Targeted delivery of pharmacological chaperones for Gaucher disease to macrophages by a mannosylated cyclodextrin carrier

Article abstract of DOI:10.1039/c3ob42530d

Gaucher disease (GD) is a rare monogenetic disorder leading to dysfunction of acid β-glucosidase (β-glucocerebrosidase; GCase) and accumulation of glucosylceramide in lysosomes, especially in macrophages (Gaucher cells). Many of the mutations at the origin of GD do not impair the catalytic activity of GCase, but cause misfolding and subsequent degradation by the quality control system at the endoplasmic reticulum. Pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the endogenous mutant enzyme represent promising alternatives to the currently available enzyme replacement and substrate reduction therapies (ERT and SRT, respectively), but unfavorable biodistribution and potential side-effects remain important issues. We have now designed a strategy to enhance the controlled delivery of PCs to macrophages that exploit the formation of ternary complexes between the PC, a trivalent mannosylated β-cyclodextrin (βCD) conjugate and the macrophage mannose receptor (MMR). First, PC candidates with appropriate relative avidities towards the βCD cavity and the GCase active site were selected to ensure efficient transfer of the PC cargo from the host to the GCase active site. Control experiments confirmed that the βCD carrier was selectively recognized by mannose-specific lectins and that the corresponding PC:mannosylated βCD supramolecular complex retained both the chaperoning activity, as confirmed in human GD fibroblasts, and the MMR binding ability. Finally, fluorescence microscopy techniques proved targeting and cellular uptake of the PC-loaded system in macrophages. Altogether, the results support that combined cyclodextrin encapsulation and glycotargeting may improve the efficacy of PCs for GD. This journal is

Full text of DOI:10.1039/c3ob42530d

Organic &
Biomolecular Chemistry
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Targeted delivery of pharmacological chaperones
for Gaucher disease to macrophages by a
mannosylated cyclodextrin carrier†
Cite this: Org. Biomol. Chem., 2014,
2, 2289
1
a
b
a
Julio Rodríguez-Lavado, Mario de la Mata, José L. Jiménez-Blanco,
a
c
d
M. Isabel García-Moreno, Juan M. Benito, Antonio Díaz-Quintana,
b
e
e
f
José A. Sánchez-Alcázar, Katsumi Higaki, Eiji Nanba, Kousaku Ohno,
g
a
c
Yoshiyuki Suzuki, Carmen Ortiz Mellet* and José M. García Fernández*
Gaucher disease (GD) is a rare monogenetic disorder leading to dysfunction of acid β-glucosidase (β-gluco-
cerebrosidase; GCase) and accumulation of glucosylceramide in lysosomes, especially in macrophages
(Gaucher cells). Many of the mutations at the origin of GD do not impair the catalytic activity of GCase,
but cause misfolding and subsequent degradation by the quality control system at the endoplasmic reti-
culum. Pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the
endogenous mutant enzyme represent promising alternatives to the currently available enzyme replace-
ment and substrate reduction therapies (ERT and SRT, respectively), but unfavorable biodistribution
and potential side-effects remain important issues. We have now designed a strategy to enhance the
controlled delivery of PCs to macrophages that exploit the formation of ternary complexes between the
PC, a trivalent mannosylated β-cyclodextrin (βCD) conjugate and the macrophage mannose receptor
(MMR). First, PC candidates with appropriate relative avidities towards the βCD cavity and the GCase active
site were selected to ensure efficient transfer of the PC cargo from the host to the GCase active site.
Control experiments confirmed that the βCD carrier was selectively recognized by mannose-specific
lectins and that the corresponding PC:mannosylated βCD supramolecular complex retained both
the chaperoning activity, as confirmed in human GD fibroblasts, and the MMR binding ability. Finally,
fluorescence microscopy techniques proved targeting and cellular uptake of the PC-loaded system
in macrophages. Altogether, the results support that combined cyclodextrin encapsulation and
glycotargeting may improve the efficacy of PCs for GD.
Received 18th December 2013,
Accepted 3rd February 2014
DOI: 10.1039/c3ob42530d
www.rsc.org/obc
Introduction
a
Dept. Química Orgánica, Facultad de Química, Universidad de Sevilla, c/Profesor
García González 1, 41012 Sevilla, Spain. E-mail: mellet@us.es;
Fax: (+34) 954624960
Lysosomal storage disorders (LSDs) are a heterogeneous group
of inherited diseases caused by genetic defects affecting lyso-
somal catabolic enzymes. In many cases, the mutation at the
origin of the disease gives rise to a protein that cannot fold
properly and is subjected to endoplasmic reticulum associated
degradation (ERAD) by the quality control system of the cell.
b
Centro Andaluz de Biología del Desarrollo (CABD), CSIC – Universidad Pablo de
1
Olavide, Carretera de Utrera Km. 1, 41013 Sevilla, Spain
c
Instituto de Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, Avda.
Américo Vespucio 49, 41092 Sevilla, Spain. E-mail: jogarcia@iiq.csic.es;
Fax: (+34) 954460165
d
Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), CSIC – Universidad de Sevilla, Ironically, the mutant protein is often functional, but it cannot
undergo trafficking to the Golgi apparatus for maturation and
e
Avda. Américo Vespucio 49, 41092 Sevilla, Spain
Division of Functional Genomics, Research Center for Bioscience and Technology,
then to the lysosome, which results in the abnormal accumu-
lation of cellular debris, mostly glycosphingolipid metabolites,
in different cell types and tissues. By mechanisms that are
not fully understood, such accumulation leads to a range of
potentially life-threatening pathologies. Incidence of each LSD
individually is rare, though altogether LSDs affect 1 in 5000 to
Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, 683-8503, Japan
f
Division of Child Neurology, Institute of Neurological Sciences, Faculty of Medicine,
2
Tottori University, 86 Nishi-cho, Yonago, 683-8504, Japan
g
Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-Kitazawa, Setagaya-ku,
Tokyo 156-0057, Japan
†
Electronic supplementary information (ESI) available: Synthesis of compounds
7
and 8, NMR and ESI-MS spectra of newly synthesized compounds, CD-chaper-
1
0 000 live births in Western countries. Gaucher disease (GD),
one binding isotherms, and plots for the inhibition of commercial glycosidases
and of the adhesion of rhMMR to yeast mannan. See DOI: 10.1039/c3ob42530d
the most prevalent LSD (about 14% of the total), is associated
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with the dysfunction of β-glucocerebrosidase (GCase, EC no.
3
3
.2.1.45), responsible for glucosylceramide (GlcCer) hydrolysis.
Over 300 mutations have been characterized in the gene encod-
ing for GCase, which translates into a large array of disease
manifestations, from visceromegaly in attenuated forms of type
1
GD to neurological affections in acute and subacute neurono-
4
pathic type 2 and 3 GD, and varied disease progression rates.
The therapeutic goal towards GD, and LSDs in general, is
restoring the balance between substrate influx and degra-
5
dation. Enzyme replacement therapy (ERT), in which patients
are regularly supplemented with an exogenous recombinant
6
GCase, is the main clinical treatment for GD nowadays. Alter-
natively, other therapeutic approaches based on more “drug-
7
able” candidates have been investigated. Substrate reduction
therapy (SRT), based on glycosphingolipid biosynthesis inhibi-
8
tors, has proven useful to reduce GlcCer influx. Both ERT and
SRT treatments address substrate accumulation but not the
protein folding defects and their potential contributions to
9
the pathophysiology of the disease. More recently, specific
ligands of the deficient enzyme capable of promoting correct
folding at the ER have been shown to restore normal traffick-
1
0
ing and, ultimately, lysosomal activity.
This so-called
pharmacological chaperone therapy (PCT) constitutes a prom-
ising therapeutic paradigm for GD and many other protein
folding disorders. Someway counter-intuitively, competitive Fig. 1 Chemical structure of the sp -iminosugar pharmacological cha-
inhibitors of the affected enzyme, used at sub-inhibitory con- perones 6S-NOI-NJ and 6S-AdB-NJ and of the trimannosylated βCD
1
1
2
3
carrier (ManS) -βCD.
centrations, can act as mutant enzyme activators through this
1
2
rescuing mechanism. In any case, the future development of
PCT requires engineering small molecular entities featuring (i)
highly selective affinity towards the mutant enzyme, not to targeted carrier must promote cell internalization before
disrupt parallel cellular metabolic machineries, (ii) favourable premature PC release.
chaperoning versus inhibitory capabilities, (iii) membrane-
Interestingly, native β-cyclodextrin (cyclomaltoheptaose;
diffusiveness to access cells and cellular compartments, and βCD) has been previously shown to facilitate transfer of amphi-
2
(
iv) recognition abilities to selectively target storage affected philic sp -iminosugars to recombinant GCase in co-crystalliza-
1
7
tissues.
tion experiments.
