Structure-activity relationships for the interaction of 5,10-dihydroindeno[1,2-b]indole derivatives with human and bovine carbonic anhydrase isoforms I, II, III, IV and VI

Article abstract of DOI:10.1016/j.ejmech.2011.12.022

Several 5,10-dihydroindeno[1,2-b]indole derivatives incorporating methoxy, hydroxyl, and halogen (F, Cl, and Br) moieties on the indene fragment of the molecule were prepared and tested against five carbonic anhydrase (CA, EC 4.2.1.1) isoforms. The inhibitory potencies of these compounds against the human (h) isoforms hCA I, II, IV, VI and bovine (b) isoform bCA III were assessed. Most of them exhibited low micromolar inhibition of these enzymes. K_I values of these compounds against hCA I and hCA II were in the range of 2.14-16.32 μM, and 0.34-2.52 μM, respectively. Isozyme hCA IV was inhibited with K_I-s in the range of 0.435-5.726 μM, while hCA VI with K_I-s of 1.92-12.84 μM bCA III was inhibited with K _I-s in the range of 2.13-17.83 μM. The structurally related compounds, 1,2-dimethoxybenzene, catechol and indole were also tested in order to understand the structure activity relationship. In silico docking studies of some derivatives within the active site of hCA I and II were also carried out in order to rationalize the inhibitory properties of these compounds and understand their inhibition mechanism.

Full text of DOI:10.1016/j.ejmech.2011.12.022

European Journal of Medicinal Chemistry 49 (2012) 68e73
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships for the interaction of 5,10-dihydroindeno[1,2-b]
indole derivatives with human and bovine carbonic anhydrase isoforms I, II, III, IV
and VI
,
e,
Deniz Ekinci a, Hüseyin Çavdar b, Serdar Durdagi c, Oktay Talaz d , Murat S¸ entürk
,
**
***
,
Claudiu T. Supuran f
*
a Ondokuz Mayıs University, Faculty of Agriculture, Department of Agricultural Biotechnology, 55139 Samsun, Turkey
b Dumlupınar University, Education Faculty, 43100 Kütahya, Turkey
c Institute for Biocomplexity and Informatics, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
Karamanoglu Mehmetbey University, Science and Art Faculty, Chemistry Department, 70100 Karaman, Turkey
d
ꢀ
e
_
ꢀ
ꢀ
Agrı Ibrahim Çeçen University, Science and Art Faculty, Chemistry Department, 04100 Agrı, Turkey
f Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 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:
Several 5,10-dihydroindeno[1,2-b]indole derivatives incorporating methoxy, hydroxyl, and halogen (F, Cl,
and Br) moieties on the indene fragment of the molecule were prepared and tested against five carbonic
anhydrase (CA, EC 4.2.1.1) isoforms. The inhibitory potencies of these compounds against the human (h)
isoforms hCA I, II, IV, VI and bovine (b) isoform bCA III were assessed. Most of them exhibited low
micromolar inhibition of these enzymes. KI values of these compounds against hCA I and hCA II were in
Received 15 September 2011
Received in revised form
25 November 2011
Accepted 14 December 2011
Available online 20 December 2011
the range of 2.14e16.32
the range of 0.435e5.726
the range of 2.13e17.83
m
M, and 0.34e2.52
M, while hCA VI with KI-s of 1.92e12.84
M. The structurally related compounds, 1,2-dimethoxybenzene, catechol and
m
M, respectively. Isozyme hCA IV was inhibited with KI-s in
m
m
M bCA III was inhibited with KI-s in
Keywords:
m
Carbonic anhydrase
Phenol inhibitor
Sulfonamide inhibitor
Docking
indole were also tested in order to understand the structure activity relationship. In silico docking studies
of some derivatives within the active site of hCA I and II were also carried out in order to rationalize the
inhibitory properties of these compounds and understand their inhibition mechanism.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Indole
1. Introduction
disorders, includingepilepsy, possibly in the treatmentofAlzheimer’s
disease, whereas several agents are in clinical evaluations as anti-
Carbonic anhydrases (CAs, EC 4.2.1.1) form a family of metal-
loenzymes that play an important role in several physiological and
pathological processes [1]. There are sixteen CA isoforms identified in
mammals (humans have only 15 isoforms) that differ in their
subcellular localization and catalytic activity [1e3]. Some of the
isozymes are cytosolic (CA I, CA II, CA III, CAVII and CA XIII), others are
membrane-bound (CA IV, CA IX, CA XII and CA XIV), two are mito-
chondrial (CA VA and CA VB), and one is secreted in saliva (CA VI).
