Carbonic anhydrase inhibitors: Design, synthesis, kinetic, docking and molecular dynamics analysis of novel glycine and phenylalanine sulfonamide derivatives

Article abstract of DOI:10.1016/j.bmc.2015.10.009

The inhibition of two human cytosolic carbonic anhydrase isozymes I and II, with some novel glycine and phenylalanine sulfonamide derivatives were investigated. Newly synthesized compounds G1-4 and P1-4 showed effective inhibition profiles with K_I values in the range of 14.66-315 μM for hCA I and of 18.31-143.8 μM against hCA II, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico docking studies were applied. Atomistic molecular dynamic simulations were performed for docking poses which utilize to illustrate the inhibition mechanism of used inhibitors into active site of CAII. These sulfonamide containing compounds generally were competitive inhibitors with 4-nitrophenylacetate as substrate. Some investigated compounds here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide, sulfanilamide or mafenide and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

Full text of DOI:10.1016/j.bmc.2015.10.009

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors: Design, synthesis, kinetic, docking
and molecular dynamics analysis of novel glycine and phenylalanine
sulfonamide derivatives
a
d
b
c,
d
⇑
_
Ismail Fidan , Ramin Ekhteiari Salmas , Mehmet Arslan , Murat Sßentürk , Serdar Durdagi ,
Deniz Ekinci e, Esra Sßentürk f, Sedat Cosßgun g, Claudiu T. Supuran h,
⇑
a Gebze Technical University, Chemistry Department, 41400 Gebze, Kocaeli, Turkey
b Yalova University, Engineering Faculty, Department of Polymer Engineering, 77100 Yalova, Turkey
c
_
Ag˘rı Ibrahim Çeçen University, Science and Art Faculty, Chemistry Department, 04100 Ag˘rı, Turkey
d Bahcesehir University, Medicinal Faculty, Department of Biophysics, 34349 Istanbul, Turkey
e Ondokuz Mayıs University, Faculty of Agriculture, Department of Agricultural Biotechnology, 55139 Samsun, Turkey
f
_
Ag˘rı Ibrahim Çeçen University, School of Health Services, 04100 Ag˘rı, Turkey
g Fatih University, Science and Art Faculty, Chemistry Department, 34500 Istanbul, Turkey
h University of Florence, Neurofarba Department, Via Ugo Schiff 6, Polo Scientifico, 50019 Sesto Fiorentino (Firenze), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of two human cytosolic carbonic anhydrase isozymes I and II, with some novel glycine and
Received 14 July 2015
Revised 5 October 2015
Accepted 6 October 2015
Available online xxxx
phenylalanine sulfonamide derivatives were investigated. Newly synthesized compounds G1–4 and P1–4
showed effective inhibition profiles with KI values in the range of 14.66–315
lM for hCA I and of 18.31–
143.8 M against hCA II, respectively. In order to investigate the binding mechanisms of these inhibitors,
l
in silico docking studies were applied. Atomistic molecular dynamic simulations were performed for
docking poses which utilize to illustrate the inhibition mechanism of used inhibitors into active site of
CAII. These sulfonamide containing compounds generally were competitive inhibitors with 4-nitro-
phenylacetate as substrate. Some investigated compounds here showed effective hCA II inhibitory effects,
in the same range as the clinically used sulfonamide, sulfanilamide or mafenide and might be used as
leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet
assayed for their interactions with such agents.
Keywords:
Carbonic anhydrase
Glycine
Phenylalanine
Sulfonamide
Docking
Enzyme inhibitor
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Our groups recently investigated the interaction of some mam-
malian CA isozymes with several types of sulfonamide derivatives,
Carbonic anhydrase (EC 4.2.1.1, CA) is a family of metalloen-
zymes that catalyze the rapid conversion of CO2 to HCOÀ3 and H+,
and involved in the biochemical process.1 CA isoforms are found
in a variety of tissues where they participate in several important
biological processes such as acid-base balance, respiration,
carbon dioxide and ion transport, bone resorption, ureagenesis,
gluconeogenesis, lipogenesis and electrolyte secretion.1–4 Many
CA isozymes involved in these processes are important therapeutic
targets with the potential to be inhibited/activated for the treat-
ment of a range of disorders such as edema, glaucoma, obesity,
cancer, epilepsy and osteoporosis.1,4
such as sulfanilamide and a series of chromone containing sulfon-
amides, benzenesulfonamides, for example, and some of their
derivatives.5 Here we extend these earlier investigations to series
of sulfonamides, some of which are widely used as prodrug or as
drugs. Sulfonamides possess many types of biological activities,
and representatives of this class of pharmacological agents are
widely used in clinic as antibacterial, hypoglycemic, diuretic,
anti-hypertensive and antiviral drugs among others.1,4–6 Recently,
a host of structurally novel sulfonamide derivatives have been
reported to show substantial antitumor activity in vitro and/or
in vivo.6–8
In the present study we have purified CA I and II (hCA I and hCA
II) from human erythrocytes and examined the in vitro inhibition
effects of above mentioned aminoacid derivatives on these
enzymes, using the esterase activity of hCA I and II, with 4-nitro-
phenyl acetate as substrate. Molecular modeling studies also
⇑
Corresponding authors. Tel.: +90 472 2156574; fax: +90 472 2156554 (M.Sß.);
tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: senturkm@gmail.com (M. Sßentürk), claudiu.supuran@unifi.it
(C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2015.10.009
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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I. Fidan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
applied for better understanding of molecular mechanisms of used
compounds.