The basket-shaped structure of CDs
Recent studies have shown that bicyclic glucose mimics features a hydrophobic cavity that can accommodate guest
with amphiphilic character and GCase inhibitory properties molecules of appropriate size and improve their water solubi-
1
8
behave as active site-directed pharmacological chaperones lity and bioavailability, which has been broadly exploited in
1
3
19
(
PCs) for GD. Among them, amphiphilic bicyclic nojirimycin pharmaceutical technology. The possibility to incorporate
2
analogues belonging to the so-called sp -iminosugar glyco- mannosyl moieties that are specifically recognized by
mimetic family have proven to be able of rescuing the mis- the macrophage mannose receptor (MMR), a C-type lectin
1
4
folded protein from ERAD and restoring trafficking to the expressed at the membrane of macrophages and dendritic
1
5
20
lysosome, where the catalytic activity is expressed. The cells,
through selective chemical functionalization
2
1
efficacy of PCT for GD may benefit from strategies enhancing methods further offers a good opportunity for optimization
delivery of the chaperone to the cells that are primarily of macrophage-targeted PC:βCD complexes for enhanced PCT
affected by glucosylceramide accumulation, namely macro- against GD. Herein, we have characterized the host–guest inter-
phages (Gaucher cells). Site-specific delivery of recombinant actions of N′-octyl- and N′-[4-(adamant-1-ylcarboxamido)butyl]-
glycosidases by cell membrane receptor-targeted carriers has iminomethylidene-6-thionojirimycin (6S-NOI-NJ and 6S-NAdB-
2
2
already proven a promising strategy for improving ERT in the NJ), bearing octyl and adamantyl moieties, respectively,
1
6
context of several LSDs, but the suitability of such an with the trivalent thiomannopyranosyl-tagged βCD derivative
approach for pharmacological chaperone delivery to macro- (ManS) -βCD (Fig. 1). We have further assessed how formation
phages remains unexplored. First, the binding affinity of of the (ManS) -βCD:PC complexes influences both the
3
3
the chaperone towards the carrier must be finely tuned interaction with glycosidases and the recognition by mannose-
to warrant efficient transfer to GCase. Second, the specific lectins. Our results demonstrate that (i) the chaperon-
2
carrier must be equipped with a ligand allowing specific recog- ing capability of the sp -iminosugars 6S-NOI-NJ and
nition by macrophages. Third, the PC-loaded macrophage- 6S-NAdB-NJ is preserved upon complexation with (ManS) -βCD
3
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Fig. 2 Schematic representation of the strategy devised for macro-
phage-specific delivery of GD pharmacological chaperones by MMR-
mediated mediated internalization of their inclusion complexes with the
(
3
ManS) -βCD carrier and further transfer to GCase.
as determined in fibroblasts from GD patients and (ii) the
PC-loaded βCD conjugates are internalized in macrophages,
following the formation of PC:(ManS) -βCD:MMR ternary com-
3
plexes, to finally deliver the PC inside the cells (Fig. 2).
Results and discussion
Selection criteria for the pharmacological chaperone and
Scheme 1 Building block-based synthesis of the CD carrier (ManS)
βCD.
3
-
cyclodextrin carrier partners
Bicyclic
5N,6S-(N′-alkyliminomethylidene)-6-thionojirimycin
2
7
derivatives have shown a strong and selective inhibitory activity in the presence of copper(II) (→1). The incorporation of
against several β-glucosidases and excellent properties as a cysteaminyl segment was next undertaken to avoid steric con-
pharmacological chaperones against several GD-associated strains during the final conjugation step and to warrant acces-
1
5a
GCase mutants.
We have chosen the N′-octyl and the N′-[4- sibility of the glycoligand to molecular recognition events in
2
2a
(adamant-1-ylcarboxamido)butyl] representatives 6S-NOI-NJ
the final conjugate. Nucleophilic displacement of the tosylate
2
2b
and 6S-NAdB-NJ
in this study because the octyl and ada- group in 1 by 2-(tert-butoxycarbonylamino)ethanethiol (N-Boc-
mantyl moieties are known to exhibit a high avidity for the cysteamine) required harsh conditions and proved trouble-
cavity of β-cyclodextrin in aqueous media, with association some, however. Alternatively, tosylate was exchanged into
2
4
−1 23
constant values (Kas) in the range 10 –10
M
.
Inclusion iodine by treatment with NaI and the resulting iodo derivative
complex formation with a βCD-based carrier is then expected 2 was next smoothly reacted with N-Boc-cysteamine to furnish
to ensure efficient transfer of the PC from the CD cavity to the adduct 3. Acid-promoted carbamate removal quantitatively
active site of GCase. In the design of (ManS)
carrier partner for site-specific delivery of the selected PCs to of 4 and the isothiocyanate-armed dendron 5 was conducted
macrophages, we kept in mind that multivalency is generally a in DMF under Et N catalysis at rt to give the corresponding
prerequisite to elicit a biologically useful affinity in carbo- thiourea adduct 6. Final catalytic deacetylation quantitatively
3
-βCD as the yielded the monocysteaminyl-functionalized βCD 4. Coupling
28
3
2
4
hydrate–protein recognition processes and that monosubsti- furnished the target (ManS) -βCD carrier, whose purity
3
tution of the βCD host by a mannosyl dendron is much less and structure were confirmed by mass spectrometry, NMR
likely to alter the guest inclusion capabilities than polysubsti- spectroscopy, and combustion analysis. The presence of the
2
5
13
tution strategies.
The synthesis of (ManS)
thiourea tether was ascertained by the C NMR resonance at
-βCD has been accomplished using 183–180 ppm (δCvS) and the typical line broadening associated
3
a modular convergent strategy that takes advantage of the high with restricted rotation at the pseudoamide NH–(CvS) bond
2
9
efficiency of the thiourea-forming reaction for macromolecule at room temperature. The unsymmetrical nature of the
2
6
conjugation (Scheme 1). First, commercial βCD was regio- cyclooligosaccharide resulted in extensive signal overlapping
1
selectively tosylated at a single primary O-6 position by reac- in the H NMR spectrum that was partially overcome by using
tion with p-toluenesulfonyl chloride in aqueous basic medium 1D TOCSY experiments.
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3
(ManS) -βCD:pharmacological chaperone complex formation
thermodynamics
To measure the affinity of the pharmacological chaperones
6
S-NOI-NJ and 6S-NAdB-NJ for the (ManS) -βCD host, NMR
3
2
titration experiments in D O were conducted. The resonances
of the βCD H-3 and H-5 protons were the most intensely
affected after complex formation, supporting inclusion of the
hydrophobic moieties of the PCs in the βCD cavity. Using
the continuous variations method and an iterative
3
0
least squares fitting procedure the corresponding association
constant (Kas) values were determined to be 399 ± 4 and
019 ± 196 M− , respectively (see the Experimental section
1
1
and ESI† for details and binding isotherm figures). Titration
isotherms were compatible with a 1 : 1 complex stoichiometry
in both cases.
Fig. 3 Activity of wild-type human GCase at the indicated concen-
trations of the PCs 6S-NOI-NJ and 6S-NAdB-NJ or their corresponding
1 : 1 complexes with (ManS) -βCD. Results are expressed relative to
3
activity of the enzyme in the absence of inhibitor (100%).
Glycosidase inhibition capabilities of (ManS)
pharmacological chaperone complexes
3
-βCD:
2
uncomplexed sp -iminosugars and the corresponding
inclusion complexes with the (ManS) -βCD carrier were equally
efficient at inhibiting glucocerebrosidase activity in cell lysates
at concentrations over 0.1 μM (Fig. 3).