Inhibitors or activators of this metalloenzyme family have several
medical applications, such as among others in the treatment of
glaucoma, as diuretics, in the management of several neurological
obesity or antitumor drugs/diagnostic tools. However, it is relatively
difficult to design agents (inhibitors or activators) with specificity or
selectivity for any of these isoforms, and as a consequence, many
pharmacological agents belonging to the class of the CA inhibitors
(CAIs)orCAactivators(CAAs)actaspromiscuousinhibitors/activators
of most isozymes with physiological/pathological relevance, and as
drugstheyshowundesiredsideeffects[3e10]. Sofarinhibitoryeffects
of different sulfonamide derivatives, anions, metal ions, phenols and
various drugs have been investigated against many CAs [11e16].
Indole moieties occur in many synthetic and natural products,
some of which are physiologically relevant or therapeutically used
[17,18]. Melatonin, an important hormone containing the indole
moiety, is known to regulate biological rhythms, and to act as
a receptor-independent free-radical scavenger, being also a broad-
spectrum antioxidant [19e22]. In recent investigations, antioxidant
activity of synthetic indole derivatives and their possible mecha-
nisms of actione have been widely studied [18]. Many of these
* Corresponding author. Tel.: þ39 055 4573005.
** Corresponding author. Tel.: þ90 338 2262431.
*** Corresponding author. Tel.: þ90 536 6605381.
E-mail addresses: otalaz@kmu.edu.tr (O. Talaz), senturkm36@gmail.com
(M. S¸ entürk), claudiu.supuran@unifi.it (C.T. Supuran).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.022

D. Ekinci et al. / European Journal of Medicinal Chemistry 49 (2012) 68e73
69
(i) against the slow cytosolic isoform hCA I, the indole derivatives
investigated here showed weak to moderate inhibitory prop-
erties. Thus, derivatives 1e12 exhibited weak inhibition of this
compounds have been demonstrated to possess potent antioxidant
activity, anticarcinogenic, antimutagenic, antibacterial, antiviral,
and anti-inflammatory activities [23].
isoform, with KI-s in the range of 12.13e16.32
mM, with
In
a recent study, the antioxidant activities of 5,10-
a comparable potency as the reference compounds EMATE or
ZNA (Table 1). The compounds 14e16 were more effective
inhibitors against hCA I, with KI-s in the range of 3.26e3.48 mM
(Fig. 2), with a comparable potency to EZA, being more
effective than EMATE, ZNA and AZA. However, compounds 13
dihydroindeno[1,2-b]indole derivatives were reported by one of
our groups [24]. For this reason, we extend the previous study by
investigating the effects of these molecules on the CA activity, with
the goal to discover new CA inhibitors (CAIs).
and 17e20 acted as strong hCA
I
inhibitors (KI-s:
2.14e2.59 M), being thus slightly more effective compared to
m
2. Results and discussion
sulfonamides/sulfamates EMATE-AZA (Table 1). Interestingly,
hydroxyl containing compounds 13e20 were much more
effective CAIs also compared to catechol 22. This trend shows
the efficacy of indole moiety to lead to interesting CAIs. The
best inhibitor was 5,10-dihydroindeno[1,2-b]indol-1-ol 13 and
this data is supported by the in silico docking data (Table 2). It
should be mentioned that presumably these phenols investi-
gated here have inhibition mechanisms distinct of those of the
sulfonamides, which are zinc ion binders within the CA active
site [1e3]. On the contrary, phenols probably anchor to the
zinc-coordinated water molecule/hydroxide ion [6].
