A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase

Article abstract of DOI:10.1021/jacs.7b07352

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

Full text of DOI:10.1021/jacs.7b07352

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Article
A Fluorescence Polarization Activity-Based Protein
Profiling Assay in the Discovery of Potent, Selective
Inhibitors for Human Non-lysosomal Glucosylceramidase
Daniel Lahav, Bing Liu, Richard J.B.H.N van den Berg, Adrianus M.C.H. van den
Nieuwendijk, Tom Wennekes, Amar T. Ghisaidoobe, Imogen Breen, Maria J. Ferraz, Chi-
Lin Kuo, Liang Wu, Paul P. Geurink, Huib Ovaa, Gijsbert A. van der Marel, Mario van der
Stelt, Rolf G. Boot, Gideon J. Davies, Johannes M.F.G. Aerts, and Herman S. Overkleeft
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07352 • Publication Date (Web): 22 Sep 2017
Downloaded from http://pubs.acs.org on September 22, 2017
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Figure 1. (A) Screening for GBA2 inhibitors (e.g. 1, 2 or 3) using activityꢀbased probe 4 as a readꢀout in a
FluoPolꢀABPP assay. (B) 3ꢀD Structure (PDB 5NCX) of the TxGH116 βꢀglucosidase bound to cyclophellitol
aziridine 5; the enzymatic nucleophile Glu441 and putative acid/base Asp593 are shown for reference. The
map shown is a maxꢀimumꢀlikelihood weighted 2FobsꢀFcalc at 0.4 eꢀ/Å3.
191x433mm (300 x 300 DPI)
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Figure 2. Optimization and validation of FluoPol-ABPP for GBA2. (A) Effect of pH. (B) Effect of probe
concentration. (C) Structures of additional established inhibitors (D) Inhibition by established inhibitors (1,
2, 6, 7 and 8). Error bars represent standard error of the mean (SEM).
60x43mm (300 x 300 DPI)
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Figure 3. FluoPol-ABPP assay with an iminosugar library (358 entries). (A) Competition at 100 nM. (B)
Examples of 2nd generation analogues of compounds 9 and 10.
84x87mm (300 x 300 DPI)
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Figure 4. Competitive ABPP experiment of HEK293T cells overexpressing GBA2 or GBA3 (and expressing
en-dogenous GBA) treated with compounds 9-12 at various final concentrations prior to cell lysis and ABPP
profiling of remaining enzyme activity.
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A Fluorescence Polarization Activity-Based Protein Profiling Assay in
the Discovery of Potent, Selective Inhibitors for Human Non-
lysosomal Glucosylceramidase
Daniël Lahav&,#, Bing Liu&,#, Richard J. B. H. N. van den Berg&, Adrianus M. C. H. van den Nieuwen-
dijk&, Tom Wennekes&, Amar T. Ghisaidoobe&, Imogen Breen†, Maria J. Ferraz§, Chi-Lin Kuo§, Liang
Wu†, Paul P. Geurink$, Huib Ovaa$, Gijsbert A. van der Marel&, Mario van der Stelt$, Rolf G. Boot§,
Gideon J. Davies†, Johannes M. F. G. Aerts§* and Herman S. Overkleeft&*
&Bioorganic Synthesis, $Molecular Physiology, §Medical Biochemistry, Leiden Institute of Chemistry, Leiden University,
Einsteinweg 55, 2333 CC Leiden, The Netherlands, $Department of Chemical Immunology, Leiden University Medical Cen-
ter, Einthovenweg 20, 2333 ZC Leiden, The Netherlands, †Structural Biology Laboratory, Department of Chemistry, The
University of York, York YO10 5DD, United Kingdom
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Supporting Information Placeholder
leaflet of the Golgi apparatus prior to translocation to the
lumen of the organelle for elongation to complex glyco-
sphingolipids. GBA2 activity is complementary to
GH30 human acid glucosylceramidase (GBA, gluco-
cerebrosidase), which processes glucosylceramide in
lysosomes.5 Genetic mutations in GBA are at the basis
of the lysosomal storage disorder Gaucher disease. Gau-
cher macrophages are loaded with massive amounts of
glucosylceramide.5 Other tissues in Gaucher, however,
have remarkably normal glucosylceramide levels, and it
is likely that GBA2 can compensate for reduced GBA
activity in these tissues.5 Glucosylceramide has been
linked to inflammation as well,6 underscoring the bio-
medical potential of compounds able to modulate gluco-
sylceramide levels.
