Ureido-substituted sulfamates show potent carbonic anhydrase IX inhibitory and antiproliferative activities against breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2012.05.083

A series of 50 sulfamates were obtained by reacting 4-aminophenol with isocyanates followed by sulfamoylation. Most of the new compounds were nanomolar inhibitors of the tumor-associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII, whereas the

Full text of DOI:10.1016/j.bmcl.2012.05.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4681–4685
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ureido-substituted sulfamates show potent carbonic anhydrase IX
inhibitory and antiproliferative activities against breast cancer cell lines
Jean-Yves Winum a, Fabrizio Carta b, Carol Ward c, Peter Mullen c, David Harrison d, Simon P. Langdon c,
Alessandro Cecchi b, Andrea Scozzafava b, Ian Kunkler e, Claudiu T. Supuran b,f,
⇑
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Breakthrough Breast Unit and Division of Pathology, Institute of Genetics and Molecular Medicine, Edinburgh EH4 2XU, UK
d Pathology, Medical and Biological Sciences Building, University of St. Andrews, North Haugh, St. Andrew, Fife KY16 9TF, UK
e Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Edinburgh EH4 2XU, UK
f Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 50 sulfamates were obtained by reacting 4-aminophenol with isocyanates followed by sulfa-
moylation. Most of the new compounds were nanomolar inhibitors of the tumor-associated carbonic anhy-
drase (CA, EC 4.2.1.1) isoforms IX and XII, whereas they inhibited less cytosolic offtarget isoforms CA I and
II. Some of these sulfamates showed significant antiproliferative activity in several breast cancer cell lines,
such as SKBR3, MCF10A, ZR75/1, MDA-MB-361 and MCF7, constituting interesting anticancer leads.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 May 2012
Revised 21 May 2012
Accepted 21 May 2012
Available online 30 May 2012
Keywords:
Carbonic anhydrase
Inhibitor
Isoform IX and XII
Antitumor agent
Sulfamate
Breast cancer
Recently we reported1 a series of ureido-substituted benzene-
sulfonamides of type A which showed potent inhibitory activity
against several isoforms of carbonic anhydrase (CA, EC 4.2.1.1) in-
volved in tumorigenesis, such as CA IX and XII. Furthermore, in
vivo, some of these compounds significantly inhibited the growth
of both primary tumors and metastases in an animal model of
breast cancer, making them interesting candidates for clinical
development.1,2 The CAs, are a superfamily of metalloenzymes
which catalyze the interconversion between CO2 and bicarbonate
by using a metal hydroxide nucleophilic mechanism.1–9 Five genet-
OSO2NH2
O
N
R
N
H
H
3a – 3bb
It should be noted that recently, CA inhibitory mechanisms
other than the binding to the metal center11 were reported for
a
-
CAs, which do not directly involve the metal ion from the enzyme
active site.12–15 For example polyamines bind to the enzyme by
anchoring to the zinc-coordinated water/hydroxide ion,13 whereas
coumarins act as prodrugs and bind at the entrance of the active
site cavity, quite distant from the metal ion, after being hydrolyzed
by the esterase activity of the CAs to 2-hydroxy-cinnamic
acids.14,15
CA inhibition has pharmacologic applications in the field of
diuretics, antiglaucoma, anticonvulsant, and recently anticancer
agents/diagnostic tools for imaging tumors.1,4,5 Diverse isoforms
of the 16 presently known in humans are involved in a variety of
diseases.1,2 The transmembrane isoforms CA IX and XII were
ically distinct CA families are known to date, the
a-, b-, -, d- and
f-class enzymes.2–4 Inhibition and activation of these enzymes
are well understood processes, with most classes of inhibitors
binding to the metal center,1–10 and activators binding at the