3
To pinpoint whether or not (ManS) -βCD complexation alters
the availability of the pharmacological chaperones to interact
with glycosidases, the inhibition capabilities of 6S-NOI-NJ and
3
6
S-NAdB-NJ free or in complex with (ManS) -βCD were first
3
(
ManS)
3
-βCD:pharmacological chaperone complex formation
profiled towards two commercial β-glucosidases (β-Glcases),
namely β-Glcase from almonds and β-Glcase from bovine liver,
thermodynamics
belonging to the same clan as that of the human GCase (clan To probe the ability of the (ManS) -βCD carrier and the corres-
3
3
1
A). Complexes were pre-formed by freeze-drying equimolecu- ponding complexes with the 6S-NOI-NJ and 6S-NAdB-NJ to
lar mixtures of each sp -iminosugar and the mannosylated CD target mannose-specific lectins, a comparative study of their
2
carrier (ManS) -βCD in water. The corresponding inhibition binding capabilities towards the model plant lectin Concana-
3
constants (K ) at the optimal pH of each glycosidase are shown valin A (Con A) by isothermal titration calorimetry (ITC) was
i
in Table 1. In control experiments, (ManS)
affect the activities of the enzymes up to mM concentrations nosides. For the measurements, the unloaded carrier and
data not shown). The inhibition mode, as determined by Line- the preformed 1 : 1 inclusion complexes with each PC were
weaver–Burk plots, was competitive in all cases. titrated into a solution of Con A at pH 7.4. ITC data for
3
-βCD alone did not first conducted. Con A specifically recognizes α-D-mannopyra-
3
2
(
The inhibitory activity of the pharmacological chaperones the binding were fitted using a single site model based on
was not significantly altered after inclusion complex for- monomeric Con A. The stoichiometry (n) determined for
mation, with K
i
values, in the μM range, that remained 2–3 the complexes of any of these species with the lectin was 1 : 1
orders of magnitude lower than the 6S-NOI-NJ : (ManS) -βCD (n = 1); i.e., the mannosyl dendron in (ManS) -βCD interacts
3
3
or 6S-NAdB-NJ : (ManS) -βCD complex dissociation constants. exclusively with one unit of Con A, irrespective of being
3
Moreover, the percentage of enzyme inhibition for a constant unloaded or loaded with the PC. Inclusion of the PC in the
concentration of 6S-NOI-NJ or 6S-NAdB-NJ was not affected by βCD cavity did not significantly affect the lectin affinity, with
increasing concentrations of (ManS)
These results are in agreement with fast dynamics of the equi- 6S-NOI-NJ] and Con A:[(ManS)
librium at play that facilitates the efficient transfer of the in the 1.6–3.6 μM range (Fig. 4 and Table 2) as compared with
S-NOI-NJ and 6S-NAdB-NJ chaperones from the CD cavity to 2.1 ± 0.7 μM for the Con A:(ManS) -βCD complex. These values
3
-βCD up to 1 : 10 ratios. dissociation constants (K
D
) for the Con A:[(ManS)
3
-βCD :
3
-βCD : 6S-NAdB-NJ] complexes
6
3
the glycosidase active site. Further experiments with are indicative of 20-to-80-fold affinity enhancements as com-
human GCase confirmed this hypothesis. Thus, the pared with reported data for methyl α-D-mannopyranoside
i 3
Table 1 Inhibition constants (K , μM) for 6S-NOI-NJ and 6S-NAdB-NJ and their corresponding 1 : 1 complexes with (ManS) -βCD towards commer-
cial glucosidases
a
Glucosidase (source, pH)
6S-NOI-NJ
3
6S-NOI-NJ : (ManS) -βCD
6S-NAdB-NJ
3
6S-NAdB-NJ : (ManS) -βCD
β-Glcase (almond, 7.3)
β-Glcase (bovine liver, 7.3)
0.76 ± 0.05
3.7 ± 0.1
0.29 ± 0.02
9.9 ± 0.1
0.45 ± 0.03
68 ± 2.0
5.8 ± 0.05
33 ± 0.02
a
i
K values were determined from the corresponding Lineweaver–Burk plots (see ESI for experimental details).
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3
3
determined by the same technique (K
the data confirm that the trivalent mannosyl ligand in the
designed PC carrier (ManS) -βCD is available to participate in
D
= 83 μM). Altogether
3
lectin receptor recognition phenomena, benefitting from the
multivalent effect, and that the carbohydrate–lectin interaction
remains equally efficient after loading with the PC cargo.
Lectins recognizing an identical sugar epitope can quite
differ structurally. Therefore, extrapolations of data on their
responsiveness towards multivalent presentations of the puta-
tive ligand must be taken with care. We have made use of
enzyme-linked lectin assay (ELLA) protocols, available for
3
4
both Con
A
and commercially available recombinant
3
5
human macrophage mannose receptor (rhMMR), to validate
the results for macrophage targeting purposes. A 15-fold
affinity enhancement against Con A for (ManS) -βCD as com-
3
pared to the monovalent reference methyl α-D-mannopyrano-
side, expressed as the ratio between the concentrations
required to achieve 50% inhibition (IC50) of the association of
horse radish peroxidase-labelled Con A (HRP-Con A) to an
immobilized ligand (yeast mannan), was determined by ELLA,
in agreement with literature data for other trivalent mannosi-
2
1a,28,36
des.
In the case of the rhMMR, the ELLA experiment
afforded a (ManS) -βCD versus methyl α-D-mannopyranoside
3
relative affinity enhancement significantly higher (72-fold),
suggesting that the disposition of the mannosyl epitopes in
the dendron is particularly appropriate to benefit from the
multivalent effect in the case of this lectin. Actually, control
experiments indicated that the affinity of (ManS) -βCD towards
3
rhMMR is analogous to that of high mannose oligosacchar-
ides, known to be preferred ligands for this receptor in
3
6
Nature, in the same experimental setup (see ESI†).
Evaluation of the chaperoning capabilities of (ManS)3-
βCD : 6S-NOI-NJ and (ManS) -βCD : 6S-NAdB-NJ complexes
3
in Gaucher fibroblasts
2
The GCase chaperoning capabilities of the sp -iminosugars
6S-NOI-NJ and 6S-NAdB-NJ before and after complexation
with (ManS) -βCD were evaluated in three different mutant
3
GD fibroblast lines, namely F213I/F213I (catalytic domain,
neuronopathic), N370S/N370S (catalytic domain, non-neuro-
nopathic) and L444P/L444P (non-catalytic domain, neuro-
nopathic), as well as in wild-type fibroblasts. Cells were
cultured in the absence and in the presence of different
amounts of the PCs (3 and 30 μM) and their corresponding
1
3
: 1 complexes with (ManS) -βCD. After 4-day incubation,
GCase activity was determined by in situ fluorimetric measure-
ment of the production of 4-methylumbelliferone (see ESI† for
details). As observed in Fig. 5A, neither 6S-NOI-NJ, 6S-NAdB-NJ
nor their complexes disrupted the activity of the wild-type
enzyme, revealing that lysosomal function is not inhibited at
the highest concentration tested (30 μM). Both 6S-NOI-NJ and
3
Fig. 4 Thermograms for the titration of Con A with the 1 : 1 (ManS) -
3
βCD : 6S-NAdB-NJ and (ManS) -βCD : 6S-NOI-NJ complexes (A and B,
6
S-NAdB-NJ enhanced the activity of GCase in a dose depen-
dent manner, up to 3-fold, in F213I/F213I mutant fibroblasts
Fig. 5B). The non-neuronopathic N370S/N370S mutant was
respectively) and non-linear regression fittings to the 1 : 1 stoichiometry
model (C and D, respectively). ΔQ represents the heat evolved after each
injection of the complex.
