, and aminocyclitol 4 with a stabilization ratio of
14.3 at 50 μM.
Enzyme Enhancement Activity in Human N370S and
L444P Lymphoblasts. The pharmacological chaperone
potential of aminocyclitol derivatives was further evaluated in
human lymphoblasts derived from Gaucher patients homo-
zygous for N370S or L444P variants, which are the two most
common mutations associated with GD.4 The enhancement of
β-glucosidase activity in intact cells induced by incubation with
the tested compounds served for this purpose as previously
reported.12,13 The fold-increase in enzyme activity and the
pharmacological chaperone concentration where maximal
GCase activity was observed is summarized in Table 2 (see
Effects of Compounds on GCase and GBA2 Activity in
Mouse Tissue Homogenates. To distinguish between the
lysosomal GBA1 and nonlysosomal GBA2 cellular β‑glucosi-
dases, the compounds were assayed for β-glucosidase inhibition
in mouse tissue homogenates. All compounds were initially
screened for GCase and GBA2 inhibition at 50 and 5 μM
(Figure 4B). NN-DNJ, a potent inhibitor of GBA2,32 was
included as a reference. As a general trend, the inhibition of
GCase in mouse tissue homogenates was higher than that
measured in intact fibroblasts. Similarly, the lysosomal GCase
inhibitory activity was usually stronger than that observed in
nonlysosomal GBA2 enzyme. The iminosugar NN-DNJ and
aminocyclitols 2, 3, and 9 displayed a significant GBA2
inhibition at 50 μM, whereas the rest of compounds showed
lower inhibition at the same concentration. Surprisingly, the
N‑nonyl aminocyclitol 2, which exhibited moderate inhibitory
activity against GCase showed 92% and 69% of GBA2
inhibition at 50 and 5 μM, respectively. The results obtained
with the compounds at lower concentrations are given in the
Supporting Information (Figure S9).
Stabilization of Recombinant GCase (Imiglucerase)
under Thermal Denaturation Conditions. Enzyme stabili-
zation under thermal denaturation conditions was used as an
indication of the potential of the target compounds to behave
as pharmacological chaperones. The recovery of recombinant
GCase activity after thermal denaturation is generally used as an
in vitro model to evaluate the potential of the tested
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N370S lymphoblasts with 1 nM of 7 led to a 1.9-fold maximal
increase in N370S GCase activity, thereafter its activity
gradually decreased with concentration (Figure 5A). This
high cellular activity is in contrast with other compounds tested
as pharmacological chaperones on cellular assays, which require
micromolar concentrations, usually at well over their Kis when
determined using isolated GCase enzyme. Prior to the present
data, the superior pharmacological chaperone activities at
nanomolar concentrations have been reported using α-C9-
DIX,17 2-O-hexyl-DIX,18 bicyclic isourea and guanidine
compounds,13 or diaminocyclitols.12 The use of very low
concentrations of chaperones is highly desirable due to the
increase in selectivity for GCase and potentially lower side
effects.