entrance of the active site cavity. The sulfonamides and their
isosteres (sulfamates, sulfamides) represent the main class of phar-
macologically relevant CA inhibitors (CAIs), with many such deriv-
atives in clinical use as antiglaucoma agents, anticonvulsants,
diuretics or antiobesity drugs.4–10
⇑
Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 457 3385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.083

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J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4681–4685
shown to be involved in tumorigenesis,2,4,5,16,17 being overexpres-
sed in a broad range of tumor types.16 Their expression negatively
correlates with the prognosis of cancer patients and is regulated by
hypoxia through the hypoxia-inducible factor (HIF) path-
ways.2,4,5,16,17 Several groups reported recently that inhibition of
CA IX/XII with sulfonamides or coumarins, in various types of tu-
mors, has significant anticancer activity in several animal models
of the disease (both alone, or in combination with radiation or
cytotoxic drugs).1,5,18
Although several aliphatic/aromatic sulfamates have been
investigated previously as CA IX/XII inhibitors,19 no detailed such
data are available for aromatic derivatives of this type. Considering
the fact that sulfonamides and sulfamates have a similar mecha-
nism of CA inhibition at the molecular level,2,20 as both these clas-
ses of CAIs are zinc binders, but their pharmacologic properties are
rather different, we report here a series of sulfamates which have
been obtained by considering sulfonamides A as lead molecules.1
Indeed, one of the most peculiar feature of the sulfonamides of
type A was their selective inhibition of the tumor-associated iso-
forms CA IX and XII over the cytosolic offtarget isoforms CA I and
II.1 This has been subsequently explained by the report of the X-
ray crystal structure of 5 of these compounds of type A,21a possess-
ing various R moieties, which allowed the observation of a very
variable orientation of the tail incorporating the RNHCONH moiety,
in various sub-pockets of the CA active site.21a Such an orientation
was considered possible due to the flexibility of the ureido linker,
and has not been observed for example in structurally related com-
pounds possessing the amide (CONH) linker instead of the ureido
(NHCONH) one.21
4-nitrophenyl-ureido compound 3m (KI of 1.23
whereas most other substitution patterns led to compound
with inhibition constants in the range of 2-6 M (Table 1).
The weak inhibition of this isoform may be considered a
positive feature of this class of CAIs (and is shared also with
the corresponding sulfonamides A reported earlier)1 since
hCA I, highly abundant in red blood cells, is undoubtedly
an offtarget when considering antitumor CAIs.23
lM),
l
(ii) The physiologically dominant cytosolic isoform hCA II was
moderately inhibited by sulfamates 3, which showed KIs in
the range of 119–902 nM (Table 1). The simplest, unsubsti-
tuted member of this family of compounds, 3a, was a med-
ium potency hCA II inhibitor (KI of 393 nM). Introducing
various substituents, such as halogens, methoxy, cyano, phe-
nyl, phenoxy, etc.) either lead to a slight increase in the
inhibitory activity or maintain he inhibition at the same lev-
els as for 3a (e.g., compounds 3b–3l). Interestingly, the
4-nitrosubstituted derivative 3m, was a weaker hCA II
inhibitor compared to the sulfamates discussed above. Other
substitution patterns leading to a loss of activity compared
to 3a were those present in 3p (3,5-dimethylphenyl), 3s
(2-bromo-4,6-difluorophenyl), 3u (1-adamantyl), 3ab (2-
biphenyl), 3ac (2-phenoxyphenyl), 3ad (3-phenoxy-phenyl),
3ai (2-ethoxyphenyl), 3am (4-isopropylphenyl), 3ao (9-flu-
orenyl), these compounds showing inhibition constants in