ABSTRACT: Human non-lysosomal glucosylceramidase
(GBA2) is one of several enzymes that controls levels of
glycolipids and whose activity is linked to several hu-
man disease states. There is a major need to design or
discover selective GBA2 inhibitors both as chemical
tools and as potential therapeutic agents. Here, we de-
scribe the development of a fluorescence polarization
activity-based protein profiling (FluoPol-ABPP) assay
for the rapid identification, from a 350+ library of
iminosugars, of GBA2 inhibitors. A focused library is
generated based on leads from the FluoPol-ABPP screen
and assessed on GBA2 selectivity offset against the
other glucosylceramide metabolizing enzymes, gluco-
sylceramide synthase (GCS), lysosomal glucosylcerami-
dase (GBA) and the cytosolic retaining β-glucosidase,
GBA3. Our work, yielding potent and selective GBA2
inhibitors, also provides a roadmap for the development
of high-throughput assays for identifying retaining gly-
cosidase inhibitors by FluoPol-ABPP on cell extracts
containing recombinant, overexpressed glycosidase as
the easily accessible enzyme source.
In addition to its interplay with GBA, GBA2 is itself
implicated in many diseases. Mutations in the GBA2
gene cause hereditary spastic paraplegia (HSP) and au-
tosomal recessive cerebellar ataxia (ARCA),7-9 indicat-
ing that compounds able to enhance GBA2 activity
could have therapeutic potential. Conversely, com-
pounds able to (partially) block GBA2 activity also hold
therapeutic value; we recently found that lowering
GBA2 activity in Niemann-Pick type C (NPC) mouse
models led to elongated life-span and improved motor
coordination.10 This finding we correlated to correction
of GBA2 activity levels in Purkinje cells, which are
amongst the most affected neuronal cells in NPC. Slow-
ing-down of neuropathological processes in NPC mice
was achieved by genetic ablation of the GBA2 gene and
alternatively by pharmacological inhibition of the gene
product, the latter with N-alkyldeoxynojirimycin deriva-
tives such as N-butyl-deoxynojirimycin or N-(5)-
INTRODUCTION
Human neutral glucosylceramidase (GBA2, non-
lysosomal glucocerebrosidase), a member of the glyco-
side hydrolase (GH) family, GH116 (www.cazy.org),
hydrolyzes glucosylceramide in the cytosol to produce
glucose and ceramide.1-3 GBA2 is located at the cyto-
solic leaflet of the endoplasmic reticulum (ER), Golgi
apparatus and endosomes.1,4 GBA2 controls levels of
glucosylceramide, which is synthesized at the cytosolic
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adamantane-1-yl-methoxy-pentyl
deoxynojirimycin
nucleophile Glu441, in an active site pocket on the en-
(respectively Zavesca (1) and AMP-DNM (2), Figure
1).10 Indeed, we and others have reported on the devel-
opment of N-alkyl-deoxynojirimycin derivatives as po-
tent GBA2 inhibitors. The most potent GBA2 inhibitor
at present is AMP-DNM 2 with a Ki of about 3 nM; this
compound however also inhibits GCS and GBA at high
nanomolar concentrations.11,12
zyme surface. The active site pocket is freely accessible
to solvent, with little steric hindrance for tagged, func-
tionalized aziridines such as 4 (Supplementary Figure
4a). Binding of tagged ABP 4 to both GBA2 and
TxGH116 can also be directly observed by fluorescent
scanning of the respective proteins after ABP labeling
(Figure 1A and Supplementary Figure 4B).
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GBA2 inhibitors thus have considerable potential both
as cellular probes / tools and as therapeutic agents. Of-
ten, though, potent GBA2 inhibitors have significant off-
targets; not only promiscuity towards GBA but also the
enzyme responsible for the biosynthesis of glucosylcer-
amide: glucosylceramide synthase (GCS). Selective in
vivo GBA2 inhibition is therefore complicated. For this
reason, and considering the pharmaceutical potential of
GBA2 as a drug target, selective GBA2 inhibitors are
desirable commodities. Similarly, in order to access
specific inhibitors, there is an urgent need for assays that
rapidly identify potential GBA2 inhibitors. We here
report on the development of a fluorescence polarization
activity-based protein-profiling (FluoPol-ABPP) assay
for fast identification of GBA2 inhibitors in extracts
from cells overexpressing GBA2. Screening of our
iminosugar library, which contains over 350 entries,
provided lead structures bearing a relatively small (com-
pared to existing nanomolar GBA2 inhibitors, including
2 and 3) apolar head groups. Building a focused library
around this theme and assessment of the inhibitory po-
tential of these against GBA2, GBA and GCS yielded
new leads for, and new insights into, future development
of potent, selective GBA2 inhibitors. These leads, com-
bined with our FluoPol-ABPP assay are a useful addi-
tion to existing reagents and tools in unraveling GBA2
functioning in health and disease.
