Aryl sulfamates are broad spectrum inactivators of sulfatases: Effects on sulfatases from various sources

Article abstract of DOI:10.1016/j.bmcl.2008.11.059

Aryl sulfamates were originally developed as inhibitors of steroid sulfatase, and have recently been shown to be powerful inactivators of a bacterial sulfatase, PaAtsA from Pseudomonas aeruginosa. We demonstrate that a simple aryl sulfamate, 3-nitrophenyl

Full text of DOI:10.1016/j.bmcl.2008.11.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 477–480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aryl sulfamates are broad spectrum inactivators of sulfatases: Effects
on sulfatases from various sources
Pavla Bojarová, Spencer J. Williams *
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Rd, 3010 Parkville, Vic., Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Aryl sulfamates were originally developed as inhibitors of steroid sulfatase, and have recently been
shown to be powerful inactivators of a bacterial sulfatase, PaAtsA from Pseudomonas aeruginosa. We dem-
onstrate that a simple aryl sulfamate, 3-nitrophenyl sulfamate, can inactivate sulfatases from various
sources including snail, limpet and abalone. In each case inactivation was time-dependent and active-site
directed, as demonstrated by protection against inactivation by substrate. These results suggest that such
easily acquired aryl sulfamates can be used as reliable biochemical reagents for the study of sulfatases
from a diverse array of sources.
Received 23 September 2008
Revised 11 November 2008
Accepted 12 November 2008
Available online 20 November 2008
Keywords:
Inactivation
Inhibitor
Kinetics
Ó 2008 Elsevier Ltd. All rights reserved.
Sulfamate
Sulfatase
Sulfatases (EC 3.1.6) are hydrolytic enzymes found in a wide
variety of lower and higher organisms.1 One group of sulfatases
cleave sulfate esters in a mechanism involving formylglycine
mains elusive, it seems likely that the reaction of an aryl sulfamate
and the FGly residue is responsible for inactivation. Even though sul-
famates are potent inactivators of estrone sulfatase and PaAtsA, it is
not known whether these easily acquired compounds are inactiva-
tors of other sulfatases, which could have widespread implications
,2
(
FGly), an active-site residue unique to these enzymes. FGly origi-
nates post-translationally from cysteine or serine found within a
conserved pentapeptide motif (C/S)X(P/A)XR.3 This sequence is
required for posttranslational modification of the first cysteine or
serine residue to FGly, and is essential for catalytic activity.
Estrone sulfatase (EC 3.1.6.1) is implicated in the progression of
–5
1,15,16
in a range of biotechnological applications.
In this work, we have shown that a representative aryl sulfa-
mate, 3-nitrophenyl sulfamate (which can be prepared in one step
from commercially available 3-nitrophenol and chlorosulfonyl iso-
6,7
17
breast cancer in post-menopausal women, and other sulfatases
cyanate ), efficiently inactivates three other sulfatases from vari-
8,9
participate in bacterial pathogenesis, and the evasion of plant
defenses by grazing insects.10 The design of sulfatase inhibitors is
therefore highly attractive for applications in medicine and bio-
technology. Many aryl sulfamates, phenolic esters of sulfamic acid
ous sources: snail (Helix pomatia), abalone entrails and keyhole
limpet (Patella vulgata). The kinetics of inactivation of these sulfat-
(
H
2
NSO
2
OH; Fig. 1), are potent inhibitors of human estrone sulfa-
and have recently been reported to be nanomolar inhibi-
NO2
1
1–13
tase
O
O
tors of a bacterial sulfatase, Pseudomonas aeruginosa arylsulfatase A
O2N
O S O
K
O S NH2
1
,14
(PaAtsA).
For both enzymes, inactivation by aryl sulfamates was
O
O
time-dependent, active-site directed and irreversible. These data
are consistent with an inactivation mechanism involving covalent
modification of the active site.
1
2
O
Several mechanistic alternatives for the inactivation of sulfatases
1,11–14
H
by aryl sulfamates have been proposed.
In recent work we
O
H2N S O
O
demonstrated that inactivation of PaAtsA by aryl sulfamates occurs
O
H2N S O
O
H
H
through cleavage of the ArO–S bond and that it exhibits >1 stoichi-
O
O
1
4
ometry. Although the exact identity of the inactivated species re-
EMATE
667COUMATE
*
Corresponding author. Tel.: +61 3 83442422; Fax: +61 3 9347 8124.
E-mail address: sjwill@unimelb.edu.au (S.J. Williams).
Figure 1. Structures of potassium 4-nitrophenyl sulfate 1 and inactivators 3-
nitrophenyl sulfamate 2, EMATE and 667COUMATE.
