Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation

Article abstract of DOI:10.1021/acschembio.7b00885

Glycosaminoglycans (GAGs) play critical roles in diverse processes ranging from viral infection to neuroregeneration. Their regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biological activity. Small-molecule modulators of these sulfotransferases could serve as powerful tools for understanding the physiological functions of GAGs, as well as potential therapeutic leads for human diseases. Here, we report the development of the first cell-permeable, small-molecule inhibitor selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase responsible for biosynthesis of chondroitin sulfate-E (CS-E). We demonstrate that the molecule specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacological tools for interrogating GAG sulfation-dependent processes and may represent a novel therapeutic approach for neuroregeneration.

Full text of DOI:10.1021/acschembio.7b00885

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Article
Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation
Sheldon T. Cheung, Michelle S Miller, Reynand Pacoma, Jason
Roland, Jian Liu, Andrew M Schumacher, and Linda C. Hsieh-Wilson
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00885 • Publication Date (Web): 03 Nov 2017
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Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sul-
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Sheldon T. Cheung, Michelle S. Miller, Reynand Pacoma, Jason Roland, Jian Liu, Andrew M. Schumach-
‡
,†
er, Linda C. Hsieh-Wilson*
†Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena,
California 91125, United States
‡Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
§Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
North Carolina 27599, United States
ABSTRACT: Glycosaminoglycans (GAGs) play critical roles in diverse processes ranging from viral infection to neuroregeneration. Their
regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biological activity.
Small-molecule modulators of these sulfotransferases could serve as powerful tools for understanding the physiological functions of GAGs, as
well as potential therapeutic leads for human diseases. Here, we report the development of the first cell-permeable, small-molecule inhibitor
selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase respon-
sible for biosynthesis of chondroitin sulfate-E (CS-E). We demonstrate that the molecule specifically inhibits GAG sulfotransferases in vitro,
decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-
mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacological tools for interrogating GAG sulfation-
dependent processes and may represent a novel therapeutic approach for neuroregeneration.
ferases is available, which has hindered efforts to design tools to
target these enzymes. Indeed, there are no cell-permeable, small-
molecule inhibitors of any GAG sulfotransferases. Prior work has
INTRODUCTION
Glycosaminoglycans (GAGs) play important roles in a myriad of
1
-4
1,5-7
cellular processes, including viral invasion, cancer metastasis,
2
2-24
1
,3,8,9
7,9-12
focused on peptide- or carbohydrate-based inhibitors
such as
development,
and spinal cord injury.
Their diverse biologi-
sulfated or iminosugars that displayed limited cell permeability due
cal activities are believed to stem from their complex structures,
which allow them to interact with a wide range of proteins.
GAGs are long, linear polysaccharides that display various patterns
of sulfation. For example, chondroitin sulfate (CS) GAGs are as-
sembled from glucuronic acid (GlcA) and N-acetylgalactosamine
2
2,23
1
,3,7,13,14
to the presence of charged groups.
Thus, despite long-standing
2
5,26
interest in inhibiting GAG sulfotransferases,
it was unclear
whether small molecules could be developed to selectively target
this important class of enzymes in a cellular context.
(
GalNAc) disaccharide units that contain at least four distinct sul-
GAG sulfotransferase inhibitors would provide valuable tools for
manipulating GAG sulfation patterns and for understanding their
roles in biology and disease. The induced expression of specific
sulfation motifs is associated with human diseases, including cer-
7,12
fation motifs: CS-A, CS-C, CS-D, and CS-E. Numerous studies
suggest that specific sulfation motifs can serve as key structural
elements for protein recognition and the regulation of downstream
1,7,14-17
27,28
29
30,31
32,33
signaling.
tain cancers, Alzheimer’s disease, osteoarthritis, malaria,
2
34
herpes, and macular corneal dystrophy, but surprisingly, pharma-
cological tools for manipulating GAG sulfation have been lacking.
The sulfation patterns of GAGs are controlled by a family of sul-
1
8-21
fotransferases.
Two major classes of the enzymes, the cytosolic
Cellular sulfation levels can be modulated genetically or by using
and membrane-associated sulfotransferases, catalyze the transfer of
a sulfuryl group from the donor 3’-phosphoadenosine-5’-
phosphosulfate (PAPS) to a variety of amine- and hydroxyl-
19,20,35-37
sodium chlorate, which inhibits PAPS synthetase.