RESULTS AND DISCUSSION
Our FluoPol-ABPP assay is based on our activity-based
glycosidase profiling methodology that capitalizes on
the two-step double displacement mechanism employed
by retaining glycosidases. N-Alkyl-cyclophellitol aziri-
dines, with the aziridine nitrogen bearing reporter groups
(fluorophores, biotin), are in class, broad-spectrum re-
taining β-glucosidase activity-based probes (ABP) re-
porting on GBA, GBA2 and the cytosolic retaining β-
glucosidase, GBA3 in human tissue (Figure 1A).13 In
order to support specific reaction and covalency, we
solved the structure of the Thermoanaerobacterium
xylanolyticum TxGH116 β-glucosidase, a close homo-
logue of human GBA2 with a conserved active site, in
complex with cyclophellitol aziridine 5 (the precursor of
ABP 4) to ascertain that cyclophellitol aziridines react
with the active site nucleophile, and thus that competi-
tors of ABP 4 are true active site inhibitors, Figure 1B.14
The aziridine indeed binds to the invariant catalytic
Figure 1. (A) Screening for GBA2 inhibitors (e.g. 1, 2 or
3) using activity-based probe 4 as a read-out in a FluoPol-
ABPP assay. (B) 3-D Structure (PDB 5NCX) of the
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GBA2 overexpressing cells and wild-type cells. IC50
TxGH116 β-glucosidase bound to cyclophellitol aziridine
5; the enzymatic nucleophile Glu441 and putative acid/base
Asp593 are shown for reference. The map shown is a max-
imum-likelihood weighted 2Fobs-Fcalc at 0.4 e-/Å3.
values of a set of known GBA2 inhibitors, both competi-
tive (Zavesca 1, AMP-DNM 2, L-ido-AMP-DNM 6,
DNM 7) and mechanism-based (cyclophellitol 8) were
next established using the FluoPol-ABPP format using
50 nM of ABP 4. Values obtained (Figure 2D) were in
line with literature values on the same compound, both
for the potent (2, 3, 6) and less potent (1, 7, 8) which
were all established using fluorogenic substrate assays,
thereby validating our FluoPol-ABPP assay.16
FluoPol-ABPP studies that have appeared in the litera-
ture make use of the tetramethylrhodamine (TMR)
dye.15 In order to stay in tune with these studies, we
therefore synthesized TMR-cyclophellitol aziridine 4
(see for synthesis details the supporting information, SI
schemes 1 and 2). GBA2 was stably overexpressed in
HEK293 cells, constituting the majority of all β-
glucosidase activity (the ratio of GBA2 levels over other
targets labeled by 4 was established by prolonged imag-
ing of the gel shown in Figure 1A, see the supporting
information, SI Figure S1). Extracts of HEK293 overex-
pressing GBA2 were treated with ABP 4 at 500 nM at
pH 7, and the protein content resolved by SDS PAGE.
Fluorescence scanning of the wet gel slab (λex 530 nm,
λem 580 nm, Figure 1A), revealed a major band at about
102 kD, which corresponds to the molecular weight of
mature GBA2. Levels of active GBA2 in the overex-
pressing cells are such that endogenous GBA and
GBA3, which are also targets of cyclophellitol aziridine
ABPs, are hardly visible, even though labelling of GBA
and GBA3 is saturating at the probe concentrations used.
GBA2 labeling with 4 can be abolished by pre-
incubation with the known GBA2 inhibitors, Zavesca 1
(100 µM final concentration), and AMP-DNM 2 (10 µM
final concentration) in a competitive ABPP experiment
(Figure 1A, lanes 2 and 3, respectively). In fluorescence
polarization assays, a fluorophore is excited at a specific
wavelength with linear polarized light. The orientation
of emitted light and thereby the degree of polarized light
depends on the movement the excited fluorophore has
undergone, which depends on its size: large molecules
(such as GBA2-bound 4) move and rotate slower than
small molecules (here: free 4). Samples are irradiated
with linear polarized light (λex 530 nm) and the degree
of loss of polarized light emitted at λem 580 nm repre-
sents the degree of inhibition of GBA2 labeling with 4.