he range of 430–900 nM (Table 1). The remaining deriva-
tives, possessing a rather wide range of substitution patterns
have an activity similar or slightly better than 3a. Overall,
these sulfamates show a moderate inhibitory action towards
hCA II, which is the main offtarget isoform among the vari-
ous mammalian CAs.23
(iii) The transmembrane isoforms hCA IX, and XII were on the
other hand effectively inhibited by this new class of sulfa-
mate CAIs, which showed KIs in the range of 6–115 nM
against hCA IX, and of 1–78 nM against hCA XII, respectively
(Table 1). SAR is rather straightforward. For example, against
hCA IX, the simplest member of the series, 3a, was a potent
inhibitor, with a KI of 16 nM. Most substitution patterns to
the second aromatic ring, such as halogens, cyano, methoxy,
penta-/tetrafluoro, nitro, 1-naphthyl, etc., lead to compounds
with smilar or slightly improved inhibitory activity (KIs in the
range of 6–17 nM). Few substitution patterns also led to a loss
of the hCA IX inhibition, such as for example those present
in sulfamates 3h (4-biphenyl), 3i (4-phenoxyphenyl); 3u
(1-adamantyl); 3ab (2-biphenyl), 3ac (2-phenoxyphenyl),
3ad (3-phenoxy-phenyl); 3ag (4-benzyloxyphenyl); 3ah
(4-methoxy-5-methylphenyl); 3ak (diphenylmethyl); 3an
(2-isopropylphenyl); 3ao (9-fluorenyl). These compounds
incorporating rather bulky tails showed KIs in the range of
24–115 nM, and with the exception of 3ad, are in fact signif-
icantly strong hCA IX inhibitors. It should be noted that small
structural changes in the nature of the moiety R present in
these ureido sulfamates has drastic consequences for their
inhibitory activity against most CA isoforms investigated so
far, but these differences are particularly obvious for the inhi-
bition of hCA IX (Table 1). The second tumor-associated iso-
form, hCA XII was even better inhibited by sulfamates 3
compared to hCA IX (Table 1). Thus starting form an excellent
inhibitor which is 3a (KI of 10 nM), various substitution pat-
terns led to an enhanced inhibitory power. Among such
derivatives are compounds 3c (4-chlorophenyl); 3g (4-cya-
nophenyl); 3j (pentafluorophenyl); 3k (4-benzylphenyl);
3m (4-nitrophenyl); 3n, 3o, 3p, 3t and 3y (incorporating
more halogens or methyl moieties, or the aminomethylphe-
nyl one), 3ae (4-acetylphenyl); 3ai (2-ethoxyphenyl); 3ap
The lead compounds used in the present drug design study
were the sulfonamides A reported earlier by our group.1 The ureido
part of those molecules incorporated a large number of moieties, of
the aliphatic, aromatic and heterocyclic type, and we have chosen
to maintain this chemical diversity also in the sulfamates reported
here, as the nature of the R moiety was the main factor influencing
in vitro and in vivo activity of sulfonamides A.1 The sulfamates
structurally related to sulfonamides A were prepared as depicted
in Scheme 1. 4-Aminophenol 1 was reacted with a large number
of commercially available isocyanates, leading to the ureido-phe-
nols 2. These intermediates were sulfamoylated with sulfamoyl
chloride, leading to the series of 50 sulfamates 3 by the reported
procedures19 applied earlier to prepare aliphatic and heterocyclic
sulfamates.22
Inhibition data against the offtarget23 cytosolic CA isozymes h
(h = human) hCA I and II, as well as the tumor-associated, target
isoforms hCA IX and XII, with the new series of sulfamates 3a–
3bb are shown in Table 1. The following structure–activity rela-
tionship (SAR) can be demonstrated for this series of compounds:
(i) The cytosolic, widespread isoforms hCA I was poorly inhib-
ited by sulfamates 3, with inhibition constants in the range
of 1.23–13.0
lM. The best inhibitor in the series was the
OSO2NH2
OH
OH
ClSO2NH2
RNCO
MeCN
DMA
O
NH
NH2
O
NH
NH
R
NH
R
1
2a-2bb
3a-3bb
Scheme 1. Synthesis of sulfamates 3a–3bb reported in the Letter.