However,
genetic approaches lack the ease and reversibility of small-molecule
inhibitors. Sodium chlorate requires millimolar concentrations and
globally affects all sulfotransferases, disrupting the sulfation of
hormones, bioamines, xenobiotics, proteins, and other glycans, and
1
8,21
containing substrates.
The cytosolic sulfotransferases act on
small-molecule substrates such as xenobiotics. With only one ex-
ception, the sulfotransferases responsible for modifying GAG poly-
saccharides are type II transmembrane proteins that reside in the
Golgi apparatus. All sulfotransferases have a structurally conserved
PAPS-binding site, but distinct acceptor binding sites. Although
numerous cytosolic sulfotransferases have been crystallized, only
limited structural and functional information on GAG sulfotrans-
inducing off-target effects such as suppression of cell proliferation
35-38
and replication.
Moreover, the toxicity of sodium chlorate due
1
8,21
to oxidation of hemoglobin and other metalloproteins limits its
39
biological applications and therapeutic potential.
Here, we sought to develop selective inhibitors for GAG
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of chemical genetic tools for manipulating and understanding
GAG-mediated events, as well as a novel potential therapeutic
strategy for neuroregeneration.
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RESULTS AND DISCUSSION
To discover molecules that inhibit Chst15, we developed a fluo-
rescence-based assay in which Chst15 activity was coupled to the
enzyme Sult1c1 (Figure 1A). Sult1c1 converts 3’-
phosphoadenosine-5’-phosphate (PAP) to PAPS by using 4-
methylumbelliferyl sulfate (MUS) as the sulfate donor, resulting in
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1,42
liberation of fluorescent 4-methylumbelliferone (MU).
We
optimized and validated the assay in 96-well plates to obtain a Z-
factor value suitable for high-throughput screening (0.62; Figure
S1). The assay was then miniaturized to a 1536-well plate format,
and a diversity set of 70,000 compounds was screened at 12.5 μM
for its ability to inhibit human Chst15. The hits obtained were
counter-screened against Sult1c1, resulting in nineteen compounds
(
renumbered 1-19) that inhibited Chst15 with little or no Sult1c1
inhibition (Figure S2A). To validate these compounds further, we
3
5
used a direct, sensitive assay that monitored [ S]-incorporation
3
5
from [ S]-PAPS into CS-A (Figure S2B). The top compounds, 5
and 19, exhibited significant dose-dependent inhibition of Chst15
with IC50 values of 23 μM and 39 μM, respectively (Figure 1B).
To further evaluate 5, we re-tested its activity and that of several
3
5
commercially available analogs using the [ S]-labeling assay (Fig-
ure S3). Although 5 effectively inhibited Chst15, none of the ana-
logs showed improved inhibition, and 5 also unexpectedly showed
cross-reactivity toward Sult1c1 when re-evaluated in the counter-
screen (12.5 μM, Figure S4). We therefore focused our attention
on 19. Thirty-one analogs of 19 were synthesized or obtained
commercially (Tables 1 and 2; Figure S5). To facilitate diversifica-
tion of the molecule, we devised two synthetic routes that allowed
for modification of the anilide or vinylbenzene moieties (Scheme
1). Initial formation of the α,β-unsaturated acid followed by an
N,N’-diisopropylcarbodiimide (DIC)-promoted amide bond cou-
pling enabled functional groups around the anilide to be altered.
Alternatively, synthesis of the cyanoacetamide in two steps permit-
ted incorporation of different vinylbenzene substituents by a piper-
idinium acetate-catalyzed Knoevenagel condensation. These routes
enabled the installation of a wide range of functionalities from a
Figure 1. Identification of two lead inhibitor scaffolds. (A) Enzyme-
coupled fluorescence assay for the high-throughput screening of small-
molecule inhibitors of Chst15. Chst15 activity was monitored by cou-
pling to Sult1c1, which converted MUS to fluorescent MU. (B) Dose-
dependent inhibition of Chst15 by compounds 5 and 19 using a [ S]-
labeling assay.
35
sulfotransferases. As a starting point, we targeted Chst15, the
chondroitin sulfotransferase responsible for sulfating the 6-O-
position of N-acetylgalactosamine in CS-A to generate CS-E (Fig-
1
9,20,40
ure 1A).