Initial experiments using fluorescence polarization as a
read-out were performed in 96-well plates. The pH op-
timum of GBA2 is reported1 to be at pH 5.8 and con-
temporary assays for measuring GBA2 inhibitor activi-
ties are performed at or close to this pH. We (Figure 2A)
established that, in a range from pH 4 to pH 8 (the range
at which GBA2 possesses measurable activity), maximal
return of polarized light occurs at pH 7. The optimal
final probe concentration in the assay conditions used
(protein concentration 0.5 mg/mL, Vfinal 75 µL), which
intrinsically depends on the amount of active GBA2
present in the extracts (which we keep constant), was
found to be 25-50 nM (Figure 2B); in this setting a suit-
able difference in polarized light emission (∆ about 180
mPol) was achieved between samples derived from
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Figure 2. Optimization and validation of FluoPol-ABPP
for GBA2. (A) Effect of pH. (B) Effect of probe concen-
tration. (C) Structures of additional established inhibi-
tors (D) Inhibition by established inhibitors (1, 2, 6, 7
and 8). Error bars represent standard error of the mean
(SEM).
We next transferred our assay to a 384-well plate format
(Vfinal 15 µL) and screened our complete iminosugar
library, containing both previously published and until
now unpublished compounds (the structures of all 358
compounds, and the synthesis of new compounds are in
the supporting information). All compounds were as-
sessed at 100 nM final concentration and the percentage
of GBA2 inhibition calculated from the relative loss of
fluorescence polarization observed. Besides N-alkyl-
deoxynojirimycin, the corresponding D-galacto- and L-
ido-configured iminosugars are known to be potent
GBA2 inhibitors and library entries adhering to this
general description are picked up in our FluoPol-ABPP
assay as well (Figure 3).16 Alternatively configured pi-
peridines, partially deoxygenated deoxynojirimycin
derivatives and hydroxylated pyrrolidines show up only
rarely, if at all, as inhibitors in our assays (for a more
detailed grouping of compound classes and GBA2 inhi-
bition see the supporting information).
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As can be seen in Table 1, compounds 9 and 10 are high-
ly potent and rather selective GBA2 inhibitors. Removal
of one methyl group, as in isopropyl derivatives 11 and
12, led to an increase of inhibitory potency against
GBA2 with concomitant (slight) decrease of inhibitory
potency against GCS. L-ido configured compounds 10
and 12 are over 100,000-fold selective for GBA2 over
GBA1, which is a significant improvement compared to
previously reported inhibitors showing a maximal
10,000-fold selectivity (see for selectivity ratios of the
compounds in Table 1 for GBA2 offset against GBA,
GBA3 and GCS, respectively the supporting infor-
mation, SI 8). As mentioned earlier, the activity against
GCS is much reduced when comparing to our previously
reported adamantyl and biphenyl compounds, respec-
tively 2 and 3. Compounds 9, 13 and 14 are about 1000-
fold more potent on GBA2 over GCS, while previously
reported compounds are at best 150-fold more selective.
While substitution of the neopentyl moiety for tetrahy-
drofuranylmethyl groups, as in 14 and 15, had a detri-
mental effect on inhibitory potency towards GBA,
GBA2 and GCS, it has no significant effect on the selec-
tivity. Based on both potency and selectivity, neopentyl
and isopropyl derivatives 9-12 are perhaps the most
potent and selective GBA2 inhibitors reported to date.
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Figure 3. FluoPol-ABPP assay with an iminosugar library
(358 entries). (A) Competition at 100 nM. (B) Examples of
2nd generation analogues of compounds 9 and 10.
Amongst the most potent GBA2 inhibitors we identified
neopentyloxypentyl-deoxynojirimycin 9 and its L-ido-
congener 10 (Figure 3). Intriguingly, for these com-
pounds the hydrophobic N-alkyl group is relatively
small compared to that of 2 and 3. We previously found
that large (as in 2) and preferably aromatic (as in 3)
alkoxy substituents are preferred by GCS, and reasoned
that a reduced size of this part of the molecule may be
favorable for achieving GBA2 selectivity over the
ceramide glucosyltransferase. Accordingly, we synthe-
sized a set of close analogues of compounds 9 and 10
and assessed their inhibitory potency against GBA2,
GBA, GBA3 and GCS (examples of 2nd generation li-
brary 11-15 in Figure 3, see for synthesis and analytical
details the supporting information).