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.059

4
78
P. Bojarová, S. J. Williams / Bioorg. Med. Chem. Lett. 19 (2009) 477–480
ases were compared with those for PaAtsA,14 demonstrating that
aryl sulfamates are broad spectrum inactivators of sulfatases and
providing new insight into the mechanism of inactivation by aryl
sulfamates.
sA14 and, unlike 4-nitrophenyl sulfamate, is relatively stable at
ꢀ
1
pH 7 (spontaneous decay rate constant at 37 °C is 0.0028 min
corresponding to decomposition of 4.2% inactivator in 15 min).
Inactivation kinetics were assessed according to Eq. 1:
,
Kinetic parameters for the hydrolysis of potassium 4-nitro-
K
i
k
inact
1
7
phenyl sulfate 1 by the four sulfatases are shown in Table 1. 4-
Nitrophenyl sulfate was chosen as upon hydrolysis it releases 4-
nitrophenol, which has a high extinction coefficient providing bet-
ter sensitivity in enzymatic assays than 3-nitrophenyl sulfate.
Nonetheless, the assays for limpet and abalone sulfatases suffered
from poor sensitivity owing to the limited amounts of these en-
E þ l ꢀ E:l ! E ꢀ P
where E, I and E–P represent enzyme, aryl sulfamate and covalent
ð1Þ
adduct, respectively; K is the dissociation constant for the en-
i
zyme-sulfamate complex; and kinact is the first-order rate constant
for conversion of the non-covalent E.I complex to the E-P complex.
Inactivation of snail sulfatase obeyed pseudo-first-order kinet-
ics (Fig. 2A), and a full kinetic analysis allowed determination of
zymes available and thus
a
-cyclodextrin was added to enhance
value of 4-nitrophe-
the sensitivity. -Cyclodextrin lowers the pK
a
a
k
i
inact and K values (Table 2). The active-site directed nature of
nol released in the hydrolytic reaction and thus increases the
amount of the ionized form present at pH values close to or below
the original pK value (7.14). Cyclodextrins have been successfully
a
inactivation was unambiguously demonstrated by protection of
snail sulfatase from inactivation using various concentrations of
substrate 1 (Fig. 2B).
applied to enhance the sensitivity of cleavage of 4-nitrophenyl gly-
cosides by glycosidases at low pH1 ; however, to the best of our
knowledge this is the first time they have been used for the anal-
Substrate 1 competes with inactivator 2 according to Eq. (2):
8,19
K
i
k
inact
E + I
+
E.I
E-P
ysis of sulfatases. Addition of
a-cyclodextrin (10 mM) resulted in
a 2.2-fold increase in the extinction coefficient (
D
e
410) from 6530
S
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ð2Þ
to 14400 M cm at pH 7 (cf.
D
e
410 = 17 000 M cm in 0.4 M
Km
glycine–NaOH buffer, pH 10). The effect of
a-cyclodextrin on the
extinction coefficient is saturable (see Supporting Information)
and does not affect the kinetic parameters of the abalone and
limpet sulfatases.
E.S
A plot of the inverse pseudo-first-order rate constant, k 1, against
ꢀ
To demonstrate the generality of aryl sulfamates as sulfatase
inactivators, we selected 3-nitrophenyl sulfamate (2; Fig. 1), which
was among the most potent inactivators identified against PaAt-
substrate concentration (Fig. 2B, inset) enabled determination of
the inhibition constant for inactivator 2 (K = 40.3
i
l
M), which com-
pares favorably with that measured by full kinetic analysis (Table
).
2
Inactivation of both abalone and limpet sulfatases was biphasic.
In the case of limpet sulfatase (Fig. 3A and B), the first-order rate
constants (k) were extracted from the initial rates of inactivation.
With abalone sulfatase (Fig. 4A–C), the biphasic character was
more pronounced and the first-order rate constants could not be
extracted reliably from the initial phase of inactivation. Therefore,
the rate constants for both the fast and slow phase of inactivation
(in a ratio of 2.2:1) were determined by fitting the data to a double
exponential decay Eq. (3):
Table 1
Kinetic parameters for hydrolysis of potassium 4-nitrophenyl sulfate 1 by sulfatases
max (mmol min 1 mg
ꢀ
ꢀ1
K
(mM)
Vmax/K
m
(l min mg
Sulfatase
V
m
ꢀ
1
ꢀ1
protein)
)
ꢀ
3
PaAtsA
6.1 ± 0.2
(0.77 ± 0.12) ꢁ 10
2.03 ± 0.07
10 ± 1
7900 ± 1300
0.157 ± 0.006
0.070 ± 0.009
0.066 ± 0.004
Snail
0.319 ± 0.003
0.72 ± 0.02
0.68 ± 0.01
a
Limpet
Abalonea
10.2 ± 0.6
a
¼ ffast ꢁ eðꢀkfastꢁtÞ þ ð1 ꢀ ffastÞ ꢁ eðꢀkslowꢁtÞ
Limpet and abalone sulfatases are inhibited by phosphate buffer: 4% and 15%
m
res
=
m
0
ð3Þ
2 4 2 4
activity, respectively, in 5 mM K HPO /KH PO , pH 7.