Previously our laboratory identified the CS-E motif
Scheme 1. Synthesis of Analogs of 19a
as an important structural determinant in chondroitin sulfate pro-
teoglycans (CSPGs) that inhibits axon regeneration after central
1
7
nervous system (CNS) injury. Blocking the CS-E motif using a
monoclonal antibody specific for CS-E promoted optic nerve re-
a, b
c
1
7
generation in vivo. As such, Chst15 may represent a novel target
for neuronal repair after spinal cord injury, stroke, or other injuries.
21
20
In this report, we describe the first cell-permeable, small-
molecule inhibitors of Chst15 and other GAG sulfotransferases.
Using a fluorescence-based, high-throughput screen (HTS), we
identified two promising scaffolds that upon structure-activity rela-
tionship (SAR) optimization led to molecules with improved po-
tency in biochemical and cellular assays. Kinetic analyses revealed a
reversible-covalent mechanism of inhibition, along with good selec-
tivity toward GAG sulfotransferases compared to cytosolic sul-
fotransferases. Notably, the most potent inhibitor decreased CS-E
sulfation levels on CSPGs and reversed the inhibitory activity of
CSPGs on axonal growth. These studies open the door to a new set
24
d
e
22
23
aReagents and conditions: (a) ethyl cyanoacetate, piperidine, AcOH,
PhMe (91%); (b) NaOH, THF (77%); (c) Ar-NH , DIC, THF (17-
6%); (d) cyanoacetic acid, DIC, THF (45%); (e) Ar-CHO, piperi-
2
5
dine, AcOH, PhMe (52-93%). See Table 1, Table 2, and Figure S5 for
specific structures.
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substitution was highlighted further upon analysis of various di-
Table 1. Relative Inhibition of Chst15 by Anilide Ana-
1
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5
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9
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a
chloro-substituted analogs (34-36), which had comparable or
greater potency than the original compound 19. In particular, com-
pound 34 showed 35% greater potency when compared to 19 in
logs
35
the [ S]-labeling assay.
We next explored the importance of the vinylbenzene substitu-
ents by synthesizing analogs derived from 34. Removal of any of
the three substituents on the vinylbenzene ring (37, 41, 44) greatly
attenuated the inhibitory potency of the compound (Table 2).
Replacement of the bromo substituent with other halogens (38-
40) decreased the activity by up to 2.5-fold, suggesting that the
stereoelectronic properties of the bromo group are well suited for
binding the enzyme. By comparison, modifications to the methoxy
group were more tolerated. For example, replacement of the meth-
oxy with a bromo group (42) led to no significant change in poten-
cy, while replacement with an ethoxy group (43) enhanced poten-
cy by 26% as compared to 19. However, neither of these analogs
exhibited improved potency when compared to 34. Meanwhile,
modification of the phenol group to aniline (45) or indazole (49,
Figure S5) decreased the potency by 3-fold and 26-fold, respective-
ly. Methylation of the phenol functionality also drastically lowered
the inhibitory potency for compounds with common scaffolds (e.g.
Relative
Inhibition
Compound
R1
R2
R3
R4
1
2
2
2
9
5
6
7
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
F
Cl
H
H
H
H
H
H
H
H
Cl
F
1.00 ± 0.01
0.19 ± 0.00
0.30 ± 0.04
0.10 ± 0.00
0.08 ± 0.00
0.10 ± 0.00
0.35 ± 0.05
0.57 ± 0.02
0.37 ± 0.00
0.07 ± 0.01
1.35 ± 0.12
0.98 ± 0.10
1.21 ± 0.09
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Br
Me
CF3
tBu
H
H
H
28
H
29
30
31
H
H
H
32
H
H
33
34
35
H
Ph
H
H
Cl
H
Cl
Cl
H
3
5 vs. 47, Table 1 and Figure S5). Thus, the hydrogen bonding and
Cl
H
electronic properties of the phenolic hydroxyl group are important
36
H
for Chst15 inhibition.
aRelative inhibition values were determined from three independent
measurements using 25 μM of inhibitor and normalized to the inhibi-
tion by compound 19.
We hypothesized that the α,β-unsaturated cyanoacrylamide moi-
ety might serve as an electrophile for nucleophilic amino acid side
chains. Indeed, cyanoacrylamide compounds have been reported to
inhibit kinases via a reversible covalent mechanism by reacting with
Table 2. Relative Inhibition of Chst15 by Vinylbenzene-
Analogs
4
3,44
noncatalytic cysteine residues. Rotating frame nuclear Overhau-
ser effect spectroscopy (ROESY) data suggested that the E-isomer
is generated in 34 after the Knoevengel condensation (Figure S6).