2.2. CA purification, assay and inhibition with some amino acid
derivatives
The purification of the two CA isozymes used here was per-
formed with a simple one step method by a Sepharose-4B-ani-
line-sulfanilamide affinity column chromatoghrapy.12 Inhibitory
effects of G1–4 and P1–4 compounds on enzyme activities were
tested under in vitro conditions; KI values were calculated from
by using the Cheng–Prusoff equation and are given in Table 1.13
We report here the first study on the inhibitory effects of amino
acid derivatives G1–4 and P1–4 on the esterase activity of hCA I
and II:
2. Results and discussion
2.1. Chemistry
The rationale of investigating sulfonamide derivatives as CA
inhibitors (CAIs) is due to the fact that the simple benzenesulfon-
amide (PhSO2NH2) has been shown to be competitive inhibitor
with both CO2 and 4-nitrophenyl acetate as substrate for CA
isoforms.1,5a Sulfonamide type inhibitors bind to CAs, with coordi-
nation to the Zn(II) ion from the enzyme active site by substituting
the fourth, non-protein ligand, a water molecule or hydroxide ion,
such as for example acetazolamide (AZA), a clinically used com-
pound since 1954.8,9
(i) Against the slow cytosolic isozyme hCA I, G3 and P2 behave
as good inhibitors, with KI values in the range of 29.62 and
63.7 lM. Thus, the natural compound derivatives of the
groups in hydrophobic benzoic moiety strongly influences
hCA I inhibitory activity. It is also interesting to note that
the P3 was much better hCA I inhibitors as compared to
the corresponding G3 and P2 from which they were
prepared. Kinetic investigations indicate that similarly to
sulfonamides and inorganic anions,4,5,8 all the investigated
compounds act as competitive inhibitors with 4-NPA as sub-
strate, that is, they bind in different regions of the active site
cavity as compared to the substrate. However the binding
site of 4-NPA itself is unknown, but it is presumed to be in
the same region as that of CO2, the physiological substrate
of this enzyme.14
The X-ray crystal structure has been extensively used for
understanding the inhibition mechanism of CAIs. For example,
for the adduct of hCA II with sulfamide,8a,9 it has been observed
that the compound binds to CA by anchoring its NH moiety to
the zinc ion of the enzyme active site, through a hydrogen bond,
as well as through a second hydrogen bond to the NH amide of
Thr199, an amino acid conserved in all
a-CAs and critically
important for the catalytic cycle of these enzymes.6–9 Only
recently, our groups investigated the interactions of some
methanesulfonates, chromone containing sulfonamides, benzene-
sulfonamides, salicylic acid derivatives, some pesticides, some nat-
ural product polyphenols and phenolic acids with all mammalian
isozymes, CA I–XV,8–11 evidencing some low micromolar/submi-
cromolar inhibitors as well as the possibility to design isozyme
selective CAIs. Indeed, the inhibition profile of various isozymes
with this class of agents is very variable, with inhibition constants
ranging from the millimolar to the submicromolar range for many
simple sulfonamides (Fig. 1).5a,10
(ii) A better inhibitory activity has been observed with G3 and 4
investigated here for the inhibition of the rapid cytosolic iso-
zyme hCA II (Table 1). Structure–activity relationship (SAR)
is thus quite sharp for this small series of these compounds:
the –COOH G1 are ineffective leads, with carboxylic acid
moieties is already a submicromolar hCA II inhibitor. The
Figure 1. Chemical structures of synthesized and used compounds for this study.
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I. Fidan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Table 1
The sulfonamide zinc-binding group is thus superior to the thiol
one (from the thioxolone hydrolysis product) for generating
CA inhibitors with a varied and sometimes isozyme-selective
inhibition profile against the mammalian enzymes. However, it is
critically important to explore further classes of potent CAIs in
order to detect compounds with a different inhibition profile as
compared to the sulfonamides and their bioisosteres and to find
novel applications for the inhibitors of these widespread enzymes.