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4681–4685
4683
Table 1
Inhibition of hCA isoforms I, II, IX and XII with sulfamates 3a–3bb, by a stopped flow CO2 hydrase assay, and selectivity ratio for the inhibition of the tumor-associated isoform CA
IX over the cytosolic, offtarget isozyme hCA II24
SO2NH2
O
N
R
N
H
H
A: lead compounds for the drug design
R = aromatic, aliphatic, heterocyclic moiety
No
R
KI (nM)
Selectivity ratio
hCA II/hCA IX
hCA I
h CA II
hCAIX
hCA XII
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3x
3y
3z
3ab
3ac
3ad
3ae
3af
3ag
3ah
3ai
3aj
3ak
3am
3an
3ao
3ap
3aq
3ar
3as
3at
3au
3ax
3ay
3az
3aw
3ba
3bb
Ph
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-I-C6H4
4-NC-C6H4
4-MeO-C6H4
4-Ph-C6H4
4-PhO-C6H4
3240
2800
2870
3050
2100
3280
2350
5400
4360
3180
6500
5460
1230
4370
3500
5600
2450
8700
1390
3240
43000
3300
4320
2450
2960
8600
9000
10540
4520
3480
6640
3320
4500
3600
8690
7510
6540
13000
3480
12500
3100
2490
4530
2470
7650
4300
3800
7640
3010
3095
393
287
291
305
186
279
413
284
319
145
286
213
450
348
286
546
431
298
641
338
467
285
280
192
119
761
815
902
348
413
285
347
486
310
459
613
547
750
344
568
435
306
448
541
317
236
438
257
421
443
16
13
12
13
10
9
15
24
27
6
16
18
6
9
5
10
9
5
8
10
6
3
12
8
1
5
7
4
2
3
2
6
18
6
5
46
4
5
2
6
46
69
78
4
9
13
6
24.6
22.1
24.3
23.5
18.6
31.0
27.5
11.8
11.8
24.2
17.9
11.8
75.0
42.7
57.2
78.0
53.9
17.5
71.2
30.7
22.2
28.5
35.0
27.4
11.9
12.9
10.5
7.8
C6F5
4-PhCH2-C6H4
PhCH2CH2
4-O2N-C6H4
4-Me2N-C6H4
2,3,4-F3C6H2
3,5-Me2C6H3
4-EtO2C-C6H4
1-naphthyl
2-Br-4,6-F2C6H2
2,4,6-Cl3C6H2
1-adamantyl
3,4-Cl2C6H3
3-Cl-C6H4
2,4-F2C6H3
2-Me-4-MeO-C6H3
2-Ph-C6H4
2-PhO-C6H4
3-PhO-C6H4
4-Ac-C6H4
3-Ac-C6H4
4-PhCH2O-C6H4
2-MeO-5-Me-C6H3
2-EtO-C6H4
4-MeC6H4-CH2
Ph2CH
7
8
17
9
11
21
10
8
7
10
59
78
115
13
18
28
36
15
10
54
30
67
75
9
16
8
7
12
18
15
9
26.8
22.9
10.2
9.6
32.4
31.0
8.5
4
10
59
18
37
26
4
29
8
3
4-iPr-C6H4
2-iPr-C6H4
fluoren-9-yl
3-MeS-C6H4
20.4
8.2
10.0
38.2
35.2
54.4
43.7
37.3
30.1
21.1
26.2
73
2-naphthyl
2-EtOOC-C6H4
3-(2,3-Dihydrobenzofuran-5-yl)
3-EtOOC-C6H4
2-NC-C6H4
1-naphthyl-CH2CH2
thiophen-2-yl-CH2CH2
(2,3-dihydrobenzo[1,4]dioxin-6-yl)
N-Boc-piperidin-4-yl
pyridin-2-yl-CH2
pyridin-2-yl-CH2 CH2
5
11
25
3
4
2
6
7
12
10
36.7
35.0
44.3
5
14
⁄Mean from 3 different assays. Errors were in the range of 10 of the reported values (data not shown).
(3-methylthiophenyl), and 3ay–3ba (incorporating various
heterocyclic moieties as substituents R). They all possess KIs
in the range of 1–5 nM. Thus, a quite large number of sulfa-
mates 3 show excellent, low nanomolar (<5 nM) inhibitory
activity against hCA XII.
(acetazolamide, ethoxzolamide, dichlorophenamide, etc)
show better hCA II than hCA IX inhibitory action.2 In
the new series reported here, only a few compounds had a
selectivity ratio in the range of 7.8–11.9 (compounds 3h,
3i, 3l, 3z, 3ac, 3ad, 3ag, 3ah, 3ak, 3an, 3ao), being thus rel-
atively little CA IX-selective. The remaining ones had selec-
tivity ratios in the range of 24–78.0, being thus highly
selective CA IX/XII inhbitors (compared to the inhibition of
CA II and I).