Time (s)
Time (s)
A
0
B
100
200
300
400
500
0
100
200
300
400
500
600
1
1
0
0
-1
-2
-3
-4
-5
-6
-7
-8
-
-
-
-
-
-
-
-
1
2
3
4
5
6
7
8
6
4
2
00
00
00
0
0
0
.05
.04
0.03
.02
0
0.01
0
0
200
400
600
0
20
40
[
sulfamate 2] (μM)
[substrate 1] (mM)
Figure 2. Inactivation of snail sulfatase by 3-nitrophenyl sulfamate 2 at pH 7. (A) Semi-logarithmic plot of inactivation by 500
1.3 M (j), 15.6 M (s), 7.8
l
M (d), 250
l
M (h), 125
l
M (ꢁ), 62.5
l
M (4),
3
l
l
lM (N), and 0 M (}) sulfamate 2. Inset: Replot of inactivation rates. (B) Semi-logarithmic plot of protection against inactivation by 2 (35 lM)
using 0 M (d), 2.5 mM (}), 5 mM (4), 10 mM (ꢁ), 15 mM (h), and 30 mM (N) potassium 4-nitrophenyl sulfate 1 as substrate; (s) denotes control. Inset: Plot of inverse
ꢀ
1
pseudo-first-order rate constant of inactivation (k ) against concentration of 1.

P. Bojarová, S. J. Williams / Bioorg. Med. Chem. Lett. 19 (2009) 477–480
479
Table 2
Inactivation parameters for inhibition of sulfatases by 3-nitrophenyl sulfamate 2
inact (sꢀ1)
K
(mM)
t
1/2 (s)
kinact/K
(s mM
ꢀ1
ꢀ1
)
Sulfatase
k
i
i
PaAtsAa
Snail
Limpet
Abalone, fast
Abalone, slow
0.063 ± 0.007
(0.13 ± 0.03) ꢁ 10
0.035 ± 0.001
0.98 ± 0.16
1.6 ± 0.3
ꢀ3
11
17
18
10
480 ± 110
1.19 ± 0.06
0.038 ± 0.006
0.043 ± 0.009
0.0411 ± 0.0004
0.038 ± 0.002
0.070 ± 0.005
0.0027 ± 0.0002
5.0 ± 1.0
257
0.0005 ± 0.0001
a
See Ref. 14.
0
.04
B
Time (s)
A
0.03
0.02
0
1
100
200
300
400
0.01
0
0
0
2
4
6
8
10 12 14
-
-
-
-
-
-
1
2
3
4
5
6
[
sulfamate 2] (mM)
C
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
1
U
P
C
Figure 3. Inactivation of limpet sulfatase by 3-nitrophenyl sulfamate 2 at pH 7. (A) Semi-logarithmic plot of inactivation by 12.5 mM (s), 8.3 mM (N), 5 mM (d), 2.5 mM (h),
1
mM (ꢁ), 0.5 mM (4), 0.25 mM (j), and 0 M (}) sulfamate 2. (B) Replot of inactivation rates. (C) Protection against inactivation by 2 (1 mM) using substrate 1 (79 mM).
Residual enzyme activity (
respectively.
res 0
vres) was assayed after 20 s from addition of 2. C denotes control, P protected and U unprotected reaction with v /v ratio 1, 0.91 and 0.66,
and are shown in Table 2. The first-order rates for protection of
limpet and abalone sulfatases by substrate 1 against 3-nitro-
phenyl sulfamate-induced inactivation could not be accurately
determined as substrate was rapidly depleted in the inactivation
reaction mixture. Evidence for substrate protection is therefore
The mode of inactivation seen for the four sulfatases deserves
1
4
comment. While inactivation of PaAtsA and snail sulfatases by
2 obeyed pseudo-first-order kinetics (Fig. 2A), limpet and abalone
sulfatases were inactivated in a biphasic manner (Fig. 3A, 4A). This
latter behavior is reminiscent of the inactivation of estrone sulfa-
tase by EMATE, COUMATE and 667COUMATE (Fig. 1).1
1–13
Two
reported as a ratio of
v
res
/v
0
at a single time point (Fig. 3C and
4D, respectively).