(
much less sensitive to these PCs; only 6S-NAdB-NJ at 3 μM con-
centration was able to promote a significant enzyme activity
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D 3
Table 2 Thermodynamic parameters and dissociation constant (K ) calculated from ITC experiments for the binding of Con A to (ManS )-βCD and
to the corresponding complexes with the pharmacological chaperones 6S-NOI-NJ and 6S-NAdB-NJ
ΔG° (kJ mol−
1
)
ΔH° (kJ mol−1
)
TΔG° (kJ mol−1
)
D
K (μM)
(
6
6
ManS)
3
-βCD
S-NOI-NJ : (ManS)
S-NAdB-NJ : (ManS)
−32.4 ± 1.0
−33.0 ± 3.1
−31.0 ± 1.3
−137.7 ± 10.5
−125.3 ± 10.9
−105.8 ± 2.4
−105.2 ± 10.5
−92.3 ± 10.9
−74.7 ± 2.4
2.1 ± 0.7
1.6 ± 0.1
3.6 ± 1.5
3
-βCD
-βCD
3
enhancement (about 2-fold; Fig. 5C). Negligible effects were derivative 8 (Fig. 7; see ESI† for synthetic details) as a model
measured in the L444P/L444P mutant with either 6S-NOI-NJ or βCD guest and an MMR ligand, respectively. Experiments were
6
S-NAdB-NJ, Fig. 5D. These results are consistent with the conducted in macrophage-like cells differentiated from
mutation-dependent responsiveness previously encountered THP-1 human monocytes, monitoring by 3D fluorescence
15a
for this family of PCs. In any case, the behavior of the two PCs microscopy. These cells mimic many characteristic features of
was largely independent of inclusion complex formation with human primary macrophages, including MMR expression.
(
ManS) -βCD, which is in agreement with a fast transfer of the
In control experiments, incubation of the cells with the
reference compound 8 (400 μM) produced an intense green
fluorescence in the cytoplasm that was virtually abolished by
excess yeast mannan (data not shown), in agreement with
MMR-mediated internalization of the conjugate. A virtually
identical result was obtained when 8 was replaced by the 1 : 1
3
PC from the βCD cavity in the carrier to the GCase active site.
Macrophage-targeting and delivery capabilities of (ManS)3-
βCD:pharmacological chaperone complexes
To confirm the potential of the (ManS) -βCD carrier to target
3
(
ManS) -βCD : 7 complex at the same concentration (Fig. 8, row
3
the MMR in macrophages, the ability to adhere to resident per-
itoneal macrophages (mice) was first probed in vitro. Adhesion
was quantified using a method adapted from that reported by
A), but not when uncomplexed 7 was incubated with the cells
under identical conditions, strongly supporting that the
(
ManS) -βCD carrier promotes internalization of the included
3
3
7
Muller and Schuber that takes advantage of the strong
enhancement in the fluorescent intensity of 6-p-toluidino-2-
naphthalenesulfonic acid (TNS) upon inclusion in the hydro-
phobic cavity of βCD and βCD derivatives to form 1 : 1
guest upon binding to the MMR. In parallel experiments,
2
an excess of the sp -iminosugar 6S-NAdB-NJ did not affect
internalization of 8, discarding the direct interaction of
the chaperone with the MMR. However, in the case of the 1 : 1
2
1a
complexes.
The cells were incubated with different con-
(
ManS)
cantly decreased upon mixing with increasing amounts of
S-NAdB-NJ prior to incubation of the cells (Fig. 8, rows B
3
-βCD : 7 complex the fluorescence intensity signifi-
centrations of the 1 : 1 TNS : (ManS)
3
-βCD complex at 4 °C to
prevent phagocytosis and the amount of complex associated
with the cell membrane was determined fluorimetrically. As
denoted in Fig. 6A, fluorescence levels increased upon incu-
6
and C). This is consistent with the partial displacement of the
fluorescent probe 7 from the carrier by the pharmacological
chaperone and the subsequent internalization of the non-fluo-
bation of the macrophages with the TNS : (ManS) -βCD
3
complex in a dose dependent manner, while the non-targeted
TNS : βCD complex, used as a control, did only produce back-
rescent (ManS) -βCD : 6S-NAdB-NJ complex through the MMR-
3
mediated route. Accordingly, internalization of the (ManS)3-
βCD : 7 complex was also abolished in the presence of excess
of yest mannan (Fig. 8, row D). A quantitative representation
of the relative intracellular fluorescent intensities for the above
experiments is shown in Fig. 8E. With the intrinsic limitations
of any indirect method, the ensemble of results support the
ground fluorescence levels. Interestingly, TNS : (ManS)
adhesion to macrophages was gradually inhibited in the pres-
ence of increasing concentrations of uncomplexed (ManS)
βCD or the corresponding 1 : 1 (ManS) -βCD : 6S-NOI-NJ and
ManS) -βCD : 6S-NAdB-NJ complexes with very similar efficien-
3
-βCD
3
-
3
(
3
cies (Fig. 6B, IC50 42, 39 and 36 μM, respectively). This result
supports that the carrier and the corresponding PC or TNS
complexes compete for the same receptor at the surface of
macrophages and is in agreement with the involvement of the
MMR in the adhesion process.
hypothesis that (ManS) -βCD specifically interacts with the
3
MMR through the trivalent mannosyl antenna and that this
interaction elicits internalization of the corresponding
inclusion complexes with PCs into the macrophages.
We have previously used a covalently attached dansyl tag to
2
15b
trace sp -iminosugar internalization in fibroblasts.
However, the presence of the relatively big fluorescent moiety
Conclusions
may significantly alter the membrane-crossing and diffusion The present study provides a proof of concept of the suitability
properties of the molecule as compared with the unlabelled of the βCD-mannosyl dendron conjugate (ManS) -βCD as a
3
2
chaperone, limiting the scope of the conclusions. In order to macrophage-targeted delivery system for sp -iminosugar-type
ascertain whether or not the PC:(ManS) -βCD complexes are amphiphilic pharmacological chaperones against Gaucher
3
internalized in macrophages following MMR binding, we have disease. The monosubstituted but multivalent conjugate struc-
instead devised competitive assays using the adamantane- ture of the carrier prototype has been purposely conceived to
equipped dansyl fluorescent probe 7 and the mannosyl keep the inclusion capabilities towards the hydrophobic
2294 | Org. Biomol. Chem., 2014, 12, 2289–2301
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3
Fig. 6 (A) Fluorimetrically determined adhesion of 1 : 1 TNS : (ManS) -
βCD (filled line, ●) and TNS : βCD (slashed line, <) complexes to the cell
membrane of mice macrophages and (B) competitive membrane-bound
TNS displacement assay using (ManS)
S-NOI-NJ : (ManS)
-βCD (dotted line, □) and 6S-NAdB-NJ : (ManS)
βCD (slashed line, Δ) complexes.
3
-βCD (filled line, ○), and the 1 : 1
6
3
3
-
Fig. 5 Chaperone activity of 6S-NOI-NJ and 6S-NAdB-NJ and their
Fig. 7 Structure of dansylated probes 7 and 8 used for confocal
microscopy monitoring of macrophage targeting and cellular uptake of
PC:CD complexes.
3
corresponding 1 : 1 complexes with (ManS) -βCD on wild type (A) and
mutant GCases (B, F213I/F213I; C, N370S/N370S; D, L444P/L444P) in
fibroblasts (in situ cell enzyme assay). Cells were cultured for four days
in the absence or presence of increasing concentrations of the com-
pounds. Lysosomal GCase activity was estimated in intact cells by moieties of the PC candidates, 6S-NOI-NJ and 6S-NAdB-NJ
measuring the rate of production of 4-methylumbelliferone (see ESI† for
details).
while warranting efficient mannose-specific lectin recognition
abilities. Both hypotheses have been first demonstrated for
commercial β-glucosidases and Con A lectin and further vali-
dated in human β-glucocerebrosidase and human MMR
(
recombinant). Complexation of the pharmacological
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combination of macrophage targeting abilities and efficient
PC transfer to GCase may provide a means for improving the
therapeutic outcome of pharmacological chaperone treatments
for Gaucher disease and warrants further in vivo evaluation.