(iv) The selectivity ratio for inhibiting the tumor-associated iso-
form hCA IX over the cytosolic offtarget one hCA II, was in
the range of 7.8–78.0 for the new sulfamates 3 reported
here. This is an excellent result, since most sulfonamide CAIs

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J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4681–4685
1.2
1
1.2
SKBR3 (100uM)
MCF10A (100uM)
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
1.2
1
1.4
MDA-MB-361 (100 uM)
ZR-75-1 (100uM)
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
Figure 1. The effect of some of the novel compounds on the proliferation of a panel of breast cancer cell lines in normoxic conditions (21% O2). SKBR3, MCF10A, ZR75/1 and
MDA-MB-361 cells were plated out at a concentration of 1 Â 103 cells per well of a 96-well plate and allowed to adhere for 48 h. Media was removed and the compounds
were added at a concentration of 100
assays.25
lM to assess efficacy in comparison with control cells (shown as 1.0). After 5 days of treatment, proliferation was assessed using SRB
MCF7 (hypoxia)
In order to investigate whether this potent in vitro anti-CA IX
effects are paralleled by effective antiproliferative action in vivo,
we used 4 breast cancer cell lines (SKBR3, MCF10A, ZR75/1 and
MDA-MB-361) which have been treated with 9 of the derivatives
of type 3 described in this paper (see Fig. 1 and 2 for the derivatives
used in the experiments) which possessed interesting in vitro po-
tency as CA IX/XII inhibitors (Table 1).
3n
3m
3c
3i
2
1.8
1.6
1.4
1.2
1
O
D
3j
3l
We had tested initially these compounds at 100 lM under
3p
3g
3b
normoxic conditions and the obtained data is shown in Figure 1
for the four cell lines mentioned above. It was encouraging to ob-
serve that the potencies of the compounds 3 were of a similar order
across these cell lines (Fig. 1). Indeed, data of Figure 1 show that
compounds 3c, 3i, 3j and 3p were highly effective as antiprolifera-
0.8
0.6
0.4
0.2
0
tive agents (at the concentration of 100 lM used in this initial
experiment) in all the breast cancer cell lines investigated, whereas
derivatives 3n, 3m, 3l, 3g and 3b showed less effective such prop-
erties. It should be noted that although moist of these compounds
are effective in vitro CA IX/XII inhibitors, there is not a straightfor-
ward correlation between the in vitro enzyme inhibitory activity
and the in vivo antiproliferative results (but several of the highly
effective inhibitors, for example 3p and 3g were also highly active
in vivo).
Subsequently, under hypoxic conditions in the MCF7 cell line,
when the expression of CA IX is highly induced by the shift from
normoxia to hypoxia,16 a dose-dependent and significant antipro-
liferative effect was observed with all the 9 compounds of type 3
0µM
3µM
10µM
30µM
Figure 2. MCF7 breast cancer cells were plated on 96-well plates at a concentration
of 3 Â 103 per well, to allow for the inhibitory effect of hypoxia on cell proliferation.
After 48 h adherence, plates were placed in a Don Whitley H35 hypoxystation set to
0.5% O2 and left to equilibrate for 6 h, before the addition of compounds of type 3
made up in hypoxic media. Compounds were tested at a range of concentrations
from 0.1–100
lM and dose responses assessed using SRB assays after 5 days of
treatment.25
MCF10A, ZR75/1, MDA-MB-361 and MCF7, constituting interesting
anticancer leads.
(Fig. 2) which started at around 10 lM concentration of inhibitor
and was increasing dramatically at 30
inhibitor.
lM concentration of
Acknowledgment
In conclusion, we report here a series of 50 sulfamates, which
were obtained by reacting 4-aminophenol with isocyanates fol-
lowed by sulfamoylation. Most of the new derivatives were nano-
molar inhibitors of the tumor-associated isoforms CA IX and XII,
whereas they inhibited less the cytosolic, offtarget isoforms CA I
and II. Some of these sulfamates showed significant antiprolifera-
tive activity in several breast cancer cell lines, such as SKBR3,
This research was financed in part by a Grant of the 7th Frame-
work Programme of the European Union (METOXIA project).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05. 083.