possible explanations for the biphasic kinetics of inactivation can
0
0
.06
.04
A
B
C
2
1
.0
.0
0
Time (s)
0
200 400 600 800 1000 1200 1400
0
.5
0
.02
0
-
-
-
-
-
0.5
0
0
1
2
3
4
5
6
0
4
8 12 16 20 24 28
-
1
[
sulfamate 2] (mM)
[sulfamate 2] (mM)
1.5
D
1.0
-
2
0
0
0
0
0
.8
.6
.4
.2
.0
2.5
-
3
3.5
-
4
1
4.5
U
P
C
Figure 4. Inactivation of abalone sulfatase by 3-nitrophenyl sulfamate 2 at pH 7. (A) Semi-logarithmic plot of inactivation by 25 mM (h), 15 mM (+), 10 mM (ꢁ), 5 mM (4),
.75 mM (*), 2.5 mM (d), 1 mM (–), 0.5 mM (j), 0.25 mM (ꢁ), 0.125 mM (s), 0.0625 mM (N) and 0 M (}) sulfamate 2. (B and C) Replot of rates of fast and slow fractions of
inactivation, respectively. (D) Protection against inactivation by 2 (0.5 mM) using substrate 1 (30 mM). Residual enzyme activity ( res) was assayed after 25 s from addition of
. C denotes control, P protected and U unprotected reaction with ratio 1, 1.01 and 0.77, respectively.
3
v
2
res 0
v /v

4
80
P. Bojarová, S. J. Williams / Bioorg. Med. Chem. Lett. 19 (2009) 477–480
be advanced. The first explanation is that the crude enzyme prep-
arations used in this work as well as that for estrone sulfatase con-
tain at least two different sulfatase enzymes (or isozymes) that
possess different parameters of inactivation. Thus, the first fast
phase of inactivation reports on an enzyme possessing a high ki-
broad-scale use as small molecule tools in dissecting sulfatase-spe-
cific processes in complex biological systems. The generality of aryl
sulfamates as sulfatase inactivators suggests their potential applica-
tion in the treatment of sulfatase-related dysfunctions aside from
therapy of hormone-dependent breast cancer.
nact/K
i
value, whereas the second slower phase reports on a differ-
ent enzyme with a lower kinact/K value. An alternative explanation
i
Acknowledgments
for biphasic inactivation kinetics is that inactivation by 3-nitro-
phenyl sulfamate results in sulfamoylation of multiple enzyme res-
This work was supported by the Australian Research Council
and the Selby Foundation.
idues through two independent processes.1
2,14
Thus, one process
leads to the loss of enzyme activity by specific sulfamoylation of
the active-site FGly residue. The second process results in direct
sulfamoylation of other enzyme residues, possibly conserved ac-
tive-site nucleophiles such as Lys or His.14 This second process
leads to a non-specifically sulfamoylated enzyme with altered
parameters of inactivation that is then inactivated by specific sul-
famoylation of the active-site FGly.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.059.
References and notes
It is also worthwhile commenting on the differences in the effi-
1
2
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Bojarová, P.; Williams, S. J. Curr. Opin. Chem. Biol. 2008, 12, 573.
i
ciency of inactivation (in terms of the kinact/K ratio) seen for the
ꢀ
1
ꢀ1
four sulfatases. This value ranges from 480 to 0.038 s mM for
sulfatases from different origins, declining in the order
PaAtsA > snail > abalone (fast fraction) ꢂ limpet sulfatase. This cor-
relates well with the relative affinity of these sulfatases for potas-
3. Bond, C. S.; Clements, P. R.; Ashby, S. J.; Collyer, C. A.; Harrop, S. J.; Hopwood, J.
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4
5
.
.
sium 4-nitrophenyl sulfate as reflected by the increasing K
from nanomolar (PaAtsA) to millimolar (abalone and limpet sulfat-
ases), Table 1. Therefore, the varying kinact/K ratio for these en-
m
value
6
7
.
.
i
zymes is likely related to differences in their affinity for the
nitrophenyl group. Indeed, closer inspection of these data reveals
a good correlation of inactivator K value with the K value for each
i m
enzyme. These data suggest that the ability of aryl sulfamates to
inactivate sulfatases is a common phenomenon.
Sulfatases mediate a wide variety of biological processes includ-
ing developmental cell signaling, hormone regulation, pathogenesis
and cellular degradation. Studies of these enzymes are limited by a
lack of specific reagents, especially inhibitors. This study demon-
strates that aryl sulfamates are time-dependent, active-site-direc-
ted inactivators of a range of sulfatases from various sources. The
8
.
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1
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m
1
able to catalyze the hydrolysis of a specific aryl sulfate, we anticipate
that the corresponding aryl sulfamate should act as an inhibitor. We
have already demonstrated that aryl sulfamates can be used as ac-
tive-site titrants for sulfatases,14 and there is great potential for their
1
7. Denehy, E.; White, J. M.; Williams, S. J. Chem. Commun. 2006, 314.
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