Experimental
General methods
I
27
6
-O-p-Toluenesulfonylcyclomaltoheptaose (1) and 2,2,2-tris-
[
5-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosylthio)-2-oxapentyl]-
2
8
ethyl isothiocyanate (5) were obtained according to literature
2
2a
22b
procedures. Iminosugars 6S-NOI-NJ and 6S-NAdB-NJ were
synthesized according to literature procedures. The synthesis
of dansylated derivatives 7 and 8 is described in the ESI.†
Optical rotations were measured at 20 ± 2 °C in 1 dm tubes on
a Jasco P-2000 polarimeter. UV spectra were recorded in 1 cm
1
13
tubes on a Jasco V-630 spectrophotometer. H (and C NMR)
spectra were recorded at 500 (125.7) MHz with Bruker 500 DRX
magnet. 1D TOCSY, 2D COSY, HMQC and HSQC experiments
were used to assist on NMR assignments. Thin-layer chromato-
graphy (TLC) was carried out on aluminum sheets coated with
silica gel 60 F254 Merck with visualization by UV light and by
2 4
charring with ethanolic 10% H SO and 0.1% ninhydrin.
Column chromatography was carried out on Silica Gel 60.
ESI mass spectra were recorded on a Bruker Daltonics
™
Esquire6000 ion-trap mass spectrometer. Elemental analyses
were carried out at the Instituto de Investigaciones Químicas
(
Sevilla, Spain).
6
I
I
38
-Deoxy-6 -iodocyclomaltoheptaose (2). To a solution of
monotosylated βCD derivative 1 (1.0 g, 0.76 mmol) in DMF
18 mL) NaI (0.57 g, 3.8 mmol, 5 eq.) was added. The mixture
(
was stirred at 80 °C overnight and then the solvent was
removed under reduced pressure. The resulting residue was
t
stirred in a mixture of BuOH–EtOH–H O (5 : 4 : 3, 10 mL), fil-
2
trated and washed with cold EtOH (10 mL), DCM (10 mL) and
acetone (10 mL), and dried to yield compound 2 in 90% yield
(
(
0.87 g). R = 0.18 (6 : 3 : 1 MeCN–H O–NH OH); [α] = +128.7
f 2 4 D
c 0.75, MeOH); H NMR (500 MHz, DMSO-d , 313 K):
6
1
Fig. 8 Fluorescence microscopy pictures (3D projections) of
THP-1 human monocytic cells differentiated into macrophage-like cells
I–VII
II–VII
δ 5.72–5.60 (m, 13H, OH-2
2
, OH-3
), 5.56 (d, 1H, JOH,3
), 4.88 (d, 1H, J1,2
=
=
I
II–VII
.3 Hz, OH-3 ), 4.89–4.83 (m, 6H, H-1
3
incubated with (ManS) -βCD:7 complex (400 μM) alone (row A) and in
I
II–VII
the presence of 6S-NAdB-NJ (400 or 800 μM, rows B and C, respect- 3.7 Hz, H-1 ), 4.57–4.43 (m, 6H, OH-6
), 3.80–3.51 (m, 24H,
ively) or yeast mannan (1 mg mL− , row D). The fluorescence of probe 7,
the nuclear staining reagent Hoechst 33342, and the merged pictures
are represented in the left, center, and right columns, respectively.
Acquired z planes were separated by 0.3 μm, and an average of 50
planes was taken for each cell. Panel (E) quantitates the intracellular
1
II–VII
II–VII
II–VII
I
H-3
, H-5
, H-6
), 3.70 (m, 1H, H-5 ), 3.63 (bd, 1H,
I
I
J6a,6b = 11.3 Hz, H-6a ), 3.54 (bd, 1H, J
(
(
= 3.0 Hz, H-6b ), 3.43
5,6a
I
II–VII
II–VII
m, 1H, H-3 ), 3.45–3.26 (m, 12H, H-2
m, 1H, H-2 ), 3.20 (t, 1H, J3,4 = J4,5 = 8.9 Hz, H-4 ); C NMR
, H-4
), 3.33
I
I
13
I–VII
fluorescence after treatment with (ManS)
3
-βCD : 7 complex (400 μM) (125.7 MHz, DMSO-d , 313 K): δ 102.7–102.1 (C-1
), 86.5
6
I
II–VII
I–VII
II–VII
I–VII
alone (control) and in the presence of 6S-NAdB-NJ (400 or 800 μM) or
yeast mannan (1 mg mL− ). Identical circular regions of interest (ROIs)
comprising 200 cells were taken for fluorescence quantification.
(
7
C-4 ), 82.3–81.9 (C-4
0.0 (C-3 ), 60.6–60.4 (C-6
), 73.6–72.4 (C-2
, C-3
C-5
),
1
I
II–VII
I
), 10.0 (C-6 ); ESIMS: m/z 1279.7
–
[M + Cl] . Anal. Calcd for C H IO : C, 40.52; H, 5.59; found:
42 69 34
C, 40.13; H, 5.62.
I
chaperones by (ManS)
3
-βCD preserves their chaperoning
6 -[2-(tert-Butoxycarbonylamino)ethylthio]cyclomaltoheptaose
potential. The corresponding PC complexes specifically recog- (3). To a solution of 2 (1.42 g, 1.14 mmol) and Cs CO (0.48 g,
2
3
nize the MMR at the surface of macrophages, the cell type that 1.48 mmol, 1.3 eq.) in dry DMF (18 mL), under an Ar atmos-
is mostly affected in GD patients, and this recognition phere, tert-butyl N-(2-mercaptoethyl)carbamate (250 μL,
phenomenon elicits macrophage internalization. The 1.48 mmol, 1.3 eq.) was added. The suspension was heated at
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Paper
I
III–VII
II–VII
7
5 °C for 3 h and the solvent was reduced until 1/3 volume. 2.3 Hz, J5,6b = 6.6 Hz, H-5 ), 3.92–3.84 (m, 17H, H-3
, H-6
= 9.5 Hz, H-3 ), 3.87 (t, 1H, J = J3,4
2,3
),
=
II
The resulting residue was poured into acetone (150 mL), fil- 3.88 (t, 1H, J
3
,4
I
II–VII
tered and purified by column chromatography (6 : 3 : 1 MeCN– 9.3 Hz, H-3 ), 3.81–3.76 (m, 6H, H-5
NHCyst), 3.58 (2d, 6H, JH,H = 6.0 Hz, H-3Pent), 3.54 (dd, 1H,
MeCN–H O–NH OH); [α] = +115.3 (c 1.0 in DMSO); H NMR H-2 ), 3.54 (dd, 1H, J = 9.5 Hz, H-2 ), 3.56–5.49 (m, 11H,
), 3.73 (m, 4H, CH
2
N,
3
H
2
O–NH
4
OH) to give 3 in 67% yield (0.98 g); R
f
= 0.47 (6 : 3 : 1 CH
2
I
1
II
2
4
D
1,2
III–VII
II–VII
I
(500 MHz, 9 : 1 DMSO-d
6
–D
2
O, 333 K): δ 4.88–4.82 (m, 6H, H-2
, H-4
2
), 3.53 (m, 1H, H-4 ), 3.47 (m, 8H, CH NH,
), 4.84 (d, 1H, J1,2 = 3.0 Hz, H-1 ), 3.86–3.59 (m, 24H, H-1Pent), 3.18 (dd, 1H, J6a,6b = 12.8 Hz, H-6a ), 2.94 (dd, 1H,
II–VII
I
I
H-1
H-3
II–VII
II–VII
II–VII
I
3
, H-5
, H-6
), 3.82 (bt, 1H, J = J5,6a = 8.6 Hz, H-6b ), 2.88 (t, 2H, J
= 7.2 Hz, CH2SCyst), 2.87–2.78 (m, 6H,
H-5Pent), 2.17–1.98 (4 s, 36H, MeCO), 1.96 (m, 6H, H-4Pent);
C NMR (125.7 MHz, CD OD, 313 K): δ 182.0 (CS),
3
4
,5
H,H
I
I
II–VII
H-5 ), 3.65, (t, 1H, J2,3 = 8.6 Hz, H-3 ), 3.42–3.26 (m, 12H, H-2
H-4
,
II–VII
I
I
13
), 3.34 (dd, 1H, H-2 ), 3.26 (t, 1H, H-4 ), 3.08 (m, 2H,
I
I
I–VII
I
CH N ), 3.07 (bd, 1H, H-6a ), 2.71 (m, 1H, H-6b ), 2.64 (m, 170.9–170.1 (CO), 102.5–102.2 (C-1
), 84.6 (C-4 ), 82.6
2
Cyst
1
3
II–VII
I–VII
I–VII
2
H, CH
2
S
Cyst), 1.43 (bs, 9H, CMe
3
); C NMR (100.6 MHz, 9 : 1 (C-1Man), 81.8–81.7 (C-4
), 73.4–73.2 (C-3
), 72.9 (C-5
),
I–VII
I
I–VII
DMSO-d
6
–D O): δ 156.0 (CO), 102.9–101.2 (C-1
2
), 86.7 (C-4 ), 72.5–72.3 (C-2
), 71.1 (C-2Man), 69.8 (C-3Man), 69.5 (C-3Pent),
), 78.6 (C ), 73.5–72.4 (C-2 , C-3 , C-5 ), 69.1 (C-5Man), 66.3 (C-4Man), 62.4 (C-6Man), 60.8–60.6 (C-6
II–VII
I–VII
I–VII
I–VII
II–VII
82.3–82.0 (C-4
,
q
II–VII
I
I
6
0.6–60.2 (C-6
), 43.0 (CH
2
N
Cyst), 39.9 (CH
2
S
Cyst, C-6 ), 29.0 C-1Pent), 44.3 (CH
2
N, CH
2
N
Cyst), 33.3 (C-6 ), 32.1 (CH
2
S
Cyst),
(
CMe
3
); ESIMS: m/z 1316.9 [M + Na]+. Anal. Calcd for 29.3 (C-4Pent), 28.0 (C-5Pent), 19.4–19.1 (MeCO); ESIMS: m/z
2
+
C H NO S: C 45.47, H 6.46, N 1.08. Found: C 45.31, H 6.46, 1314.8 [M + 2 Na] . Anal. Calcd for C101H158N O S : C, 46.93;
4
9
83
36
2 64 5
N 0.96.