2.1. Chemistry
The general synthetic method to prepare 5,10-dihydroindeno
[1,2-b]indoles is shown in Scheme 1, exemplified for compounds
2 and 13 [24]. The synthesis involves a straightforward reaction
sequence: (i) Fisher indolization of 2,3-dihydro-1H-inden-1-one,
methoxyindene-1-ones or 5-halo-2,3-dihydro-1H-inden-1-ones,
followed by: (ii) deprotection of the methoxy groups to afford the
desired hydroxyl-substituted 5,10-dihydroindeno[1,2-b]indoles
(1e20). Initially, the indolization reactions were performed with
O
SO2NH2
SO2NH2
N
S
N
O
H2NO2SO
C2H5O
Zonisamide
EMATE
R1
Ethoxzolamide
O
N N
S
R2
R3
N
H
N
H
SO2NH2
R
R
21
; R: OMe
23
AZA
N
H
1-20
R4
22; R: OH
the use of phenylhydrazine, and the corresponding indene-1-ones,
in acetic acid in the presence of trifluoroacetic acid (TFA) as
a catalyst. O-Demethylation in compounds 2e9 was accomplished
with boron tribromide in CH2Cl2 at room temperature. The struc-
(ii) against the ubiquitous and dominant rapid cytosolic isozyme
hCA II, compounds 1e9, acted as weak inhibitors, with KI-s in
the range of 1.94e2.52
and 20 were moderate inhibitors with KI-s in the range of
0.536e0.813 M. However, the remaining four derivatives 10,
12, 17 and 19 acted as strong hCA II inhibitors (KI-s:
0.343e0.496 M), with a comparable potency as the reference
mM (Table 1). Compounds 11, 13e16, 18
tures of the synthesized compounds were approved by IR, 1H, 13
C
m
NMR data, and elemental analysis. Detailed synthetic procedures
for the preparation and identification of the compounds can be
found in Talaz et al. [24].
m
compounds EMATE-AZA (Table 1). The most potent inhibitor
was the bulky 5,10-dihydroindeno[1,2-b]indole-2,3-diol 19 (KI
2.2. CA inhibition studies
0.343 mM). In silico docking studies support the experimental
results regarding this compound (Table 2), which is also much
more effective than the structurally related compound cate-
chol 22 (KI 9.9 mM). It is interesting to note that halogens (F, Cl,
Br) increase the hCA II inhibition efficacy of the molecules
while this trend was not seen against hCA I. The hydroxyl-
substituted compounds 13e20 were more effective CAIs
compared to catechol 22;
The 10-dihydroindeno[1,2-b]indole derivatives 1e20, 1,2-
dimethoxybenzene 21, catechol 22 and indole 23, as well as stan-
dard, clinical used CAIs, such as, ethoxzolamide (EZA), zonisamide
(ZNA) and acetazolamide (AZA) (Fig. 1) have been tested for the
inhibition of two cytosolic ubiquitous isozymes of human origin,
that is, hCA I and hCA II, the membrane-bound isoform hCA IV, the
secretory isozyme hCA VI and the bovine muscle one, bCA III
(Table 1). EMATE, a sulfamate with CA inhibitory properties was
also included in this study [25].
(iii) the indole derivatives 1e20 showed variable inhibitory prop-
erties against the membrane-bound isoform hCA IV.
Compounds 1e9 exhibited weak inhibitory action (KI-s of
The following should be noted regarding CA inhibitory data of
Table 1:
4.215e5.726
moderate inhibitors (KI-s: 0.532e1.032
m
M) whereas derivatives 10e17,19 and 20 acted as
mM). The most potent

70
D. Ekinci et al. / European Journal of Medicinal Chemistry 49 (2012) 68e73
OMe
OH
OMe
PhNHNH2
AcOH-THF
BBr3
CH2Cl2
N
H
N
H
O
4-methoxy-2,3-dihydro-1H-inden-1-
2
13
one
Scheme 1. Synthesis of 1-methoxy-5,10-dihydroindeno[1,2-b]indole (2) and 5,10-dihydroindeno[1,2-b]indol-1-ol (13).
inhibitor was 5,10-dihydroindeno[1,2-b]indole-1,4-diol 18,
which exhibited an inhibition constantof0.435 M, being much
more effective than catechol 22 (KI: 10.9 M). Methoxy deriv-
atives 2e6 were more effective CAIs (KI-s: 4.082e5.726 M)
than 1,2-dimethoxybenzene 21 (KI: 14.32 M) indicating that
the indole moiety increases the hCA IV inhibitioryactivity of the
investigated derivatives (Table 1). Halogens slightly increased
the activity more than the hydroxy derivatives, except for the
two dihydroxy derivatives 17 and 18;
2.3. In silico studies
m
m
In this study, fully flexible docking methodology for both
receptor residues and docked ligands was employed, by using the
GOLD docking algorithm [26e28]. The tested derivatives were
docked at the binding site of the targets hCA I and hCA II. GOLD
fitness and ChemScore docking scores of docked inhibitors at hCA-I
and CA-II targets and corresponding binding interactions for
the most potent inhibitors were tabulated at Tables 2 and 3,
respectively.