The effect of compounds 1−8 on L444P GCase activity,
which characterize a more severe disease phenotype than
N370S, were measured using GD type 2 patient-derived
lymphoblast cell line. After a three-day incubation, amino-
cyclitols 1 and 2 showed no effect or inhibited GCase at high
concentrations. However, 3−8 showed a moderate increase in
GCase activity (1.2−1.5-fold) at low concentrations. It is worth
noting the impressive potency of 7, which produced a 40%
increase in GCase activity of the L444P lymphoblast cell line at
a 10 picomolar concentration (Figure 5B). Under similar
conditions, NN-DNJ was found to be completely inactive. The
activity of the aminocyclitols on L444P GCase enhancement of
activity is a remarkable feature because this mutation is highly
averse to chaperone treatment. Several reports describe the use
of pharmacological chaperones for different GCase variants
that, however, failed in L444P cell assays.15,21,23,33 This lack of
activity has been attributed to the distant location of the
mutation site from the enzyme catalytic center. To our
knowledge, only isofagomine,14 ambroxol,34 and α-1-C-octyl-
1-deoxynojirimycin31 have been reported to increase activity in
L444P Gaucher cells. However, with the exception of our
previous reports on aminocyclitol compounds,12,13 all these
compounds must be used at micromolar concentrations to
increase GCase activity significantly in cells. The amino-myo-
inositol derivatives here described increased enzyme activity
20−40% in L444P and 80−100% in N370S lymphoblasts at
nanomolar or even subnanomolar concentrations, as shown in
Figure 5 (see also Supporting Information Figures S12 and
S13). It is apparent that, in addition to their high enzyme
affinities, these compounds reflect favorable cell permeability,
Table 2. Enzyme Stabilization Ratio of Imiglucerase after
Thermal Denaturation at 48 °C and Maximum Observed
Increase in GCase Activity Using Pharmacological
Chaperones and NN-DNJ
stabilization
N370S GCase activity
L444P GCase
b
a
b
compd
ratio
increase
activity increase
cd
,
1
1.4
no activity
no activity
no activity
no activity
d
2
2.3
3
5.0
1.8 0.1 (500 nM)
1.7 0.1 (10 μM)
1.4 0.2 (1 μM)
2.0 0.2 (1 μM)
1.9 0.1 (1 nM)
1.8 0.1 (5 μM)
no activity
1.2 0.1 (100 nM)
1.5 0.2 (1 μM)
1.2 0.1 (5 nM)
1.4 0.2 (50 nM)
1.4 0.2 (0.01 nM)
1.3 0.1 (100 nM)
d
4
14.3
5
5.2
2.0
6
7
29.0
2.2
8
e
9
1.2
ND
d
NN-DNJ
7.6
1.4 0.1 (5 μM)
no activity
a
Defined as the ratio of relative enzymatic activities (inhibitor vs
control) at a given inhibitor concentration and incubation time.
Tabulated values for 1 μM inhibitor and 60 min incubation time.
b
N370S and L444P lymphoblasts were incubated with test compounds
for 3 days before being used for enzyme assay. Data in parentheses
correspond to the concentration of the tested compound. Experiments
were performed in triplicate, and the mean
SD is shown. The
relative activity was obtained by normalizing the activity corresponding
to each compound concentration tested to the activity of untreated
c
d
cells. See ref 24. Tabulated values for 50 μM inhibitor and 60 min
e
incubation time. ND: not determined.
also Supporting Information Figures S12 and S13). The
iminosugar NN-DNJ, which is a known chaperone for
N370S, but not L444P GCase, was used as a control.23
In the analysis of N370S GCase activation, aminocyclitols 1,
2, and 9 showed no β-glucosidase activity enhancement effects
at all concentrations tested. In contrast, aminocyclitols 3−8
increased enzyme activity over a range of concentrations.
Treatment with 10 μM of 4 or 5 μM of 8 caused 1.7- and 1.8-
fold increase in the GCase activity of N370S cell line,
respectively, whereas aminocyclitols 3, 5, and 6 maximally
increased the activity between 1.4- and 2.0-fold at lower
concentrations (1 or 0.5 μM). Using similar conditions, NN-
DNJ gave a maximal enhancement of 1.4-fold at 5 μM. The
N,O-dinonyl aminocyclitol 7 was inhibitory at concentrations
higher than 100 nM in accordance with the potent inhibition
on isolated enzyme observed. However, treatment of the
Figure 5. Aminocyclitols 6 and 7 increase N370S (A) and L444P (B) GCase activity in cells derived from patients with GD. N370S or L444P GCase
patient-derived lymphoblasts were cultured for 3 days in the absence or presence of increasing concentrations (nM) of compounds before GCase
activity was measured. Experiments were performed in triplicate, and each bar represents the mean SD. Enzyme activity is normalized to untreated
cells, assigned a relative activity of 1.
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EtOAc gradient) to afford 180 mg (0.26 mmol, 90% yield) of 16. [α]D25
+7.6 (c 1.0, CHCl3). IR (film): υ = 3089, 3064, 3031, 2925, 2855,
subcellular distribution, and cell metabolism properties required
for pharmacological chaperones.