H, 6.16; N, 1.08; S, 6.20; found: C, 47.11; H, 6.34; N, 0.89;
S, 5.87.
6 -[2-[N′-[2,2,2-Tris[5-α-D-mannopyranosylthio)-2-oxapentyl]-
I
39
6
-(2-Aminoethylthio)cyclomaltoheptaose hydrochloride (4).
I
Compound 3 (94 mg, 72 μmol) was treated with a 1 : 1 TFA–
water mixture (3 mL) at rt for 1 h. The solvents were then evap- ethyl]-thioureido]ethylthio]cyclomaltoheptaose ((ManS)
3
-βCD).
orated under reduced pressure and acid traces were removed The trivalent mannosyl carrier (ManS) -βCD was obtained by
3
by co-evaporating with water several times. The residue was treating a solution of compound 6 (59 mg, 23 μmol) in dry
finally dissolved in diluted aq. HCl and freeze-dried to quanti- MeOH (3 mL) with methanolic NaOMe (1 m, 27 μL, 0.1 eq.) at
tatively furnish compound 4 as hydrochloride salt (89 mg). rt for 1 h. The reaction mixture was neutralized with Amberlite
1
+
[
(
5
3
J
α]D = +113.6 (c 0.9, H O); H NMR (500 MHz, D O): δ 5.04 IR-120 (H ) ion exchange resin, the resin filtered-off and the
2
2
II
I
d, 1H, J1,2 = 3.7 Hz, H-1 ), 4.99 (d, 1H, J1,2 = 3.7 Hz, H-1 ), solvent evaporated under reduced pressure to yield (ManS)
3
-
O)
III–VII
I
.00–4.97 (m, 5H, H-1
), 3.95 (bt, 1H, J4,5 = 9.4 Hz, H-5 ), βCD in 97% yield (46 mg). [α]
D
1
= +27.5 (c 1.0, H
2
O); UV (H
2
III–VII
III–VII
III–VII
.91–3.72 (m, 20H, H-3
, H-5
, H-6
), 3.86 (t, 1H, λmax = 262 nm (εmM = 2308); H NMR (500 MHz, D O, 323 K):
2
3
,4
II
I
II–VII
2,3 = J = 9.4 Hz, H-3 ), 3.85 (t, 1H, J2,3 = J3,4 = 9.4 Hz, H-3 ), δ 5.53 (bs, 3H, H-1Man), 5.33 (m, 6H, H-1
.78 (m, 2H, H-6 ), 3.77 (m, 1H, H-5 ), 3.61–3.44 (m, 12H,
), 3.58 (dd, 1H, H-2 ), 3.57 (m, 2H, H-2 , H-3
H-4 ), 3.47 (t, 1H, J4,5 = 9.4 Hz, H-4 ), 3.16 (t, 2H,
), 5.29 (d, 1H,
II
II
I
3
H-2
J1,2 = 3.0 Hz, H-1 ), 4.29 (bs, 3H, H-2Man), 4.19–3.99 (m, 31H,
III–VII
III–VII
II
I
II–VII
II–VII
II–VII
, H-4
, H-5
, H-6
, H-1Pent, H-3Man, H-4Man, H-5Man,
I
II
3
I
H,H
J =
H-6Man), 4.10 (t, 1H, J2,3 = J3,4 = 9.1 Hz, H-3 ), 4.09 (m, 1H,
I
II–VII
II–VII
6
.4 Hz, CH
2
N
Cyst), 3.07 (dd, 1H, J5,6a = 2.9 Hz, J6a,6b = 14.0 Hz, H-5 ), 3.97–3.85 (m, 12H, H-2
, H-4
), 3.93 (m, 2H,
I
I
I
3
H-6a ), 2.91 (dd, 1H, J
CH
8
C-5
= 6.5 Hz, H-6b ), 2.86 (t, 2H, CH N ), 3.90 (dd, 1H, H-2 ), 3.76 (t, 6H, JH,H = 5.4 Hz,
5
,6a
2 Cyst
1
3
I–VII
I
2
S
Cyst); C NMR (125.7 MHz, D
2
O): δ 101.9–101.7 (C-1
), 73.0–71.1 (C-2
), H-3Pent), 3.75 (t, 1H, H-4 ), 3.65 (s, 2H, CH
2
NH), 3.45 (bd, 1H,
I
II–VII
I–VII
I–VII
I
3
3.8 (C-4 ), 81.3–81.1 (C-4
, C-3
,
J
6a,6b = 11.2 Hz, H-6a ), 3.15 (t, 2H, JH,H = 6.5 Hz, CH SCyst),
2
I–VII
II–VII
I
I
), 60.5–60.2 (C-6
), 38.4 (C-6 ), 32.5 (CH NCyst), 29.7 3.07 (dd, 1H, J5,6b = 7.3 Hz, H-6b ), 2.99–2.87 (m, 6H, H-5Pent),
2
Cyst); ESIMS: m/z 1194.8 [M − Cl] .
+
13
(
CH
2
S
2
2.14 (m, 6H, H-4Pent); C NMR (125.7 MHz, D O, 323 K):
I
I–VII
I
6
-[2-[N′-[2,2,2-Tris[5-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl- δ 182.3 (CS), 102.6–102.1 (C-1
thio)-2-oxapentyl]ethyl]thioureido]ethylthio]cyclomaltoheptaose 81.3–80.9 (C-4
6). To a solution of 4 (94 mg, 72 μmol) and Et
44 μmol, 2 eq.) in dry DMF (1 mL), a solution of 5 in dry DMF 60.5–60.2 (C-6
), 85.3 (C-1Man), 85.0 (C-4 ),
II–VII
I–VII
), 73.6–71.4 (C-2Man, C-3Man, C-5Man, C-2
,
I–VII
I–VII
(
1
3
N (20 μL, C-3
, C-5
), 70.4 (C-3Pent), 67.2 (C-4Man), 61.5 (C-1Pent),
II–VII
I
2 2
), 44.7 (CH N, CH NCyst), 34.2 (C-6 ),
(
1 mL) was added. The reaction mixture was stirred at room 33.8 (CH2S ), 29.3 (C-4Pent), 28.0 (C-5Pent); ESIMS: m/z 1062.3
Cyst
2
+
3 134 2 52 5
temperature for 72 h preserving the pH at 8–9 with Et N. The [M + 2 Na] . Anal. Calcd for C77H N O S C, 44.46; H, 6.49;
reaction mixture was then concentrated and the residue was N, 1.35; S, 7.71; found: C, 44.25, H, 6.22; N, 1.03; S, 7.42.