Specifically, correlations between the amide and alkene protons of
34 were detected, which is only possible in the E-configuration.
Reduction of this E-olefin using Pd/C abolished the dose-
dependent effects of 34 (50, Figure S5).
a
Compound
R5
R6
R7
Relative Inhibition
To investigate the electrophilic potential of 34, we incubated it
with a surrogate nucleophile, β-mercaptoethanol (BME, Figure
S7A). Consistent with the formation of a covalent thioether ad-
3
4
OMe
OMe
OMe
OMe
OMe
H
OH
OH
OH
OH
OH
OH
OH
OH
H
Br
H
1.35 ± 0.12
0.13 ± 0.00
0.40 ± 0.03
0.49 ± 0.01
0.88 ± 0.02
0.32 ± 0.00
0.97 ± 0.10
1.26 ± 0.16
0.00 ± 0.00
0.32 ± 0.01
37
43
3
3
4
8
9
0
F
duct, we observed a decrease in the UV-visible absorption band
corresponding to the cyanoacrylamide moiety (λmax = 378 nm;
Figure S7B). Upon ten-fold dilution of the sample treated with
BME into phosphate buffered saline (PBS), the absorbance was
restored as compared to dilution into PBS containing BME, sug-
gesting that formation of the adduct was reversible (Figure S7C).
Further evidence for a reversible covalent adduct was obtained by
Cl
I
41
Br
Br
Br
Br
Br
4
4
4
2
3
4
Br
OEt
OMe
OMe
1
H NMR spectroscopy (Figure S7D). Incubation of 34 with BME
led to partial loss of the olefinic proton ( = 7.83 ppm) and appear-
ance of an aliphatic β-proton ( = 4.49 ppm). Peak integrations
indicated that ~57% of the population had formed the thioether
adduct, and this population was reduced to ~37% upon two-fold
dilution into PBS.
45
NH2
aSee footnote from Table 1.
large pool of commercially available anilines and benzaldehydes.
We compared the inhibitory activities of the analogs using the
We also examined whether 34 inhibits Chst15 through a reversi-
ble covalent mechanism by pre-incubating the inhibitor with
Chst15 for various lengths of time. Longer pre-incubation times
lead to greater inhibition of the enzyme, consistent with a covalent
mechanism of action (Figure S8A). However, unlike the inhibition
typically observed with irreversible covalent inhibitors, the activity
of Chst15 was rapidly restored in ten-fold dilution experiments
3
5
[
S]-labeling assay. Removal of either aromatic ring on 19 (21, 23,
4
6, Scheme 1 and Figure S5) abolished activity, and changing the
anilide ring from di- to mono-substitution (25-33) greatly reduced
its potency (Table 1 and Figure S5). Altering the position of mono-
substitution (31, 32) did not change this trend, nor did substitu-
tion with larger groups such as tert-butyl (30), phenyl (33), or
fused-phenyl substituents (48, Figure S5). The importance of di-
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teins with iodoacetamide probe 51 (25 μM) in the presence or
absence of 34 (10 and 25 μM) for 2 h at room temperature, and
then reacted them with 5-carboxytetramethylrhodamine-azide
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
3
3
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3
3
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3
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4
4
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5
5
5
5
5
5
5
5
6
(
azido-TAMRA). Importantly, no decrease in the labeling of any of
the proteins by 51 was observed, as visualized by in-gel fluores-
cence (Figure S9B). These data indicate that compound 34 does
not react non-specifically with some of the most reactive cysteine
residues in the proteome. As further evidence, we incubated Neu7
astrocytes in the presence or absence of 34 (25 μM) for 24 h, fol-
lowed by treatment with 51 for 1 h. Again, the labeling of proteins
by iodoacetamide probe 51 was unchanged upon treatment with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4 (Figure S9B). Together, our studies provide strong evidence
that inhibitor 34 is selective for sulfotransferases over the myriad of
reactive cysteine-containing proteins in the proteome.