1
2106, 1497, 1454, 1360, 1137, 1065, 1028, 734, 697 cm−1. H NMR
It is not yet clear how the optimization of concentrations or
the inhibitory potency of compounds for enzyme enhancement
in cell culture will translate to optimal chaperone doses in vivo.
What is remarkable is that the aminocyclitols reported here
afford potent GCase pharmacological enhancement of activity
at the lowest concentrations reported to date. Moreover, this
activity is extended to the L444P mutation, which produces a
severe neuronopathic form of GD that lacks an effective clinical
treatment. Further optimization of these amino-myo-inositols
and its ethers on other mutations and in animal models that
might lead to clinical candidates for therapy is warranted. In
addition, the structural determinants of the binding of these
high affinity inhibitors and the reasons for the activity
enhancement of a GCase enzyme mutated at an amino acid
distant from the active site are also of interest. These studies are
currently in progress.
(δ, 500 MHz, CDCl3): 0.91 (t, 3H, J = 6.8 Hz), 1.08−1.44 (m, 12H),
1.46−1.70 (m, 2H), 3.17 (dd, 1H, J = 10.3, 1.9 Hz), 3.41 (dd, 1H, J =
9.8, 1.8 Hz), 3.53 (t, 1H, J = 9.2 Hz), 3.67−3.71 (m, 1H), 3.88−3.92
(m, 2H), 3.99−4.04 (m, 2H), 4.73−4.97 (m, 8H), 7.26−7.42 (m,
20H). 13C NMR (δ, 100 MHz, CDCl3): 14.3, 22.8, 26.2, 29.5, 29.6,
29.8, 30.4, 32.1, 63.4, 73.1, 73.9, 75.7, 76.1, 77.1, 80.2, 81.6, 81.7, 84.5,
127.7−128.6, 138.15, 138.22, 138.56, 138.71. HRMS calculated for
C43H53N3O5Na, 714.3883 [M + Na]+; found, 714.3898.
General Procedure for the Reduction of Azides 12−13 and
15−16: Synthesis of 22 as a Representative Example. A solution
of the azide 16 (120 mg, 0.17 mmol) in anhydrous THF (4 mL) was
added dropwise under argon a solution of LiAlH4 (14 mg, 0.34 mmol)
in anhydrous THF (4 mL) at 0 °C. After stirring for 3 h at room
temperature, the mixture was cooled down to 0 °C and quenched with
dropwise addition of aqueous saturated Na2SO4 solution. The solution
was diluted with EtOAc, dried over MgSO4, and filtered through a plug
of Celite, which was washed three times with EtOAc. The combined
filtrates and washings were concentrated under reduced pressure to
give amine 22 (90 mg, 0.13 mmol, 80% yield), which was used in the
next reaction without further purification. [α]2D5 −47.1 (c 1.0, CHCl3).
IR (film): υ = 3080, 3060, 3033, 2924, 2854, 1497, 1454, 1362, 1090,
CONCLUSIONS
■
The synthesis of some N-alkyl amino-myo-inositol derivatives
has confirmed that this configuration results in compounds
with high affinity for the active site of GCase, the lysosomal
enzyme mutated in GD. Moreover, when an O-alkyl group is
present, the potency of the compounds is further increased,
leading to compounds with positive enzyme activity enhance-
ment in Gaucher L444P and N370S cells at subnanomolar
concentrations. The compounds obtained are highly selective
for GCase and reveal advantageous enzymatic and cellular
properties to support further studies as pharmacological
chaperones for GD.
