Tuning glycosidase inhibition through aglycone interactions: Pharmacological chaperones for Fabry disease and GM1 gangliosidosis

Article abstract of DOI:10.1039/c2cc32065g

Competitive inhibitors of either α-galactosidase (α-Gal) or β-galactosidase (β-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp²-iminosugars. Selected compounds were

Full text of DOI:10.1039/c2cc32065g

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COMMUNICATION
Tuning glycosidase inhibition through aglycone interactions:
pharmacological chaperones for Fabry disease and GM1 gangliosidosisw
a
bc
b
a
d
d
´
M. Aguilar-Moncayo,z T. Takai,z K. Higaki,* T. Mena-Barragan, Y. Hirano, K. Yura,
L. Li,be Y. Yu,be H. Ninomiya,f M. I. Garcıa-Moreno,a S. Ishii,g Y. Sakakibara,h K. Ohno,e
E. Nanba,b C. Ortiz Mellet,*a J. M. Garcı
a Fernandez* and Y. Suzuki*
´
i
j
´
´
Received 21st March 2012, Accepted 8th May 2012
DOI: 10.1039/c2cc32065g
Competitive inhibitors of either a-galactosidase (a-Gal) or
b-galactosidase (b-Gal) with high affinity and selectivity have been
accessed by exploiting aglycone interactions with conformationally
locked sp2-iminosugars. Selected compounds were profiled as
potent pharmacological chaperones for mutant lysosomal a- and
b-Gal associated with Fabry disease and GM1 gangliosidosis.
by administration of an inhibitor of the substrate-producing
enzyme.3 Pharmacological chaperones (PCs) constitute a promising
new treatment paradigm and several compounds are currently
being evaluated for LSDs.4 In this approach, a ligand of the target
glycosidase promotes those conformational changes that are
required for efficient folding, restoring trafficking.5 Although
somewhat counterintuitive, competitive inhibitors can increase
steady-state lysosomal levels of active enzymes through this
rescue mechanism. At the massive lysosomal substrate concen-
tration, the inhibitor would be replaced from the active site of
the enzyme and the metabolic activity recovered.
Inappropriate performance of any of the multiple hydrolytic
enzymes involved in lysosomal catabolic pathways is at the origin
of a range of pathologies known as lysosomal storage disorders
(LSDs).1 Many of the mutations associated with these diseases
translate into enzymes that retain partial catalytic activity in vitro
but exhibit impaired cellular trafficking as a consequence of
aberrant folding. Among the therapeutic approaches under
investigation are the parenteral infusion of exogenous enzymes
(enzyme replacement therapy, ERT)2 and the reduction in the influx
of the accumulated substrate (substrate reduction therapy, SRT)
With few exceptions,6 PCs under study mimic the glycone
moiety of the putative substrate. This is the case of the
iminosugars 1-deoxynojirimycin (1, DNJ) or 1-deoxygalacto-
nojirimycin (2, DGJ).7 The fact that the same glycone moiety
is shared for enzymes acting on anomeric substrates frequently
leads to an insufficient selectivity of these types of inhibitors
for clinical application, however.8 Incorporation of alkyl
substituents into iminosugar frameworks has been shown to
improve the affinity towards certain glycosidases and their
drugability as PCs, but fails to provide a tool for switching
between enzymes with reverse anomeric selectivity.9 Recently,
we demonstrated that replacing the sp3 amine-type nitrogen
typical of classical iminosugars by a pseudoamide-type (urea,
thiourea, isourea, isothiourea) nitrogen atom (sp2-iminosugars)
provided excellent opportunities to shape the glycosidase inhibitory
properties.10 For instance, 5-N,6-S-[N0-(n-octyliminomethylidene)]-
6-thionojirimycin (3, 6S-NOI-NJ) and the D-galacto configured
analogue 6S-NOI-GNJ (4) behaved as potent, competitive and
selective inhibitors of lysosomal b-glucosidase (b-glucocerebrosi-
dase, GCase).11 Both epimers 3 and 4 were able to increase the
GCase activity in GCase-deficient Gaucher disease (GD, the LSD
with the highest prevalence) fibroblasts, being particularly efficient
in the case of neuronopathic-associated mutations (Fig. 1).11
The outstanding selectivity of the sp2-iminosugars 3 and 4 for
b-glucosidase relies on a subtle configurational–conformational
switch mechanism controlled by aglycone interactions. In the
unbound state, the anomeric hydroxyl adopts the axial orienta-
tion (a-configuration) dictated by the generalized anomeric
a
´
´
´
Dpto. Quımica Organica, Fac. de Quımica, Universidad de Sevilla,
c/ Professor Garcıa Gonzalez 1, E-41012 Sevilla, Spain.