In a final in situ target engagement experiment we per-
formed competitive ABPP on live cells. Cells overex-
pressing GBA2 or GBA3 and containing endogenous
GBA levels, were treated with compounds 9-12 at vari-
ous final concentrations, lysed followed by treatment
with ABP 4. As can be seen from the images of the SDS
PAGE gels (Figure 4) all compounds are cell permeable
and selectively block GBA2 over GBA and GBA3 at the
concentrations tested.
Table 1: Inhibition values and selectivity ratios of deoxynojirimycin derivatives on GCS, GBA, GBA2 and GBA3
Compound
GCSa
GBA1b
GBA2b
GBA3b
>1000
27.6
27.8
66.1
27.6
119
GCS/GBA2c GBA1/GBA2c GBA3/GBA2c
50
675
0.326
153
43
2071
80
>3067
7886
1
0.15
0.008
0.15
5.05
2.77
3.87
0.729
6
0.28
25.3
12.4
3.8
0.0035
0.0027
0.0083
0.0051
0.0077
0.0075
0.0095
0.0059
0.0536
0.1771
2
3
9370
1494
745
10296
7964
3
18
6
9
990
360
516
77
5412
120
15584
1440
128737
1237
1716
689
15455
17067
18632
19661
3358
10
11
12
13
14
15
10.8
1223
7.3
128
177
116
1017
989
>282
53
92
180
>50
122
145
819
a Inhibition value for in situ assay is given as IC50 (µM)
b Inhibition value for in vitro assay is given as Ki (µM)
c Relative GBA2 offset (ratio)
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GBA2 inhibition. Conceptually, with this study we show
that FluoPol-ABPP, as a recently proposed strategy for
HTS screening of various enzymes, is also eminently
feasible for searching for glycosidase inhibitors, espe-
cially considering that the list activity-based glycosidase
probes targeting a variety of retaining glycosidases is
growing.
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EXPERIMENTAL SECTION
Cell culture Human Embryonic Kidney 293T (HEK293T)
cells (Sigma) were cultured in DMEM high glucose (Gib-
co) supplemented with 10% NBS and 100 units/mL peni-
cillin/streptomycin (Gibco) at 37°C and 5% CO2. RAW
264.7 (American Type culture collection) were cultured in
RPMI (Gibco) supplemented with 10% FCS, 1 mM Gluta-
max and 100 units/mL penicillin/streptomycin (Gibco) at
37°C and 5% CO2.
Figure 4. Competitive ABPP experiment of HEK293T
cells overexpressing GBA2 or GBA3 (and expressing en-
dogenous GBA) treated with compounds 9-12 at various
final concentrations prior to cell lysis and ABPP profiling
of remaining enzyme activity.
Transient and stable overexpression of human GBA2
and GBA3.
Primers used for transient GBA2 expressing HEK293T
were designed based on NCBI reference sequence
NM_020944.2. The full-length coding sequences were
cloned into pcDNA3.1/Myc-His (Invitrogen, Life Technol-
ogies, Carlsbad, CA. Sub-confluent HEK293T cells were
transfected with empty pcDNA3.1 or GBA2pcDNA3.1
(Plasmid:PEI ratio 1:3). Media was refreshed 24 hours later
and cells collected 72 hours after transfection in PBS buff-
er. Cells were centrifuged at 1000 rpm for 10 minutes, after
which the supernatant was removed. Cell pellets were snap-
freezed with liquid nitrogen and stored at -80°C.
CONCLUSIONS
Collectively, this work describes the development of an
effective FluoPol-ABPP assay for the rapid screening of
GBA2 inhibitors. Fluorescence polarization (FluoPol) is
often used for the analysis of the potency of small mole-
cules to inhibit enzymes17 but has to the best of our
knowledge not been used in the context of glycosidase
inhibitor assays. This holds certainly true for the combi-
nation of FluoPol and ABPP18, as we show here. Fluo-
Pol-ABPP can be conducted on target enzymes in com-
plex mixtures, as long as the ABP used modifies with
considerable selectivity the target enzyme. This can be
achieved even with broad-spectrum ABPs such as the
one used here, simply by bringing the target enzyme to
overexpression. Target selectivity is easily established
by running an SDS PAGE on the ABP labeled sample.