We next investigated the specificity of 34 against a series of cyto-
solic and GAG sulfotransferases. Importantly, we observed a clear
dichotomy in the ability of 34 to inhibit GAG sulfotransferases
compared to cytosolic sulfotransferases (Figure 2C). While 34
inhibited Chst15 and other closely related GAG sulfotransferases
such as the chondroitin 4-O-sulfotransferase Chst11, the chon-
droitin 2-O-sulfotransferase Ust, and the heparan 3-O-
sulfotransferase Hs3st1 (IC50 values 2.0-2.5 μM), its activity against
the cytosolic sulfotransferases Sult1e1, Sult2b1a, and Sult2b1b,
which modify steroidal substrates, was significantly lower (IC50
values 19-42 μM). Moreover, 34 showed no activity against the
cytosolic sulfotransferase Sult1c1 used in the enzyme-coupled assay
at the concentrations tested (3-100 μM). Inhibition of other GAG
Figure 2. Kinetic analysis and specificity of compound 34. Pseudo
first-order studies using the enzyme-coupled fluorescence assay
revealed a competitive and mixed mode of inhibition for the sub-
strates (A) PAPS and (B) CS-A, respectively. (C) Compound 34
displayed 8- to 18-fold greater specificity for GAG sulfotransferases
compared to cytosolic sulfotransferases.
sulfotransferases by 34 is consistent with the high sequence ho-
40,46,47
mology (25-81%) shared by GAG sulfotransferases
and our
observations that 34 engages the PAPS binding pocket.
even at high concentrations of 34 (up to 100 μM). These observa-
tions suggest dissociation of the enzyme-inhibitor complex, thus
supporting its reversibility (Figure S8B).
To study the mechanism further, we performed pseudo first-
order kinetic analyses with compound 34. When the CS-A sub-
strate concentration was varied under pseudo first-order conditions
with respect to PAPS, we obtained K
i
and K ’ values of 1.43 μM and
i
2
.45 μM, respectively, and observed a shift in both Vmax and K
m
indicative of a mixed mode of inhibition (Figure 2A, Table S1, Fig-
ure S10). However, when the PAPS concentration was varied un-
der pseudo first-order conditions with respect to the CS-A acceptor
substrate, we observed a competitive mode of inhibition with a K
i
value of 0.77 μM (Figure 2B, Table S1, Figure S10). Taken togeth-
er, these results suggest that inhibitor 34 competitively interacts
with the PAPS binding site and also likely engages part of the ac-
ceptor binding site.
Due to its electrophilic mechanism of action, 34 could potential-
ly react with numerous proteins that contain reactive cysteine resi-
dues. Cravatt and co-workers have previously identified reactive
cysteine-containing proteins using the iodoacetamide-alkyne probe
51 (Figure S9A), which alkylates reactive cysteine residues and
allows for visualization of the proteins following copper-catalyzed
Figure 3. Treatment of Neu7 astrocytes with 34 reduces expression of
CS-E on cell-surface and secreted CSPGs. (A) Representative images
of Neu7 astrocytes treated with 34 or chlorate and co-stained with a
CS-E-specific antibody (green) and the nuclear stain DAPI (blue). (B)
Quantification of the average fluorescence from anti-CS-E im-
munostaining under each condition. Values represent the mean ±
S.E.M. from three experiments. n = 20-100 cells per experiment. (C)
CS-A, CS-E, and uCS disaccharide composition of secreted CSPGs
from Neu7 astrocytes treated with 34 or sodium chlorate. n = 3. *P <
0.05, **P < 0.01, ***P < 0.001, n.s. = not significant. For B, P values are
calculated relative to the vehicle control. (Scale bar: 100 μm.)
4
5
azide-alkyne cycloaddition (CuAAC). Among the most highly
reactive proteins were thioredoxin, protein arginine methyltrans-
ferase 1 (PRMT1), aldehyde dehydrogenase 1A (ALDH1A), ace-
tyl-CoA acetyltransferase 1 (ACAT1), and glutathione S-
transferase omega 1 (GTSO1). We incubated each of these pro-
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the CSPGs from Neu7-conditioned media, digested them with
1
2
3
4
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chondroitinase ABC (ChABC), and analyzed their disaccharide
composition by reverse-phase ion-pair high-performance liquid
chromatography (RPIP-HPLC). CSPGs secreted from Neu7 as-
trocytes treated with 25 μM of 34 displayed a 62.9 ± 17.0% increase
in unsulfated chondroitin (uCS) content compared to CSPGs
secreted from untreated astrocytes, indicating a decrease in overall
sulfation (Figure 3C, Table S2). This overall decrease was accom-
panied by a specific 67.8 ± 10.9% reduction in the CS-E sulfation
motif on secreted CSPGs. Although other CS sulfation motifs were
also affected, the CS-E motif was reduced to the largest extent
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(
Figure S12). By comparison, CSPGs secreted from sodiumchlo-
rate-treated cells also displayed lower sulfation compared to un-
treated cells, albeit to a lesser degree and only in the presence of
high concentrations of sodium chlorate (39.7 ± 15.6% increase in
uCS; 31.6 ± 10.9 % decrease in CS-E).