´
´
E-mail: mellet@us.es; Fax: +34-954-624960
b Division of Functional Genomics, Research Center for Bioscience and
Technol., Fac. of Medicine, Tottori University, 86 Nishi-cho,
Yonago, 683-8503, Japan. E-mail: kh4060@med.tottori-u.ac.jp;
Fax: +81-859-386470
c Division of Human Genome Science, Dept. of Molecular and Cellular
Biology, School of Life Science, Fac. of Medicine,
Tottori University, 86 Nishi-cho, Yonago, 683-8503, Japan
d Center for Informational Biology, Ochanomizu University,
2-1-1 Otsuka, Bunkyoku, Tokyo 112-8610, Japan
e Division of Child Neurology, Inst. of Neurological Sciences,
Fac. of Medicine, Tottori University, Nishi-cho, Yonago 683-8504, Japan
f Dept. of Biomedical Regulation, School of Health Science,
Fac. of Medicine, 86 Nishi-cho, Yonago, 683-8503, Japan
g Department of Matrix Medicine, Fac. of Medicine, Oita University,
Oita 879-5593, Japan
h Dept. of Biosciences and Informatics, Fac. of Science and Technol.,
Keio University, 3-14-1 Hiyoshi, Kohoku-Ku, Yokohama 223-8522,
Japan
i
´
Inst. de Investigaciones Quımicas (IIQ), CSIC,
Americo Vespucio 49, Isla de la Cartuja, E-41092 Seville, Spain.
´
E-mail: jogarcia@iiq.csic.es; Fax: +34-954-460565
j International University of Health and Welfare Graduate School,
2600-1 Kita Kanemaru, Otawara 324-8501, Japan.
E-mail: SuzukiY@iuhw.ac.jp; Fax: +81-287-24-3229
4
effect, with the piperidine ring in the C1 chair conformation
and the imine group in the E-configuration. Crystallographic
evidence indicated that 3 and 4 are accommodated in the active
w Electronic supplementary information (ESI) available: Experimental
protocols and copies of NMR spectra. See DOI: 10.1039/c2cc32065g
z These authors equally contributed to this work.
c
6514 Chem. Commun., 2012, 48, 6514–6516
This journal is The Royal Society of Chemistry 2012

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This situation will force distinct orientations of the aglycone
moiety that will be anticlock- and clockwise displaced with
respect to the scenario found for GCase-bound 4, thus enabling
galactosidase binding (Scheme 1, right).
DGJ thioureas 5–7 were accessed in one step, with total
chemoselectivity and good yield, by direct coupling of the
parent iminosugar 2 with isothiocyanates. Attempts to promote
further cyclization by treatment with triflic anhydride or mesyl
chloride in dry N,N0-dimethylformamide13 led to extensive hydro-
lysis of the sulfonylating reagents. Only traces of the target
isothioureas 8–10 were detected. To our delight, the transforma-
tion proceeded smoothly in methanol under acid catalysis without
the need for any additional additive. The reaction can be effected
in one pot from iminosugar 2 by sequential isothiocyanate
coupling–intramolecular acid-promoted cyclisation, which makes
this general strategy very well-suited for library generation and
optimization schemes. Dynamic NMR experiments and NOE
data fully supported the predicted conformational assignment
indicated in Scheme 1.