One potential caveat of FluoPol-ABPP is the covalent
and irreversible nature of the ABP used. Most studies on
FluoPol-ABPP inhibitor screening entail the assessment
of covalent inhibitors.18 Here, we show that competitive
inhibitors can also be identified using FluoPol-ABPP as
an initial screen, after which more in-depth kinetics
studies can be executed on selected compounds using
conventional assays using fluorogenic substrates. Here,
we identified a few compounds from our 358 compound
collection that possess considerable GBA2 selectivity,
and based on these lead structures we prepared a small
focused library to add to our growing library of imino-
sugar glycosidase inhibitors. In-depth analysis revealed
that, indeed, modulating the size of the hydrophobic
nitrogen substituent yields inhibitors more potent, and
crucially also more selective, for GBA2 compared to
existing inhibitors. From a technical point of view, the
FluoPol-ABPP assay can be adapted to allow high-
throughput screening of large compound selections on
Stable GBA2 expressing HEK293T cells were generated as
follows. The PCR-amplified human GBA2 (GBA2, acc. nr:
NM_020944.2) coding sequence (using the following pri-
mers:
sense
5'-GGGGACAAGTTTGTACA
AAAAAGCAGGCTTAACCACCATGGGGACCCAGGA
T CCAG-3' and antisense 5'- GGGGACCACTTTGTAC
AAGAAAGCTGGGTTTCACTCTGGGCTCAGGTTTG-
3') was cloned into pDNOR-221 and sub-cloned in pLen-
ti6.3/TO/V5-DEST using the Gateway system (Invitrogen).
Correctness of the construct was verified by sequencing.
HEK293T cells were transfected with pLenti6.3-GBA2 in
combination with the envelope and packaging plasmids
pMD2G, pRRE and pRSV. Subsequently, culture superna-
tant containing viral particles was collected and used for
infection of HEK293T cells. Selection using blasticidin for
several weeks rendered cells stably expressing human
GBA2 as determined by activity assays. For stable expres-
sion of GBA3 in HEK293T cells, the PCR-amplified
GBA3 (GBA3, acc. nr: NM_020973.4) coding sequence
(using the following oligonucleotides: sense 5'-
GAATTCGCCGCACCATGGCTTTCCCTGCAGGATTT
G-3' and antisense 5'- GCGGCCGCAGATGTGCTTCAA
GGCCATTG-3') was cloned into pcDNA3.1/Zeo and
transfected into HEK293T cells using FuGene® 6 Transfec-
tion Reagent (Promega Benelux, Leiden, The Netherlands).
Selection using Zeocin for several weeks rendered cells
stably expressing human GBA3 as determined by activity
assays.
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Preparation of lysates. Cell pellets were re-suspended in
FluoPol-ABPP screen of the iminosugar library. The
screen, using the optimized conditions as described above,
was conducted in a 384-well black-bottom plate (Greiner)
with reaction volumes set at 15 µl. Concentration of the
iminosugars during the screen was 100 nM. The FluoPol
signal was measured on a PHERAstar (BMG Labtech).
Resulting polarization signals were processed as described
above. Residual enzyme activities were plotted against the
corresponding iminosugar.
lysis buffer (20 mM hepes, 2 mM DTT, 0.25 M sucrose, 1
mM MgCl2, 2.5U/ml benzonase, pH 7.0). The homogenate
was incubated on ice for 30 minutes after lysis and homog-
enization using SilentCrusher (Heidolph). Ultracentrifuga-
tion was performed at 32.000 rpm for 30 minutes at 4°C.
Supernatant fractions were aliquoted in appropriate vol-
umes, after the total protein concentration was determined
via a Bradford assay, using BSA (Sigma) for standards and
BioRad Quickstart Bradford Reagents. Aliquots were snap-
freezed with liquid nitrogen and stored at -80°C.
Gel-Activity Based Protein Profiling experiments. Pre-
pared lysate was diluted in assay buffer (20mM Hepes,
2mM DTT at pH 7.0) until appropriate final protein con-
centration was reached. Samples were incubated with 500
nM probe at 37°C for 1 hour (Vfinal=20 µL). Protein content
was denatured using Laemli Buffer (4x) at 100°C for 3
minutes. Reactions were resolved by 12.5% SDS-PAGE
electrophoresis and wet slabs were scanned for fluores-
cence (Molecular Imager Gel Doc XR, Biorad). For the in
situ competitive ABPP experiments stable GBA2 and
GBA3 overexpressing HEK293T cells were incubated for 1
hour at 37°C with various concentrations of the selected
inhibitors. Cells were washed 2 times with PBS and then
harvested by scraping in 25 mM potassium phosphate buff-
er (pH 6.5) containing 0.1% Triton X-100 (v/v). Cells were
lysed via snap-freezing in liquid nitrogen. Protein concen-
tration was determined via BCA assay (Thermo Fischer).