Having shown that 34 can effectively inhibit CS-E sulfation lev-
els on both surface-expressed and secreted CSPGs, we next exam-
ined whether this mode of inhibition could be exploited to stimu
late axonal growth. Sensory dorsal root ganglion neurons (DRGs)
adjacent to the rat spinal cord were grown on a substratum coated
with the CSPGs secreted from Neu7 astrocytes treated with or
without 34. The effects of the CSPGs on neurite outgrowth were
determined by immunostaining for the neuronal-specific marker,
βIII-tubulin, and quantifying the average neurite length. To our
delight, we observed a significant, dose-dependent promotion of
neurite outgrowth (30.7 ± 2.5% at 25 μM of 34) when DRGs were
grown on CSPGs secreted from treated astrocytes compared to
untreated astrocytes (Figure 4A). The observed effects could stem
from direct inhibition of Chst15 and/or indirect effects on other
GAG sulfotransferases such as Chst11, which creates the substrate
for Chst15. These results highlight not only the ability of 34 to
block known functions of CSPGs but also the potential value of
pan-specific GAG sulfotransferase inhibitors. In many cases, the
relative contributions of various sulfation motifs and GAG sub-
classes have not been fully investigated; thus, pan-specific inhibi-
tors may be ideal and enable validation of therapeutic hypotheses.
Figure 4. Treatment of Neu7 astrocytes with 34 attenuates CSPG-
mediated inhibition of sensory neurite outgrowth and repulsion.
(A) Representative images and quantification of DRG neurite out-
growth recovery for CSPGs purified from treated astrocytes relative
to CSPGs purified from untreated astrocytes. Data represent the
mean ± S.E.M. from three experiments. n = 20-80 cells per experi-
ment. (B) Representative images and quantification of neurite
crossing for DRG neurons grown in the presence of a boundary
(shown in red) of CSPGs purified from treated or untreated astro-
cytes. Data represent the mean ± S.E.M. from five experiments. n =
20-40 cells per experiment. *P < 0.05, **P < 0.01, n.s. = not signifi-
cant. For A, P values are calculated relative to growth on CSPGs
from untreated astrocytes. (Scale bars: 50 μm.)
CNS injury induces tissue damage and leads to formation of a
glial scar, which consists predominantly of reactive astrocytes and
CSPGs. The glial scar serves as a major barrier to axon regenera-
9-11
tion. We used an in vitro boundary assay to simulate the glial scar
and assess the ability of 34 to reverse the inhibitory activity of
CSPGs. In this assay, purified CSPGs from Neu7 astrocytes treated
with or without 34 were mixed with Texas Red dye and spotted
onto glass coverslips. DRG neurons were plated onto the co-
verslips, and the percentage of neurites that crossed the CSPG
boundary was quantified. Whereas DRG neurites were strongly
repelled by CSPGs from untreated astrocytes, they crossed more
freely into boundaries of CSPGs from astrocytes treated with 34
The ability to inhibit GAG sulfation after CNS injury could have
important implications for neural regeneration. After CNS injury,
reactive astrocytes enter the site of injury and secrete CSPGs as a
9
-11
means to prevent further damage. Previous studies in our labora-
tory have established that the CS-E sulfation motif is upregulated
1
7
on these CSPGs and inhibits the growth of sensory axons. Thus,
we first examined whether 34 could modulate CS-E expression
4
8
levels on CSPG-secreting Neu7 astrocytes. Indeed, astrocytes
treated with 10 μM or 25 μM of 34 showed a significant, dose-
dependent decrease in cell-surface CS-E expression, as indicated by
(
Figure 4B). Thus, compound 34 rendered CSPGs more permis-
4
9
sive to axonal growth, demonstrating its ability to modulate sul-
fation-dependent cellular processes.
immunostaining with a CS-E-specific antibody (Figure 3A and
B). Notably, no cytotoxicity was observable at the concentrations
3
tested when analyzed by the trypan blue exclusion assay (up to 25
μM). By comparison, sodium chlorate did not reduce CS-E levels
to as large an extent, even at doses as high as 25 mM. Similar results
were obtained using NIH3T3 fibroblasts, where significant loss of
CS-E expression was observed upon treatment with 34, and 34 had
superior potency compared to sodium chlorate (Figure S11).