1
1069, 1024, 732, 696 cm−1. H NMR (δ, 500 MHz, CDCl3): 0.92 (t,
3H, J = 6.7 Hz), 1.24−1.33 (m, 12H), 1.50−1.63 (m, 2H), 2.46 (d,
1H, J = 9.5 Hz), 3.40−3.64 (m, 4H), 3.80−3.83 (m, 1H), 3.98−4.07
(m, 2H), 4.67−5.04 (m, 8H), 7.24−7.35 (m, 20H). 13C NMR (δ, 100
MHz, CDCl3): 14.3, 22.8, 26.3, 29.5, 29.6, 29.8, 30.6, 32.1, 54.3, 72.9,
73.6, 75.85, 75.86, 76.0, 78.1, 82.3, 83.0, 83.3, 85.1, 127.6−128.7,
138.4, 138.72, 138.73, 138.9. HRMS calculated for C43H56NO5,
666.4158 [M + H]+; found, 666.4156.
General Procedure for Reductive Amination: Synthesis of 28
as a Representative Example. A solution of the amine 22 (89 mg,
0.14 mmol) in MeOH (5 mL) under an atmosphere of argon was
treated successively with NaBH3CN (18 mg, 0.28 mmol), AcOH (8
μL), and nonanal (24 μL, 0.14 mmol). After stirring for 4 h at room
temperature, the mixture was quenched with water (0.2 mL) and the
solvents were removed under reduced pressure. The resulting residue
was dissolved in Et2O (20 mL) and washed with water (15 mL). The
aqueous phase was extracted with Et2O (3 × 20 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
evaporated to dryness to give a residue, which was purified by flash
chromatography (20:1 to 5:1 hexane/EtOAc gradient) to give 28 (85
mg, 0.11 mmol, 75% yield). [α]2D5 −18.4 (c 1.0, CHCl3). IR (film): υ =
3088, 3062, 3029, 2955, 2924, 2854, 1497, 1467, 1454, 1362, 1133,
EXPERIMENTAL SECTION
■
Chemistry: General Methods. Solvents were distilled prior to use
and dried by standard methods. FT-IR spectra are reported in cm−1.
1
Unless otherwise stated, H and 13C NMR spectra were obtained in
CDCl3 solutions at 500 MHz (for 1H) and 100 MHz (for 13C).
Chemical shifts (δ) are given in ppm relative to the residual solvent
peak (CDCl3: 1H, δ = 7.26 ppm; 13C, δ = 77.16 ppm), and the
coupling constants (J) are reported in hertz (Hz). Optical rotations
were measured with a Perkin-Elmer model 341 polarimeter, and
specific rotations are reported in 10−1 deg cm2 g−1. ESI/HRMS spectra
were recorded on a Waters LCT Premier mass spectrometer. The
purity of all tested compounds was >97% as determined by HPLC on
an Alliance 2695 system using a Kinetex C18 (4.6 mm × 50 mm, 2.6
μm) column under the following chromatography conditions: mobile
phase A, H2O with 0.2% HCO2H; mobile phase B, CH3CN with 0.2%
HCO2H; flow rate, 1.0 mL min−1; injection volume, 25 μL; elution
gradient, 0.0−2.9 min, 5−90% B; 2.9−3.4 min, 90% B; 3.4−4.6 min,
90−100% B; 4.6−6.0 min, 100−5% B; 6.0−10.0 min, 5% B. An
evaporative light scattering detector (ELSD, model PL-ELS 1000,
Polymer Laboratories) was used with the following parameters: a gas
flow rate of 1.5 L min−1, a nebulizer temperature of 80 °C, and an
evaporator temperature of 90 °C.
1
1088, 1070, 732, 696 cm−1. H NMR (δ, 500 MHz, CD3COCD3):
0.85−0.90 (m, 6H), 1.20−1.39 (m, 24H), 1.39−1.51 (m, 2H), 1.57−
1.63 (m, 2H), 2.50−2.70 (m, 2H), 2.75−2.90 (m, 1H), 3.48 (t, 1H, J =
9.1 Hz), 3.56 (dd, 1H, J = 9.8, 1.5 Hz), 3.64 (t, 1H, J = 9.4 Hz), 3.71
(dt, 1H, J = 9.0, 6.4 Hz), 3.84−3.88 (m, 1H), 3.91 (t, 1H, J = 9.5 Hz),
3.97 (dt, 1H, J = 8.9, 6.4 Hz), 4.14 (br s, 1H), 4.71−4.94 (m, 8H),
7.22−7.42 (m, 20H). 13C NMR (δ, 100 MHz, CD3COCD3): 14.4,
23.34, 23.37, 27.0, 28.1, 29.2, 29.9, 30.1, 30.3, 30.4, 30.5, 31.2, 32.6,
32.7, 48.4, 62.0, 73.0, 73.8, 75.3, 75.9, 76.0, 82.7 (2), 83.4, 85.6, 128.0−
129.0, 140.00, 140.30, 140.35, 140.45. HRMS calculated for
C52H74NO5, 792.5567 [M + H]+; found, 792.5578.