m
m
(iv) a quite weak inhibition profile of the secretory isoform hCA VI
has been observed for the investigated 5,10-dihydroindeno
[1,2-b]indole derivatives. The only effective inhibitor was
5,10-dihydroindeno[1,2-b]indole-1,2-diol 17 having a KI of
We have performed docking studies of compounds 1e23 within
the hCA I and II active sites, by using as templates hCA I and II
adducts for which the X-ray crystal structures have been reported
1.92
mM, which is comparable with those of the reference
in complexes with activators (e.g., hCA I e L-His, PDB code 2FW4)
compounds EMATE-AZA (KI-s: 0.34e2.42
m
M) (Table 1).
[29] or inhibitors (the hCA II e phenol, PDB file not available in the
data base, courtesy of Prof. D.N. Christianson, Univ. of Pennsylvania,
USA) [30]. Sulfonamides EMATE-AZA together with compounds
1e23 have been docked to the active sites of the two isoforms, hCA I
and II. The results of the docking experiments are presented in
Tables 2 and 3. Pearson r values between converted binding ener-
gies of compounds and docking scores are found as 0.30 for hCA I,
and 0.44 for hCA II. Top docking poses derived for the best inhibi-
tors 13 (against hCA I) and 19 (against hCA II) are shown in some
details in Figs. 1 and 2. It may be observed that as for phenol [6] or
hydroxycinnamic acids/lacosamide [29e32] e for which the X-ray
crystal struyctures in adduct with hCA II have been reported e the
indoles investigated here do not coordinate to the metal ion but are
bound more towards the exit of the cavity, interacting eventually
with the zinc-coordinated water molecule. This is a binding mode
within the CA active site, which was less investigated to date,
except for polyamines [11] and a natural product phenolic COOMe
ester [12], which both anchor to the zinc-bound water/hydroxide,
similar to the simple phenol C6H5OH.
However, it is critically important to note that the indole
moiety strongly influences the activity since 1,2-
dimethoxybenzene 21 and catechol 22 were much less effec-
tive than the methoxy derivatives 2e6 and hydroxy deriva-
tives 13e20. Halogeno-containing derivatives 10e12 were also
ineffective, showing that electronegative atoms do not affect
the activity against hCA VI (Table 1);
(v) compounds 1e9 acted as weak inhibitors (KI-s in the range of
10.34e17.83
Compounds 10e16 were moderate inhibitors with KI-s in the
range of 8.51e9.62 M, with a comparable potency as the
reference compounds (i.e., EMATE, EZA or ZNA) (KI-s:
8.51e12.42 M). The remaining four derivatives 17e20 acted
as strong inhibitors with KI-s in the range of 2.13e5.17 M,
mM) against bovine enzyme bCA III (Table 1).
m
m
m
being more effective than the clinically used sulfonamides.
In general, when compred with 1,2-dimethoxybenzene 21 (KI:
4.24 mM) and catechol 22 (KI: 3.48 mM), the indole moiety did
not strongly affect the inhibitory potency of the compounds
containing it, but halogens and hydoxyl groups slightly
increased the activity of the compounds (Table 1).
3. Conclusions
Several 5,10-dihydroindeno[1,2-b]indole-based compounds
have been assayed for the inhibition of the physiologically relevant
human CA isozymes hCA I and II, hCA IV and hCA VI, as well as the
bovine enzyme bCA III. Towards hCA I these compounds showed
inhibition constants in the range of 2.141e16.32
in the range of 0.343e2.52 M, hCA IV was inhibited with KI-s in the
range of 0.435e5.726 M, whereas hCAVI was inhibited with KI-s in
the range of 1.92e12.84 M and bCA III with KI-s in the range of
2.13e17.83 M. In general, the compounds had comparable inhib-
mM, against hCA II
m
m
m
m
itory activity with the clinical used sulfonamides EMATE, EZA, ZNA
and AZA. Molecular docking studies of derivatives 1e20 within CA I
and II were also carried out and supported the kinetic assays. These
compounds probably bind between the phenol-binding site and
the coumarin-binding sites within the CA cavity, a region less
explored at the moment for designing inhibitors/activators of these
enzymes.
Fig. 1. Top docking pose of compound 13 within the active site of hCA I. For details of
the interactions see Table 3.