Finally, we evaluated the potential to use inhibitor 34 in vivo by
profiling its absorption, distribution, metabolism, and elimination
(ADME) properties. 34 showed good stability in human, mouse
and rat liver microsomes based on intrinsic clearance (Table S3).
The in vivo pharmacokinetic (PK) properties of 34 were also eval-
uated directly in rats. After intravenous injection of compound 34
at a dose of 3.0 mg/kg, the compound had a moderate clearance of
To characterize the effects of 34 on secreted CSPGs, we purified
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direct application of the compound, as opposed to intravenous
injection, may be more efficacious for future use in vivo. Taken
together, our studies demonstrate that the sulfotransferase inhibi-
tor 34 can diminish the inhibitory effects of CSPGs, and they high-
light its promise to be used for the stimulation of neuronal repair.
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GAG sulfotransferase inhibitors have the potential to be power-
ful pharmacological tools for elucidating the diverse physiological
functions of GAGs and serve as potential therapeutic leads for spi-
nal cord regeneration and human diseases. We report here the dis-
covery of the first cell-permeable, small-molecule inhibitor selective
for GAG sulfotransferases. Inhibitor 34 was derived from a high-
throughput screen targeted against the CS-E sulfotransferase
Chst15 and potently inhibited the sulfation of surface-expressed
and secreted CSPGs. Importantly, by decreasing sulfation levels on
CSPGs, compound 34 reversed the growth inhibitory effects of
CSPGs on sensory axons. These studies demonstrate, for the first
time, the ability to modulate a class of enzymes for which, despite
high interest, no cell-permeable, small-molecule inhibitors exist.
Furthermore, they highlight the promise of using a sulfotransferase
inhibitor-based strategy, and potentially 34 or its derivatives, for
the stimulation of neuronal growth after injury, stroke, or neuro-
degenerative disease. Finally, compound 34 provides a lead scaffold
that may be further developed to target individual sulfotransferases
with high specificity. Owing to the major lack of tools for studying
sulfation, we envision that chemically-tuned small molecules capa-
ble of modulating specific GAG sulfation patterns will find exten-
sive use as tools in manipulating and understanding GAG-
dependent physiological and pathological processes.
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METHODS
Detailed experimental procedures are provided in the Supporting In-
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14) Rogers, C. J., Clark, P. M., Tully, S. E., Abrol, R., Garcia, K. C.,
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Experimental methods, compound characterizations, sup-
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AUTHOR INFORMATION
Corresponding Author
lhw@caltech.edu
(
17) Brown, J. M., Xia, J., Zhuang, B., Cho, K. S., Rogers, C. J., Gama,
C. I., Rawat, M., Tully, S. E., Uetani, N., Mason, D. E., Tremblay, M. L.,
Peters, E. C., Habuchi, O., Chen, D. F., and Hsieh-Wilson, L. C. (2012)
A sulfated carbohydrate epitope inhibits axon regeneration after injury.
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Notes
The authors declare no competing financial interests.
(
18) Chapman, E., Best, M. D., Hanson, S. R., and Wong, C. H. (2004)
ACKNOWLEDGMENT
Sulfotransferases: structure, mechanism, biological activity, inhibition,
and synthetic utility. Angew. Chem. Int. Edit. 43, 3526-3548.
We would like to thank the Beckman Institute Laser Resource Center
at Caltech for use of the Fluorolog 3 instrument, A. Fisher for providing
APS kinase, L. Pedersen for providing Hs3st1 and Sult2b1a/b vectors,
and H. Geller for providing the Neu7 astrocyte cell line. This work was
supported by grants from the NIH (R01 GM093627 (L.C.H.-W.) and
NIH/NRSA 5T32 GM07616 (S.T.C.)).
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