Fig. 1 Structures of the iminosugars DNJ and DGJ and of the
sp2-iminosugars 6S-NOI-NJ and 6S-NOI-GNJ. The configurational–
conformational switch process experienced by the latter upon binding
to b-glucocerebrosidase (GCase) is schematically depicted.
site of the enzyme in the b-configuration, however, with the
N0-alkyl chain in the Z-orientation (Fig. 1).12 Actually, GCase
possesses a hydrophobic pocket at the entrance of the active
site that is perfectly matched by the N0-substituent in this
particular conformation. In contrast, overlapping of the active
sites of a-glycosidase or a- and b-galactosidase revealed steric
clashes that hampered binding.
Compounds 5–10 were first evaluated for their inhibitory
properties against a panel of commercial glycosidases including
a-Gal from coffee, belonging to the same CAZy family (GH27)
as that of human a-Gal A, and b-Gal from Escherichia coli
(E. c.) and bovine liver, both classified in the same clan GH A as
human lysosomal b-Gal (Table 1).14 Thioureas 5 and 7 and
isothioureas 9 and 10, showing the strongest inhibition potency
for a-Gal and b-Gal, respectively, were selected for further
evaluation against human lysosomal glycosidases. They exhibited
high affinity and total specificity for human a-Gal and b-Gal,
respectively, among several lysosomal enzymes. Inhibition data
are collected in Table 1 in comparison with data for DGJ (2) and
N-octyl-4-epi-b-valienamine (NOEV), the best performing PCs
for FD and GM1 gangliosidosis reported to date.15 Most
interestingly, the inhibition potency was about ten-fold higher
at neutral than at acidic pH, a favourable feature considering
that mutant enzyme rescue by pharmacological chaperones
requires strong binding at the ER but dissociation of the complex
once in the lysosome.
The above results strongly suggest that fine tuning of aglycone
interactions has the potential to become a powerful strategy for
elaborating selective active-site directed PCs for LSDs. To test this
hypothesis, we have focused on the lysosomal a- and b-galacto-
sidases (a-Gal and b-Gal), whose deficiency results in the LSDs
known as Fabry disease (FD) and GM1 gangliosidosis (GM1),
respectively. Two series of conformationally locked sp2-iminosugar
galactomimetic probes, namely the monocyclic and bicyclic DGJ
derivatives 5–7 and 8–10 (Scheme 1, left), were synthesized and
shown to have remarkable glycosidase selectivities, which translated
into potent pharmacological chaperoning activities in mutant
proteins associated with FD and GM1, respectively.
The above-mentioned X-ray and docking data revealed that the
anomeric hydroxyl in 4 was not directly involved in strong
contacts with amino acid residues at the active site of glycosidases.
Its removal should broaden the available conformational space
for N-substituents. On the other hand, the dissymmetry in the
substitution pattern at C-1 and C-5 was expected to promote
preferred E,Z- and E-configurations at the thiourea segment
and the cyclic isothiourea group, in 5–7 and 8–10, respectively.
Table 1 Inhibitory activity of compounds 5–10 against commercial
(Ki, mM) and human (H. s., lysosomal) glycosidases (IC50, mM).a For
comparative purposes, values of the reference compounds DGJ (2)
and NOEV are also included
Commercial
enzymes (Ki, mM)
Human enzymes
(pH 5.0/7.0; IC50, mM)
a-Gal
(coffee)
b-Gal
(E. c.)
b-Gal
(bov.)
a-Gal
H. s.
b-Gal
H. s.
27
0.016
n.d.
0.0019
0.044
0.029
18
12.5
n.d.
5.2
189
18
13
7.3
0.65
616
0.87
526
98
0.06/0.01
n.i.
4.5/0.2
n.d.
37/5.0
n.d.
n.i.
25/n.d.b
0.13/0.02
n.i.
n.d.
n.i.
n.d.
32/3.1
6.0/0.5
NOEV15
5
6
7
8
9
10
25
6.9
2.9
0.2
134
81
n.i.