purified by column chromatography (MeCN → 3 : 1 MeCN–
NMR titration experiments
H
H
2
2
O) to give 6 in 50% yield (93 mg). R
f
= 0.66 (6 : 3 : 1 MeCN–
1
O–NH OH); [α]
4
D
= +57.8 (c 1.0, MeOH); H NMR (500 MHz, Association constants (Kas) were determined in D
by measuring the proton chemical shift variations in the H
.6 Hz, J2,3 = 2.8 Hz, H-2Man), 5.30 (t, 3H, J3,4 = J4,5 = 9.8 Hz, NMR spectra of a solution of the βCD derivative (ManS) -βCD
H-4Man), 5.23 (dd, 3H, H-3Man), 5.04 (d, 1H, J1,2 = 3.7 Hz, H-1 ), in the presence of increasing amounts of the corresponding
2
O at 313 K
1
CD OD, 313 K): δ 5.39 (bs, 3H, H-1Man), 5.36 (d, 3H, J1,2
1
=
3
3
II
III–VII
I
5
4
.01–4.98 (m, 5H, H-1
), 5.00 (d, 1H, J1,2 = 3.3 Hz, H-1 ), iminosugar (6S-NOI-NJ or 6S-NAdB-NJ). The chemical shifts of
.41 (ddd, 3H, J5,6a = 4.9 Hz, J5,6b = 2.1 Hz, H-5Man), 4.30 the diagnostic signals obtained at 10–11 different CD:iminosu-
(dd, 3H, J6a,6b = 12.1 Hz, J5,6a = 5.2 Hz, H-6aMan), 4.16 (dd, 3H, gar concentration ratios were used in an iterative least-squares
J5,6b = 5.2 Hz, H-6bMan), 3.97 (ddd, 1H, J4,5 = 9.3 Hz, J5,6a
=
fitting procedure. For a detailed description, see ESI.†
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Isothermal titration calorimetry (ITC)
the corresponding 4-methylumbelliferyl β-D-glycopyranoside
solution in 0.1 M citrate buffer (pH 4). The liberated 4-methyl-
umbelliferone was measured with a fluorescence plate reader
ITC experiments were performed in a multichannel thermal
activity monitor (TAM) isothermal heat conduction micro-
calorimeter (Thermometric AB 2277/201, Järfälla, Sweden)
equipped with a 1.1 mL titration vessel. The calorimeter
was thermostated at 25 ± 0.5 °C. The vessel was loaded
with 0.8 mL of protein solution using a Hamilton syringe,
thermostated at 25 °C and continuously stirred at 60 rpm.
The CD derivative 3 was injected by a computer controlled
syringe pump (Hamilton Microlab M). Injections were made
over a period of 10 s with intervals of 6 min. The experiment
was monitored and analyzed using Digitam 4.1 software
(
excitation 340 nm; emission 460 nm; Infinite F500, TECAN
Japan, Kawasaki, Japan). For enzyme inhibition assay, cell
lysates from normal skin fibroblasts were mixed with
the 4-methylumbelliferyl β-D-glycopyranoside substrates in
the absence or presence of increasing concentrations of the
pharmacological chaperones 6S-NOI-NJ and 6S-NAdB-NJ or
their corresponding 1 : 1 complexes with the mannosylated
carrier (ManS) -βCD.
3
Cell culture and GCase activity enhancement assay
(Thermometric). To minimize dilution artifacts, the ligand
Human skin fibroblasts were cultured in Dulbecco’s modified
Eagle’s medium–10% foetal bovine serum at 37 °C under a
humidified atmosphere containing 5% CO . One control cell
2
was dissolved in the same dialysis buffer as the protein. The
errors are provided by the software from the best fit of the
experimental data to the model of equal and independent
sites, and correspond to the standard deviation in the fitting
of the curves. A separate experiment was performed to deter-
mine the heat of dilution of the ligand in the dialysis buffer.
Reported data are the mean of two measurements made
with different protein preparations; experiments were repeated
more than twice if the obtained parameters were not
congruent.
line (H37) and three lines of GD cells that carried the GlcCer-
ase mutations F213I/F213I, L444P/L444P, and N370S/N370S,
4
0
respectively, were used. The culture medium was replaced
every 2 d with fresh medium. For enzyme activity enhancement
assay, cells were cultured in the presence of different concen-
trations of the pharmacological chaperones 6S-NOI-NJ and
6S-NAdB-NJ or their corresponding 1 : 1 complexes with the
mannosylated carrier (ManS) -βCD for 5 days and harvested by
3
scraping. Cytotoxicity of the compounds was monitored by
measuring the lactate dehydrogenase activities in the cultured
supernatants (LDH assay kit, Wako, Tokyo, Japan).
Inhibition assays against commercial glycosidases
Inhibition constant (K ) values were determined by spectropho-
i
tometrically measuring the residual hydrolytic activities of the
β-glucosidases (from bovine liver or almonds; Sigma) against Enzyme-linked lectin assays (ELLA)
p-nitrophenyl β-D-glucopyranoside. Each assay was performed
Nunc-Inmuno plates (MaxiSorp™) were coated overnight with
in phosphate buffer at the optimal pH for the enzymes. The
reactions were initiated by addition of enzyme to a solution of
the substrate in the absence or presence of various concen-
trations of iminosugar or iminosugar:CD complex (prepared
by freeze-drying equimolecular mixtures of each partner). The
mixture was incubated for 10–30 min at 37 °C and the reaction
was quenched by addition of 1 M Na CO . Reaction times were
yeast mannan at 100 μL per well diluted from a stock solution
−1
of 10 μg mL in 10 mM phosphate buffer saline (PBS, pH 7.3
2+
2+
containing 0.1 mM Ca and 0.1 mM Mn ) at rt. The wells
were then washed three times with 300 μL of washing buffer
(
containing 0.05% (v/v) Tween 20) (PBST). The washing pro-
cedure was repeated after each of the incubations throughout
the assay. The wells were then blocked with 150 μL per well of
2
3
appropriate to obtain 10–20% conversion of the substrate in
order to achieve linear rates. The absorbance of the resulting
1% BSA/PBS for 1 h at 37 °C.
For determination of Con A binding affinity, the wells were
mixture was determined at 405 nm. Approximate values of K
were determined using a fixed concentration of a substrate
around the KM value for the different β-glucosidases) and
various concentrations of an inhibitor. Full K determinations
i
filled with 100 μL of serial dilutions of peroxidase labeled Con
−1
−5
−1
A from 10 to 10 mg mL in PBS, and incubated at 37 °C
for 1 h. The plates were washed and 50 μL per well of 2,2′-azi-
nobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
(
i
and enzyme inhibition mode were determined from the slope
of Lineweaver–Burk plots and double reciprocal analysis.
Representative examples of the Lineweaver–Burk plots, with
typical profile for competitive inhibition mode, are shown in
the ESI (Fig. S11–S13†).
−1
salt (ABTS) (0.25 mg mL ) in citrate buffer (0.2 M, pH 4.0 with
0
.015% H
2
O
2
) was added. The reaction was stopped after
SO and the absor-
20 min by adding 50 μL per well of 1 M H
2
4
bances were measured at 415 nm. Blank wells contained
citrate-phosphate buffer. The concentration of lectin that dis-
played an absorbance between 0.8 and 1.0 was used for inhi-
bition experiments. In order to carry out the inhibition
Lysosomal enzyme activity assay
Lysosomal enzyme activities in cell lysates were determined as experiments, each inhibitor was added in a serial of 2-fold
40
described previously. Briefly, cells were scraped in ice-cold dilutions (60 μL per well) in PBS with 60 μL of the desired
0
.1% Triton X-100 in water. After centrifugation (6000 rpm for lectin–peroxidase conjugate concentration on Nunclon™
1
5 min at 4 °C) to remove insoluble materials, protein concen- (Delta) microtiter plates and incubated for 1 h at 37 °C. The
trations were determined using a protein assay rapid kit above solutions (100 μL) were then transferred to the mannan-
Wako, Tokyo, Japan). The lysates were incubated at 37 °C with coated microplates, which were incubated for 1 h at 37 °C. The
(
2298 | Org. Biomol. Chem., 2014, 12, 2289–2301
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Paper
plates were washed and the ABTS substrate was added (50 μL Determination of mice macrophage adhesion
per well). Color development was stopped after 20 min and the
absorbances were measured.