General Procedure for Debenzylation by Catalytic Hydro-
genation: Synthesis of Aminocyclitol 7 as a Representative
Example. In a glass pressure flask, the benzylated amino compound
28 (80 mg, 0.10 mmol) was dissolved in a mixture of THF (3 mL) and
concentrated HCl (4 drops). Pd/C (40 mg, 5−15% Pd on activated C,
water-wet) was then added. The flask was repeatedly filled and
evacuated with hydrogen and vigorously stirred at room temperature
for 24 h under H2 (2 atm). The reaction mixture was next filtered
through a plug of Celite to separate the catalyst, and the filter was
washed three times with MeOH. The combined filtrates and washings
were concentrated to afford 43 mg (0.09 mmol, 92% yield) of 7 as the
hydrochloride salt. [α]D25 −18 (c 1.0, CH3OH). 1H NMR (δ, 500 MHz,
CD3OD): 0.88−0.96 (m, 6H), 1.25−1.42 (m, 24H), 1.60−1.80 (m,
General Procedure for the Alkylation of Azido Alcohols 11
and 14: Synthesis of 16 as a Representative Example. To a
solution of the azido alcohol 12 (163 mg, 0.29 mmol) in DMF (4 mL)
was added NaH (20 mg, 60% dispersion in mineral oil, 0.50 mmol) at
0 °C and stirred for 15 min. Then nonyl iodide (114 μL, 0.58 mmol)
was added, and the mixture was stirred at 0 °C for additional 30 min.
The reaction was quenched by addition of few drops of water. The
mixture was diluted with 40 mL of Et2O and 40 mL of water. The
organic layer was separated and the aqueous layer extracted with Et2O
(3 × 40 mL). The combined organic layers were dried over MgSO4,
filtrated, and concentrated under reduced pressure. The resulting
residue was purified by column chromatography (20:1 to 6:1 hexane/
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4H), 3.04−3.09 (m, 2H), 3.13 (dd, 1H, J = 10.7, 2.6 Hz), 3.19 (t, 1H, J
= 9.1 Hz), 3.50 (dd, 1H, J = 9.9, 2.2 Hz), 3.54−3.64 (m, 2H), 3.72
(dd, 1H, J = 10.5, 9.3 Hz), 3.98−4.05 (m, 1H), 4.16 (dt, 1H, J = 9.1,
7.2 Hz). 13C NMR (δ, 100 MHz, CD3OD): 14.5 (2), 23.74, 23.75,
27.1, 27.2, 27.8, 30.3, 30.4, 30.51, 30.53, 30.76, 30.80, 31.5, 33.0, 33.1,
46.9, 60.9, 71.3, 73.6, 74.8 (2), 76.6, 77.1. HRMS calculated for
C24H50NO5, 432.3689 [M + H]+; found, 432.3674.
stopped by lysing the cells with 1.8 mL of glycine/NaOH buffer (100
mM, pH 10.6). Liberated 4-methylumbelliferone was measured
(excitation 355 nm, emission 460 nm) with a SpectraMax M5
fluorometer (Molecular Devices Corporation) in 24-well format. All
determinations were performed in triplicate. Data are reported in
Figures 3B, 4A, and Supporting Information.