D. Ekinci et al. / European Journal of Medicinal Chemistry 49 (2012) 68e73
71
Table 1
Inhibition constants (KI values, mM) of hCA I, hCA II, bCA III, hCA IV and hCAVI with compounds 1e23 and standard CAIs, obtained by regression analysis graphs and an esterase
assay method with 4-NPA as substrate [35].
Compounda
R1
R2
R3
R4
hCA I
hCA II
bCA III
hCA IV
hCA VI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
OMe
H
H
H
OMe
H
OMe
H
OMe
OMe
OeCH2eO
F
Cl
Br
H
OH
H
H
OH
H
OH
OH
H
H
H
OMe
H
H
OMe
H
H
H
H
H
H
OMe
H
OMe
H
H
H
H
H
H
H
OH
H
OH
H
OH
14.73
12.13
12.63
13.44
12.32
14.21
13.25
13.27
16.32
13.83
14.28
13.26
2.141
3.26
2.36
2.03
2.12
2.29
1.94
2.52
2.27
2.31
2.44
0.496
0.524
0.472
0.648
0.734
0.785
0.813
0.447
0.561
0.343
0.536
0.50c
9.9b
17.83
16.31
14.86
15.63
10.34
11.21
15.84
14.32
12.71
8.63
8.72
8.51
8.84
9.08
9.42
9.62
2.13
4.34
4.736
4.215
4.442
4.627
4.082
5.726
4.573
4.852
5.341
0.617
0.778
0.532
0.875
0.912
0.943
1.032
0.543
0.435
0.842
0.765
14.32
10.9b
2.33
12.84
12.53
11.66
12.01
9.87
10.82
12.43
10.23
9.64
7.61
7.96
7.84
8.31
8.54
8.98
9.21
1.92
4.17
4.95
4.31
H
OMe
OMe
H
H
H
H
H
H
OH
H
H
H
OH
OH
H
H
H
H
H
H
OH
H
H
H
3.29
3.48
2.47
2.51
17
18
19
20
21
22
OH
H
2.59
5.17
4.63
4.24
3.48
H
2.46
10.4c
4003b
27.4
234.7
606b
3.63
1.86
1.58
23 (Indole)
EMATE
Ethoxzolamide (EZA)
Zonisamide (ZNS)
Acetazolamide (AZA)
32.5
0.23
0.32
1.07
17.6
12.27
3.75
14.8
12.42
9.443
8.51
1.04
0.84
38.45
0.578
2.42
0.34
36.2
0.37
83.4
a
Errors in the range of 5 % of the reported values.
From ref. [15].
From ref. [16].
b
c
4. Experimental
4.3. Purification of carbonic anhydrase isoenzymes from human
and bovine by affinity chromatography
4.1. Chemicals
Purification of hCA I and hCA II were previously described [15].
Fresh citrated human whole blood obtained from the Blood Center
of the Research Hospital at Atatürk University. Cells were washed
Sepharose-4B, protein assay reagents, 4-nitrophenylacetate and
chemicals for electrophoresis were purchased from SigmaeAldrich
Co. All other chemicals were of analytical grade and obtained from
Merck.
Table 2
Molecular docking binding scores of the compounds 1e23, EMATE, EZA, and ZNS
within hCA I and II active sites.
4.2. Synthesis
Compounds
GOLD fitness
score
GOLD ChemScore
hCA I (kcal/mol)
Detailed synthetic procedures for the preparation of the
compounds 1e20 can be found in Talaz et al. [24].