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4681–4685
4685
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22. 4-Aminophenol 1 (1 equiv.) was added to a solution of isocyanate (1 equiv.) in
15–20 ml of acetonitrile. The mixture was stirred at room temperature until
the complete formation of the product (TLC monitoring). The resulting
precipitate was then filtered and washed with ethyl acetate several times.
Sulfamates were then prepared by reacting the requisite phenol (1 equiv.) with
sulfamoyl chloride (3 equiv.) in N,N-dimethylacetamide (Okada, M.; Iwashita,
S.; Koizumi, N. Tetrahedron Lett. 2000, 41, 7057). Sulfamoyl chloride was
prepared from chlorosulfonyl isocyanate and formic acid as described
previously: Appel, R.; Berger, G. Chem. Ber. 1958, 91, 1339). After completion
of the reaction (TLC monitoring), the mixture was diluted with ethyl acetate
and washed several times with water. The organic extract was dried (MgSO4)
and concentrated under vacuum. The residue was purified either by
crystallization from ether/pentane or by chromatography on silica gel.
4-[(4-Fluorophenyl)ureido]phenyl sulfamate (3b): mp 180–182 °C; 1H NMR
(DMSO-d6, 400 MHz) d 8.8 (s, 1H), 8.75 (s, 1H), 7.9 (s, 2H), 7.5 (m, 4H), 7.2
(m, 4H); 13C NMR (DMSO-d6, 400 MHz) d 159.4, 157, 153.4, 145.3, 138.9, 136.8,
123.5, 120.9, 120.2, 116.2, 116.1, 116, 115.9; MS ESI+ m/z 348 (M+Na)+. ESIÀ m/
z 324 (MÀH)À.
4-[(4-Chlorophenyl)ureido]phenyl sulfamate (3c): mp 200–201 °C; 1H NMR
(DMSO-d6, 400 MHz) d 8.8 (s, 2H), 7.9 (s, 2H), 7.5 (2d, 4H, J = 8.7 Hz), 7.3 (d,
2H, J = 8.7 Hz), 7.2 (d, 2H, J = 8.7 Hz); 13C NMR (DMSO-d6, 400 MHz) d 153.2,
145.4, 139.4, 138.8, 129.5, 129.4, 126.3, 123.5, 121.4, 120.6, 120.3, 120.1, 116;
MS ESI+ m/z 364 (M+Na)+. ESIÀ m/z 340 (MÀH)À.
23. Supuran, C. T. Carbonic anhydrases: off-targets, add-on activities, or emerging
novel targets ? In Polypharmacology in Drug Discovery; Peters, J. U., Ed.; Wiley:
Hoboken, 2012; pp 457–489.
24. Khalifah, R.J. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO2 hydration
activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5
for
a-CAs) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalyzed CO2 hydration reaction
for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions
up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and
enzyme solutions were preincubated together for 15 min at room temperature
prior to assay, in order to allow for the formation of the E–I complex. The
inhibition constants were obtained by non-linear least-squares methods using
PRISM 3, as reported earlier,1 and represent the mean from at least three
different determinations. All CA isoforms were recombinant ones obtained in
house as reported earlier.19–21
25. Wells were fixed with 25% cold TCA for 1 h at 4 °C and the plates washed 10
times with distilled H2O and allowed to dry. A 50 ll volume of SRB stain
(Sulphorhodamine B (Sigma–Aldrich, UK) in 1% v/v acetic acid) was added to
each well for 30 min at room temperature, and each plate washed 4 Â with 1%
v/v acetic acid to remove unbound dye. After drying the plates, bound SRB was
solubilised by adding 150 ll of SRB assay buffer (10 mM Tris Base, pH 10.5) to
each well for 1 h at room temperature before reading on a BioHit BP800 plate
reader at 540 nM.
18. Dubois, L.; Peeters, S.; Lieuwes, N. G.; Geusens, N.; Thiry, A.; Wigfield, S.; Carta,
F.; McIntyre, A.; Scozzafava, A.; Dogne, J. M.; Supuran, C. T.; Harris, A. L.;
Masereel, B.; Lambin, P. Radiother. Oncol. 2011, 99, 424.