Labelling of the glucosidases was conducted using 500 nM
ABP 4 for 30 minutes at 37°C on 20 µg protein. Protein
content was denatured using Laemli Buffer (5x) at 100°C
for 5 minutes. Reactions were resolved by 10% SDS-PAGE
electrophoresis and wet slabs were scanned as described
above.
Optimization of the FluoPol-ABPP assay. Optimal probe
concentration on FluoPol signal was determined by varying
probe concentrations from 25 nM to 500 nM probe at a
constant lysate concentration (0.5 mg/ml) and neutral pH.
FluoPol-ABPP assays were also performed at different pH
values. Hepes was replaced by 20 mM citric acid-citrate
buffer for reactions at pH≤6. The pH-experiments were
performed at optimal probe concentration (50 nM). Compe-
tition experiments were conducted by 30 minutes pre-
incubation of compounds in the lysates at 37°C (2.5 %
DMSO). All reactions (Vfinal 75 µL) contained Bovine
Gamma-Globulin (Sigma) and Chaps (Sigma), respectively
0.5 mg/ml and 1 mg/ml, were carried out in 96-wells plates
(flat-black bottom, Greiner). FluoPol-signals were moni-
tored on an Infinite M1000Pro (Tecan) using λex 530 nm
and λem 580 nm. Mock containing lysates were used as
reference samples, samples without probe as blanks to
correct for background polarization and GBA2 containing
lysates without inhibitor as controls. All samples were
corrected for background polarization and the residual
enzyme activity was calculated based on the polarization
signal from the controls. Polarization signals were plotted
against time or inhibitor concentration and processed in
GraphPad Prism 6.0. IC50 values were calculated via non-
linear regression using mentioned software (N=2, n=3).
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Analysis of selected compounds as inhibitors of enzy-
matic activity of GCS, GBA, GBA2 and GBA3. Enzyme
assays of GCS, GBA1, GBA2 and GBA3 were carried out
as previously described (ref 16 and 19) and inhibition by
selected compounds was assessed. Briefly, for GBA1, pure
recombinant human enzyme (Cerezyme from Genzyme)
was used. Activity was measured with with 3.7 mM 4-
methylumbeliferone (4MU)-β-D-glucopyranoside (Sigma)
in 150 mM McIlvaine buffer pH 5.2 supplemented with
0.2% sodium taurocholate (w/v), 0.1% Triton X-100 (v/v),
0.1% bovine serum albumin (BSA) (w/v) for 30 min. The
reaction was stopped with excess 1M NaOH-Glycine (pH
10.3), liberated 4MU fluorescence was measured with a
fluorimeter LS55 (Perkin Elmer) using λEx 366 nm and λEm
445 nm. For GBA2 measurements, cellular homogenates of
a stable HEK293T over-expressing GBA2 cell line pre-
incubated for 30 min with an inhibitor of GBA1 (1 mM
conduritol β epoxide CBE from Sigma) were used. Activity
was measured with 3.7 mM 4MU-β-D-glucopyranoside in
150 mM McIlvaine pH 5.8, 0.1% BSA (w/v) for 1 h. Reac-
tions were stopped as described above for GBA1. For
GBA3 measurements, cellular homogenates of a stable
HEK293T over-expressing GBA3 cell line also pre-
incubated for 30 min with an inhibitor of GBA1 (1 mM
conduritol β epoxide CBE from Sigma) were used. Reac-
tions were conducted in 100 mM hepes pH 7.0, 0.1% BSA
(w/v) for 1 h. All IC50 values were determined in triplicate
and the inhibitors tested were pre-incubated for 30 minutes
at 37°C. Ki values were determined in duplicate using a
range from 0.05 till 5 mM 4MU-β-D-glucopyranoside in
appropriate buffer containing inhibitor. Incubation time and
temperature was performed as described above, but without
the pre-incubation step. Observed fluorescence was curve-
fitted against inhibitor or substrate concentrations using
GraphPad Prism 6.0 in order to obtain IC50 or Ki values.