hCA I
hCA II
hCA II (kcal/mol)
1
63.10
69.06
64.38
66.39
68.12
71.05
71.46
67.79
65.15
63.34
64.75
63.91
65.39
68.17
63.77
64.39
66.60
66.90
64.76
66.56
59.32
51.57
58.15
67.31
75.96
67.84
51.28
54.35
53.33
51.54
55.47
59.00
57.09
56.44
51.07
50.92
51.53
51.95
51.78
51.35
49.13
53.56
54.12
54.38
51.08
54.28
41.79
39.87
43.59
58.69
62.67
56.11
ꢀ9.76
ꢀ9.75
ꢀ8.54
ꢀ9.71
ꢀ8.46
ꢀ8.87
ꢀ8.14
ꢀ8.30
ꢀ9.22
ꢀ8.55
ꢀ8.93
ꢀ9.20
ꢀ8.75
ꢀ9.69
ꢀ9.27
ꢀ8.13
ꢀ8.28
ꢀ8.36
ꢀ7.95
ꢀ9.28
ꢀ7.48
ꢀ7.24
ꢀ8.36
ꢀ9.24
ꢀ8.07
ꢀ7.10
ꢀ9.19
ꢀ10.20
ꢀ10.15
ꢀ9.98
2
3
4
5
6
7
8
9
ꢀ10.42
ꢀ9.31
ꢀ8.97
ꢀ9.69
ꢀ10.48
ꢀ11.35
ꢀ10.45
ꢀ10.99
ꢀ9.48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
EMATE
EZA
ZNS
ꢀ9.36
ꢀ11.22
ꢀ11.45
ꢀ10.01
ꢀ8.72
ꢀ11.31
ꢀ10.50
ꢀ9.72
ꢀ9.04
ꢀ9.92
ꢀ10.44
ꢀ11.04
ꢀ10.89
Fig. 2. Top docking pose of compound 19 within the active site of hCA II. For details of
the interactions see Table 3.

72
D. Ekinci et al. / European Journal of Medicinal Chemistry 49 (2012) 68e73
Table 3
4.5. Protein determination
Binding interactions of compounds 13 and 19 within the hCA I and hCA II active sites,
respectively.
Protein during the purification steps was determined spectro-
photometrically at 595 nm according to the Bradford method, using
bovine serum albumin as the standard [37].
Compounds
H-bonds
Close contacts
13
19
H2O 273
Thr199, H2O423
Leu198, Phe91, Gln92, His119, H2O17
Ile91, Trp209, His119, Gln92, H2O438
4.6. SDS polyacrylamide gel electrophoresis
three times by centrifugation at 1000 ꢁ g at 4 ꢂ 6 ꢃC, for 20 min in
four volumes of 25 mM Na2HPO4 (pH ¼ 7,4) buffer. Supernatant and
fluffy coat were removed. The erythrocytes were lysed in 10
volumes of 5 mM Na2HPO4 (pH ¼ 7,4) buffer, containing 1 mM
EDTA. After 20 min the haemolysate was centrifuged at 10000 ꢁ g
for 60 min. The particulate fraction was washed four times in the
same buffer. The membranes were centrifuged down at 15000 ꢁ g
for 60 min pH was adjusted to 8.3 with solid Tris. Sepharose-
4Beanilineesulfanilamide affinity column equilibrated with
25 mM TriseHCl/0.1 M Na2SO4 (pH 8.3). The affinity gel was washed
with 25 mM TriseHCl/25 mM Na2PO4 (pH 8.3). Finally, human
carbonic anhydrase IV (hCA IV) isozyme was eluted with 25 mM
TriseHCl/0.5 M NaClO4 (pH 7.4). Fresh non-citrated human whole
blood obtained from the Blood Center of the Research Hospital at
Atatürk University. The blood samples were centrifuged at
5000 rpm for 15 min and precipitant were removed. The serum was
isolated. The pH was adjusted to 8.7 with solid Tris. Sepharose-
4Beanilineesulfanilamide affinity column equilibrated with
25 mM TriseHCl/0.1 M Na2SO4 (pH 8.7). The affinity gel was washed
with 25 mM TriseHCl/22 mM Na2SO4 (pH 8.7). The human carbonic
anhydrase (hCA VI) isozyme was eluted with 0.25 M H2NSO3H/
25 mM Na2HPO4 (pH ¼ 6,7) [33]. All procedures were performed at
4 ꢃC. Bovine CA III was obtained from flank steak and purified using
a modification of the method of Tu et al. (1986) [34]. 100 g of cubed
flank steak was homogenized in small batches in a blender with
a total of 150 mL of buffer A (10 mM Tris/H2S04 buffer, pH 8.0,
containing 1 mM mercaptoethanol). The resulting product was
centrifuged at 4000 ꢁ g for 30 min, and the supernatant pooled.
This crude enzyme solution was filtered through Whatman No. 1
filter paper. The clear protein solution was passed through a 2 ꢁ 50-
cm gel exclusion column using the buffer A. The active fractions, as
determined and concentrated to lyophilizer. This solution was
applied to 2.5 ꢁ 50-cm ion exchange column and eluted with the
buffer A. The active fractions comprising the major protein peak
were pooled and concentrated in 1 mM mercaptoethanol.