For evaluation of the interaction with macrophages, the pro-
cedure reported by Muller and Schuber for mannosylated
liposomes was adapted. Briefly, resident peritoneal macro-
phages were obtained from female Balb/c mice (6 to 8 weeks
old) and maintained in Dulbecco’s modified Eagle’s medium
37
For determination of recombinant human MMR (rhMMR)
binding affinity, the wells were filled with 100 μL of serial
−
1
dilutions of rhMMR from a 10 mg mL stock solution in PBS
2
+
2+
(
pH 7.3 containing 0.1 mM Ca and 0.1 mM Mn ), and incu-
(
DMEM) supplemented with 10% decomplemented fetal calf
bated at 37 °C for 1 h. The plates were washed three times with
−1
serum (FCS) containing heparin (5 U mL ). The cell number
PBST as described above and 100 μL of a solution of biotinyl-
6
−1
was adjusted to 10 cells mL and the suspension was plated
final volume 1 mL) in multiwell plates. After 2 h under a
humidified atmosphere of 5% CO in air (final pH 7.4), non-
−
1
ated anti-human MMR antibody (0.2 mg mL ; R&D Systems)
in PBS was added in each well, and the plates were
further incubated for 1 h at 37 °C. The complex NeutrAvidin®-
biotinylated HRP was preformed separately by successively
adding to Tris buffer (9.6 mL, 50 mM, pH 7.6) a solution of
(
2
adherent cells were eliminated by rinsing the dishes three
times with PBS. The adherent cells, 24 h after their isolation,
were fed with fresh serum-less DMEM and incubated with
−
1
NeutrAvidin® (100 μg mL in Tris buffer, 1.2 mL; Thermo
Scientific) and a solution of biotin-conjugated HRP (25 μg
different amounts of TNS : (ManS) -βCD complex. TNS : βCD
3
complex was used as control. After the incubation time, the
medium was pipetted-off and the cells washed four times with
cold PBS (4 °C). TNS associated with the cells was determined
fluorimetrically (JASCO fluorimeter, model FP-715, excitation
at 308 nm and monitoring emission at 443 nm) after cell diges-
tion in 1 mL PBS containing 0.1% of emulphogene BC-720,
and scraped with a rubber policeman. Standard fluorescence
curves were established under the same conditions with
−
1
mL in Tris buffer, 1.2 mL; Thermo Scientific). The mixture
was shaken for 30 min at rt and the solution was immediately
transferred into the plates (60 μL per well). After 1 h at 37 °C,
these plates were washed twice with Tris (250 μL per well)
−
1
and ABTS (0.25 mg mL , 50 μL per well) in citrate buffer
0.2 M, pH 4.0 with 0.015% H ) was added. After 5 min at rt,
(
2 2
O
the optical density was measured at 415 nm. Blank wells
were processed with anti-human MMR antibody as well as
NeutrAvidin®-biotinylated HRP. The concentration of rhMMR
that displayed an absorbance between 0.8 and 1.0 was used for
inhibition experiments. For the competitive lectin binding
aliquots of the initial TNS : (ManS) -βCD and TNS : βCD prep-
3
arations in order to correlate the measured fluorescence inten-
sity with the amount of CD derivative. Experiments were
performed in duplicate, and results did not differ more
than 5%.
inhibition experiment, (ManS)
3
-βCD was mixed in a serial of
2
-fold dilutions (60 mL per well) in HEPES buffer (20 mM,
pH 7.4) with 60 mL of the appropriate rhMMR concentration
in PBS buffer on Nunclon® (Delta) microtitre plates and incu-
bated for 1 h at 37 °C. The above solutions (100 μL) were then
Human macrophage internalization monitoring by
fluorescence microscopy
transferred to the mannan-coated titer plates, which were incu- THP-1 human monocytic cells were cultured in RPMI medium
bated for 1 h at 37 °C. The plates were washed and the solution supplemented with penicillin, streptomycin, and 10% fetal
of biotinylated anti-human MMR antibody in PBS (100 μL) was bovine serum at 37 °C in a humidified 5% CO atmosphere.
2
6
added in each well, and the plates were further incubated for Cells were seeded onto 6-well plates at 1.5 × 3 10 cells per
1
h at 37 °C. Then the NeutrAvidin® solution was transferred well. THP-1 monocytic cells were differentiated into macro-
into the plates (60 μL per well). After 1 h at 37 °C, these plates phage-like cells by phorbol 12-myristate 13-acetate (PMA;
were washed twice with Tris (250 μL per well) and ABTS Sigma-Aldrich) incubation at a final concentration of 100 ng
−
1
was added (50 μL per well). Optical density at 415 nm was mL for 3 d and it was followed by 1 d in PMA-free medium
determined after 5 min.
before treatments.
The percentage of inhibition was calculated as follows:
THP-1 macrophages were grown on 1 mm (Goldseal No. 1)
glass coverslips for 24 h in RPMI containing 10% fetal bovine
serum. After treatment, cells were rinsed once with PBS, fixed
in 3.8% paraformaldehyde for 5 min, and permeabilized in
%
Inhibition ¼ ½Aðno inhibitorÞ ꢀ Aðwith inhibitorÞꢁ=Aðno inhibitorÞ ꢂ 100
0.1% saponin for 5 min. For nuclei staining, glass coverslips
Results in triplicate were used for plotting the inhibition were then rinsed with PBS for 3 min, incubated for 1 min with
curves for each individual ELLA experiment. Typically, the IC50 PBS containing Hoechst 33 342 (1 mg mL− ) and washed with
values (concentration required to achieve 50% inhibition PBS (three 5 min washes). Finally, the coverslips were mounted
of the lectin association to the coating polysaccharide) onto microscope slides using Vectashield Mounting Medium
obtained from several independently performed tests were (Vector Laboratories, Burlingame, CA, USA). 3D projections
in the range of ±15%. Nevertheless, the relative inhibition were performed using a Delta-Vision system (Applied Pre-
values calculated from independent series of data were highly cision; Issaquah, WA) with an Olympus IX-71 microscope
reproducible. The inhibition for methyl α-D-gluco- and manno- (Shinjuku, Japan) with 100× objective/1.35 NA and filters set
pyranoside were included as positive and negative controls, for DAPI, fluorescein isothiocyanate provided by Applied Pre-
1
respectively.
cision. Acquired z planes were separated by 0.3 μm, and an
This journal is © The Royal Society of Chemistry 2014
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Paper
Organic & Biomolecular Chemistry
average of 50 planes was taken for each cell. The 3D stacks
were subjected to Quick Projection using the Softworx soft-
R. Khanna, B. A. Wustman and K. J. Valenzano, J. Med.
Chem., 2013, 56, 2705.
ware. This allows confirming that the fluorescence is located 11 (a) Q.-J. Fan, Biol. Chem., 2008, 389, 1; (b) F. E. Cohen and
inside the cell and not just in the cell membrane. Quantifi- J. W. Kelly, Nature, 2003, 426, 905.
cation of fluorescence signal was performed in 200 cells using 12 For recent contributions see: (a) M. Shanmuganathan and
ImageJ software (National Institutes of Health, Bethesda,
Maryland, USA).
P. Britz-McKibbin, Biochemistry, 2012, 51, 7651;
(b) J. Castilla, R. Rísquez, D. Cruz, K. Higaki, E. Nanba,
K. Ohno, Y. Suzuki, Y. Díaz, C. Ortiz Mellet, J. M. García
Fernández and S. Castillón, J. Med. Chem., 2012, 55, 6857;
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Acknowledgements
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This work was supported by the Spanish Ministerio de Econo-
mía y Competitividad (MINECO), contract numbers SAF2010-
1
5670 and CTQ2010-15848, the Fondo Europeo de Desarrollo
Regional (FEDER), the Fondo Social Europeo (FSE), the Minis-
try of Health, Labour and Welfare of Japan (H17-Kokoro-019,
H20-Kokoro-022), the Junta de Andalucía and the Fundación
Ramón Areces. JRL is grateful to the Spanish Ministerio de
Economía y Competitividad for a pre-doctoral FPI fellowship.
The CITIUS (University of Seville) is gratefully acknowledged
for technical assistance.
1
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