IC50 values for GCS were determined in situ with 6-[N-
methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl aminododec-
anoyl]sphingosine (NBD-ceramide) as substrate. RAW
264.7 cells were grown to confluence in 6-well plates and
pre-incubated for 1h with an inhibitor of GBA1 activity
(300 µM CBE), followed by 1h incubation at 37°C with 1
µM C6-NBD-ceramide and in the presence of a range of
inhibitor concentrations. The cells were washed 3x with
PBS and harvested by scraping. After lipid extraction (de-
scribed by Bligh and Dyer20), the C6-NBD lipids were
separated and detected by High Performance Liquid Chro-
matography (λEx 470 nm and λEm 530 nm). IC50 values were
determined in duplicate from the titration curves of ob-
served formed C6-NBD-glucosylceramide and data was
curve-fitted via GraphPad Prism 6.0.
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2013, 92, 245–251.
(8) Martin, E., Schüle, R., Smets, K., Rastetter, A., Bou-
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ASSOCIATED CONTENT
khris, A., Loureiro, J. L., Gonzalez, M. A., Mundwiller, E.,
Deconinck, T., Wessner, M., Jornea, L., Oteyza, A. C.,
Durr, A., Martin, J. J., Schöls, L., Mhiri, C., Lamari, F.,
Züchner, S., De Jonghe, P., Kabashi, E., Brice, A., Stevan-
in, G. Am. J. Hum. Genet. 2013, 92, 238–244.
(9) Sultana, S., Truong, N. Y., Vieira, D. B., Wigger, J. G.
D., Forrester, A. M., Veinotte, C. J., Berman, J. N., van der
Spoel, A. C. Zebrafish 2016, 13, 177–187.
(10) Marques, A. R. A., Aten, J., Ottenhoff, R., Van
Roomen, C. P. A. A., Moro, D. H., Claessen, N., Veloz, M.
F. V., Zhou, K., Lin, Z., Mirzaian, M., Boot, R. G., De
Zeeuw, C. I., Overkleeft, H. S., Yildiz, Y., Aerts, J. M. F.
G. PLoS One 2015, 10, 1–18.
(11) Overkleeft, H. S., Renkema, G. H., Neele, J., Vianel-
lo, P., Hung, I. O., Strijland, A., van der Burg, A. M.,
Koomen, G., Pandit, U. K., Aerts, J. M. F. G. J. Biol.
Chem. 1998, 273, 26522–26527.
(12) Wennekes, T., van den Berg, R. J. B. H. N., Boot, R.
G., van der Marel, G. A., Overkleeft, H. S., Aerts, J. M. F.
G. Angew. Chem. Int. Ed. 2009, 48, 8848–8869.
(13) Kallemeijn, W. W., Li, K.-Y., Witte, M. D., Marques,
A. R. A., Aten, J., Scheij, S., Jiang, J., Willems, L. I.,
Voorn-Brouwer, T. M., van Roomen, C. P. A. A., Otten-
hoff, R., Boot, R. G., van den Elst, H., Walvoort, M. T. C.,
Florea, B. I., Codée, J. D. C., van der Marel, G. A., Aerts,
J. M. F. G., Overkleeft, H. S. Angew. Chem. Int. Ed. 2012,
124, 12697–12701.
(14) Charoenwattanasatien, R., Pengthaisong, S., Breen, I.,
Mutoh, R., Sansenya, S., Hua, Y., Tankrathok, A., Wu, L.,
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11, 1891–1900.
Supporting Information.
Synthetic procedures for ABP 4 and (unpublished) deox-
ynojirimycin analogues, H and 13C NMR spectra, IR and
1
9
LC-MS data; Figures validating the FluoPol-ABPP method
and additional figures for the screen showing a preference
for gluco-and ido- configured iminosugars; Compound
structures and inhibitory potencies on GCS, GBA1 and
GBA2. This material is free of available free of charge via
the ACS Publications website.
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AUTHOR INFORMATION
Corresponding Authors
*j.m.aerts@chem.leidenuniv.nl;
h.s.overkleeft@chem.leidenuniv.nl
Author Contributions
#DL and BL contributed equally to this work
ACKNOWLEDGMENT
We thank the Netherlands Organization for Scientific Re-
search (NWO-CW, TOP-PUNT grant to HSO), the China
Scholarship Council (CSC, PhD fellowship to BL) and the
European Research Council (ERC-2011-AdG-290836
“Chembiosphing”, to HSO, ERC-2012-AdG-32294 “Gly-
copoise”, to GJD; GJD holds the Royal Society Ken Mur-
ray Research Professorship) for financial support.
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