Scheme 1 Synthesis of the conformationally locked mono (5–7) and
bicyclic DGJ derivatives (8–10). The 3D molecular models of the new octyl
thiourea (7, in green) and isothiourea (10, in grey) derivatives, overlapped
with that of the GCase-bound reducing derivative 6S-NOI-DGJ (4, in
orange), illustrate the sharp differences in the relative orientations of
the N0-substituent in their ground state conformations.
a
No inhibition (n.i.) was observed for 5–10 on a-glucosidase (baker yeast),
amyloglucosidase (Aspergillus niger), isomaltase (yeast), a-mannosidase
(Jack bean) and b-mannosidase (Helix pomatia) at concentrations up to
2 mM, or on human lysosomal a-Glc, b-Glc (GCase) or N-acetyl-b-D-
b
glucosaminidase at concentrations up to 0.1 mM. Not determined.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 6514–6516 6515

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Notes and references
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Fig. 2 Effect of thioureas 5 and 7 on a-Gal activity in mutant R301G
FD fibroblasts (left) and of isothiourea 9 on b-Gal activity in mutant
R201C GM1 fibroblasts (right). Each bar represents the mean ꢀ SEM
of three determinations each done in triplicate.
5 For recent examples, see: (a) C. Decrooq, D. Rodrıguez-Lucena,
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K. Ikeda, N. Asano and P. Compain, ChemBioChem, 2012,
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7 (a) Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond,
The effects of compounds 5 and 7 on a-Gal were further
evaluated in human skin fibroblasts derived from Fabry
patients homozygous for the R301G mutation, known to lead
to folding defects in the enzyme but not compromising the
catalytic site. The parent iminosugar 2 (DGJ) was used as a
control. About 3-fold increases in enzyme activity were
reached after 4 days of incubation with thioureas 5 and 7 at
3 mM, equalling the results obtained with DGJ at its optimal
concentration of 20 mM. By increasing the concentrations of
the PCs to 30 mM, a maximal enhancement of 5-fold was
achieved (Fig. 2). Neither 5 nor 7 exhibited cell toxicity at
concentrations up to 500 mM.
The N0-octylisothiourea 10 turned to be toxic to human
fibroblasts at concentrations over 100 mM and was discarded at
this stage. The N0-butyl analogue 9 was nontoxic at concentra-
tions up to 640 mM. When used at concentrations higher than
100 mM, it led to a 6-fold b-Gal activity enhancement in
fibroblasts from patients homozygous for the juvenile GM1
mutation R201C, comparable to that achieved with the reference
compound NOEV.15 Although a biologically useful chaperoning
activity was only observed from 20 mM concentration, the
chaperon:inhibitor balance was very favourable, since the mutant
enzyme activity continued to increase with the concentration of 9
up to 240 mM (Fig. 2).
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¨
In summary, we have succeeded in the design and synthesis
of sp2-iminosugar ligands that can discriminate between lyso-
somal a- and b-galactosidases from which pharmacological
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GM1 mutants have been identified. Since the general approach
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sponding glycosidases.
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´ ´
´
´
´
a
´
This study was supported by the Spanish MICIN (contract
numbers SAF2010-15670 and CTQ2010-15848; co-financed
´
a
´
by FEDER), the Fundacio
Andalucıa (Project P08-FQM-03711), the Ministry of Educa-
n Ramon Areces, the Junta de
´ ´
A. H. Futerman and C. Ortiz Mellet, Org. Biomol. Chem., 2011,
9, 4160; (b) M. Aguilar-Moncayo, T. M. Gloster, J. P. Turkenburg,
´
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Bioorg. Med. Chem., 2002, 10, 1967.
a-Moreno, C. Ortiz Mellet, G. J. Davies and J. M. Garcıa
´ ´
tion, Culture, Science, Sports and Technology of Japan
(MEXT, 22390207 and 23591498), and the Ministry of
Health, Labour and Welfare of Japan (H17-Kokoro-019,
H20-Kokoro-022, H19-Nanji-Ippan-002, H22-Nanji-Ippan-002),
and a grant from Japan Science and Technology Agency
(AS232Z00009G). K.Y. was supported by Targeted Proteins
Research Program (TPRP; MEXT).
´
´
´
c
6516 Chem. Commun., 2012, 48, 6514–6516
This journal is The Royal Society of Chemistry 2012