Measurement of L444P or N370S GCase Activity in
Lymphoblasts Derived from Patients with GD. Lymphoblasts
were seeded at a density of 2 × 105 cells per well in 1 mL of
supplemented RPMI-1640 medium in 12-well plates. Cells were
incubated in the absence or presence of various concentrations of
compounds for 3 days before GCase activity was measured. After
washing with PBS twice, the cell pellets were lysed in water by
sonication. All enzyme activation measurements were made using
aliquots of homogenate (10 μL) and 6 mM 4-methylumbelliferyl-β-D-
glucopyranoside (50 μL) in 0.1 M citrate phosphate buffer, pH 5.2,
containing 0.25% sodium taurocholate, 0.1% Triton X-100, and
incubated at 37 °C for 2 h. The enzyme reactions were stopped with
150 μL of glycine/NaOH buffer (200 mM, pH 10.6), and fluorescence
was measured (excitation wavelength 355 nm, emission wavelength
460 nm) with a SpectraMax M5 fluorometer (Molecular Devices
Corporation) in 96-well format. The nonspecific GCase activity was
evaluated by addition of conduritol B epoxide (500 μM) to control
wells and was shown to account for about 2% of the total activity in
control cells. Data are reported in Table 2, Figure 5, and Supporting
Information.
Recombinant GCase Inhibition Assay. Imiglucerase (Genzyme)
activity was determined with 4-methylumbelliferyl-β-D-glucopyrano-
side as previously reported.35 Briefly, enzyme solutions (25 μL from a
stock solution containing 0.1 mg mL−1) in the presence of 0.25% (w/
v) sodium taurocholate and 0.1% (v/v) Triton X-100 in McIlvaine
buffer (100 mM sodium citrate and 200 mM sodium phosphate buffer,
pH 5.2 or pH 7.0) were incubated at 37 °C without (control) or with
inhibitor at a final volume of 40 μL for 30 min. After addition of 60 μL
of substrate (4 mM, McIlvaine buffer, pH 5.2 or pH 7.0), the samples
were incubated at 37 °C for 10 min. Enzymatic reactions were stopped
by the addition of aliquots (150 μL) of glycine/NaOH buffer (100
mM, pH 10.6). The amount of 4-methylumbelliferone formed was
determined with a SpectraMax M5 fluorometer (Molecular Devices
Corporation) at 355 nm (excitation) and 460 nm (emission).
IC50 values were determined by plotting percent activity versus log
[I], using at least five different inhibitor concentrations. The type of
inhibition and Ki values for more active inhibitors were determined by
Lineweaver−Burk or Dixon plots of assays performed with different
concentrations of inhibitor and substrate. Data are reported in Table 1,
Figure 3A, and Supporting Information.
Thermal Stabilization Assay. Thermal stabilization assay was
performed as described in the literature23 with slight modifications.
Briefly, imiglucerase aliquots (48 μL, 2 mg mL−1) were incubated with
three different concentrations of test compound at 48 °C.
Subsequently, 150 μL of 0.1 M acetate-phosphate buffer (pH 5.0)
and 100 μL of 4-methylumbelliferyl-β-D-glucopyranoside (4 mM, 0.1%
Triton X-100 and 0.2% sodium taurocholate in McIlvaine buffer, pH
5.2) were added at different times and incubated for additional 10 min
at 37 °C. This was followed by the addition of 300 μL of glycine/
NaOH buffer (100 mM, pH 10.6), and the liberated 4‑methyl-
umbelliferone was measured (excitation wavelength 355 nm, emission
wavelength 460 nm). Enzyme activity was reported relative to
unheated (37 °C) enzyme. Each experiment was performed in
triplicate. Data are reported in Table 2 and Supporting Information.
Cell Lines and Culture. Wild-type fibroblasts and lymphoblasts
derived from patients with GD homozygous for N370S GCase
(GM10873) or L444P GCase (GM08752) were obtained from
Eucellbank and Coriell Cell Repositories, respectively. Fibroblast cells
were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS,
Invitrogen), 100 U mL−1 of penicillin, and 100 μg mL−1 of
streptomycin at 37 °C in 5% CO2. Culture medium was replaced
every 3−4 days, and all cells used in this study were between the 14th
and 30th passages. Lymphoblast cell lines were cultured in RPMI-1640
medium (Gibco) supplemented with 15% FBS, 100 U mL−1 of
penicillin, and 100 μg mL−1 of streptomycin at 37 °C in 5% CO2.