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzymes. It was carried out in 10% and 3%
acrylamide for the running and the stacking gel, respectively,
containing 0.1% SDS according to Laemmli method [38].
4.7. In silico docking studies
Crystal structures of CAs in complex with activators (e.g., hCA I e
L-His, PDB code 2FW4) [29] or inhibitors (the hCA II e phenol
adduct PDB code unavailable as the coordinates were not
submitted) were used for the molecular docking calculations of
compounds 13e20 within the active sites of the hCA I and II.
Explicit water molecules from the X-ray structures were kept for all
the calculations. Before the docking simulations, the target struc-
ture were submitted to the protein preparation module of the
Schrodinger molecular modeling package [25,27]. Compounds
13e20 were constructed using the Schrodinger’s Maestro module
and then geometry optimization was performed for these ligands
using Polak-Ribiere conjugate gradient (PRCG) minimization
(0.0001 kJÅꢀ1 molꢀ1, convergence criteria). Protonation states of
ligands and residues were tested using LigPrep and Protein Prep-
aration modules under Schrodinger package [25e27,39] at neutral
pH (experimentally the compounds have been tested at pH of 7.4).
Genetic Optimisation for Ligand Docking (GOLD) program is used
[39]. In docking the maximum number of generic algorithm runs
was set to 20 for each compound. The default generic algorithm
parameters were selected (100 population size, 5 number of
islands, 100,000 number of generic operations and 2 for the niche
size). Default cutoff values of 2.5 Å (dH-X) for hydrogen bonds and
4.0 Å for van der Waals distance were employed. The two scoring
functions used, were the GoldScore fitness function and the
ChemScore [39]. The GoldScore Fitness function is a molecular
mechanics-like function with four terms:
GoldScore Fitness ¼ Shb ext þ Svdw ext þ Shb int þ Svdw int
4.4. CA inhibition
where Shb ext is the proteineligand hydrogen bond score and
Svdw ext is the proteineligand van der Waals score. Shb int is the
contribution to the Fitness due to intramolecular hydrogen bonds
in the ligand; Svdw int is the contribution due to intramolecular
strain in the ligand.
Carbonic anhydrase activity was assayed by following the change
in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-
nitrophenylate ion over a period of 3 min at 25 ꢃC using a spectro-
photometer according to the method described by Verpoorte et al.
[35] The enzymatic reaction, in a total volume of 3.0 mL, contained
On the other hand, the ChemScore function estimates the free
energy of binding of the ligand to a protein as follows:
1.4 mL 0.05
M TriseSO4 buffer (pH 7.4), 1 mL 3 mM 4-
nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A
reference measurement was obtained by preparing the same
cuvette without enzyme solution. The inhibitory effects of
compounds 1e23, EMATE, etoxzolamide, zonisamide and AZA were
examined. All compounds were tested in triplicate at each concen-
tration used. Different inhibitor concentrations were used. Different
inhibitor concentrations were used. Control cuvette activity in the
absence of inhibitor was taken as 100%. For each inhibitor an Activity
%-[Inhibitor] graph was drawn. To determine KI values, three
different inhibitor concentrations were tested; in these experi-
ments, 4-nitrophenylacetate was used as substrate at five different
concentrations (0.15e0.75 mM). The LineweavereBurk curves were
drawn [36].
D
Gbinding ¼
D
þ
G0
þ
D
GhbondShbond þ
DGmetalSmetal
GrotHrot
D
GlipoSlipo þ
D
where Shbond, Smetal, and Slipo are scores for hydrogen-
bonding, acceptor-metal, and lipophilic interactions, respectively.
Hrot is a score representing the loss of conformational entropy of
the ligand upon binding to the protein.
The final ChemScore value is obtained by adding in a clash
penalty and internal torsion terms, which militate against close
contacts in docking and poor internal conformations. Covalent and
constraint scores may also be included:
ChemScore ¼ Gbinding þ Eclash þ Eint
D

D. Ekinci et al. / European Journal of Medicinal Chemistry 49 (2012) 68e73
73
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Acknowledgments
This study was financed by Turkish Republic Prime Ministry
State Planning Organization (DPT), (Project no: 2010K120440) and
Agri Ibrahim Cecen University Scientific Research Council, (Project
no: Agri BAP-2010/K-10) for (MS) and by an FP7 EU grant to CTS
(Metoxia).
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