Culture medium was replaced every 2−3 days, and all cells used in this
study were between the 5th and 16th passages. Total protein was
determined using the Micro BCA protein assay kit according to the
manufacture’s instructions (Pierce, Thermo Scientific).
GCase Inhibition in Intact Human Fibroblasts. The intact cell
GCase assay was performed as previously described.23 Briefly, cells
were plated into 24-well assay plates and incubated at 37 °C under a
5% CO2 atmosphere overnight. The media were then replaced with
fresh media with or without a test compound and incubated at 37 °C
in 5% CO2 for 24 h. The enzyme activity assay was performed after
removing media supplemented with the corresponding compound.
The monolayers were washed with 100 μL of phosphate buffered
saline (PBS) solution. Then, 80 μL of PBS and 80 μL of 200 mM
acetate buffer (pH 4.0) were added to each well. The reaction was
started by the addition of 100 μL of 5 mM 4-methylumbelliferyl-β-D-
glucopyranoside (200 mM acetate buffer, pH 4.0) to each well,
followed by incubation at 37 °C for 2 h. Enzymatic reactions were
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental protocols and compound characterization
data for all new compounds, as well as experimental procedure
for glycosidase inhibition assays, GCS inhibition in cell
homogenates, cytotoxicity, GCase and GBA2 inhibition in
mouse tissue homogenates, stabilization ratios after thermal
denaturation of recombinant GCase (Imiglucerase), and the
effects of compounds on L444P or N370S GCase activity in
GD lymphoblasts. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 34-934-006-108. Fax: +34-932045904. E-mail:
amadeu.llebaria@cid.csic.es.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Spanish MICINN (projects
CTQ2008-01426/BQU and CTQ2011-29549-C02-01) and
“Generalitat de Catalunya” (grant 2009SGR-1072). A.T. is
grateful to MICINN for a predoctoral fellowship. The authors
thank Dr. J. Casas and Dr. A. Delgado for helpful discussions, E.
Dalmau and Dr. R. Per
́
ez for analytical analysis, Dr. M. Egido-
Gabas and N. Guillem for experimental contributions, G. Twigg
́
for excellent technical assistance, and Genzyme Corp. for a
generous supply of imiglucerase (Cerezyme).
ABBREVIATIONS USED
■
GD, Gaucher disease; GCase or GBA1, β-glucocerebrosidase;
PCT, pharmacological chaperone therapy; ER, endoplasmic
reticulum; NN-DNJ, N-nonyl-deoxynojirimycin; GCS, gluco-
sylceramide synthase; GBA2, nonlysosomal β-glucocerebrosi-
dase; CC50, concentration of compound required to induce
50% cytotoxicity
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Journal of Medicinal Chemistry
Article
Southall, N.; Goldin, E.; Westbroek, W.; Stubblefield, B. K.; Sidransky,
E.; Aungst, R. A.; Lea, W. A.; Simeonov, A.; Leister, W.; Austin, C. P.
Evaluation of Quinazoline Analogues as Glucocerebrosidase Inhibitors
with Chaperone Activity. J. Med. Chem. 2011, 54, 1033−1058.
(c) Tropak, M. B.; Kornhaber, G. J.; Rigat, B. A.; Maegawa, G. H.;
Buttner, J. D.; Blanchard, J. E.; Murphy, C.; Tuske, S. J.; Coales, S. J.;
Hamuro, Y.; Brown, E. D.; Mahuran, D. J. Identification of
Pharmacological Chaperones for Gaucher Disease and Character-
ization of Their Effects on β-Glucocerebrosidase by Hydrogen/
Deuterium Exchange Mass Spectrometry. ChemBioChem 2008, 9,
2650−2662.
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