Although there are several studies regarding the interactions of
sulfonamide derivatives with CA isoenzymes, it is critically impor-
tant to explore further classes of potent CAIs in order to detect
compounds with a different inhibition profile to find novel applica-
tions for these CAIs (Figs. 2 and 3).
hCA I and II inhibition data with studied compounds, by an esterase assay with 4-
nitrophenylacetate as substrate14
Compound
KI*
(lM)
hCA I
hCA II
Benzenesulfonamide
p-Toluenesulfonamide
Sulfanilamide
Mafenide
G1
G2
G3
G4
P1
39.96a
118.47a
42.87a
41.91a
120.1
105.9
29.62
NE
1.103a
8.138a
0.628a
0.612a
143.8
56.40
18.31
19.32
103.9
97.4
315
63.7
P2
P3
P4
14.66
100.4
56.0
NE
2.2.1. Molecular dynamic (MD) simulations
In this section we tried to study the influence of mafenide and
G4 into the active site of CAII on protein structure. For this purpose,
three independent MD simulations were set up including of a
control system (ligand-free structure) and two complexes.
Root-mean-square deviation (RMSD) of backbone atoms of CAII
in complexes with mafenide and G4 were calculated based on
MD trajectory frames. The evolution of RMSD of each system
during 10 ns MD simulations was profiled in Figure 4. Studying
through this profile, we have observed the structural stabilities of
backbone atoms along simulations. Mafenide-bound CAII is so
mobile, but inversely, apo and G4-bound systems exist in a stable
*
Mean from at least three determinations. Errors in the range of 3–8% of the
reported value (data not shown).
a
Ref. 5a.
best hCA II inhibitor in this series of derivatives were G3 and
G4, which with a KI of 18.31–19.33 lM. It must be stressed
that KIs measured with the esterase method are always in
the micromolar range because hCA I and II are weak
esterases.8,14
Figure 2. G1 compound was docked into carbonic anhydrase I (CAI).
Figure 3. G1 compound was docked into carbonic anhydrase II (CAII).
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I. Fidan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 4. RMSD evolution of backbone atoms of mafenide, G4-bound CAII and apo system during 10 ns MD simulations.
Figure 5. RMSF values for individual backbone atoms of residues along MD simulations.
dynamic. CAII atoms were increasingly influenced upon binding of
mafenide, which may be connected to increasing of entropy terms.
Increasing of the entropy term in this system is describing the
more favorable binding of mafenide. In addition, root-mean-square
fluctuations of individual backbone atoms of residues were calcu-
lated along the simulation for each system. The results were pro-
filed in Figure 5, the values revealed the stability of each amino
acid. Studying through these calculations, we have found that
structural stability of mafenide-bound system is low in most of
the domains in CAII. And also, in some part of the apo form, atoms
have increasingly fluctuated.
to the CO2 hydrase assay.14 These findings point out that substi-
tuted compounds may be used as leads for generating potent CAIs
eventually targeting other isoforms which have not been assayed
yet for their interactions with such agents.
4. Experimental
4.1. Chemicals
Benzenesulfonamide, sulfanilamide, p-toluenesulfonamide,
mafenide, Sepharose 4B, protein assay reagents, 4-nitrophenylac-
etate were obtained from Sigma–Aldrich Co. Glycine,
nine, p-toluenesulfonyl chloride were purchased from Sigma
Aldrich and used as received. Glycine and -phenylalanine methyl
ester hydrochlorides, glycine and -phenylalanine benzyl ester
hydrochlorides were obtained from Sigma–Aldrich. Glycine and
-phenylalanine piperidinylamides were synthesized from
L-phenylala-
3. Conclusions
L
G1–4 and P1–4 used in this study affect the activity of CA
isozymes due to the presence of the different functional groups
(for instance –COOH, –COOCH3, and –COOPh) present in their
aromatic scaffold. Our findings here indicate thus another class
of possible CAIs of interest, in addition to the well-known
sulfonamides/sulfamates/sulfamides, the bearing bulky moieties
in their molecules. Indeed, some amino acid derivatives com-
pounds investigated here showed effective hCA I and II inhibitory
activity, in the low micromolar range, by the esterase method
which usually gives KI-s an order of magnitude higher as compared
L
L
BOC-protected derivatives according to the reported procedure.15
All other chemicals and solvents were analytical grade and
obtained from Merck. Column chromatography was performed
using silica gel 60 (43–60 nm, Merck). Thin layer chromatography
was performed using silica gel plates (kieselgel 60 F254, 0.2 mm,
Merck). Characterization of synthesized compounds was per-
formed using 1H NMR, 13C NMR spectroscopy (Varian 400 MHz).
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I. Fidan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Elemental analysis data was obtained using a Leco CHNS-932
apparatus (Michigan, USA).
C, 60.17; H, 5.37; N, 4.39; S, 10.04 Found: C, 59.61; H, 5.79; N, 4.73;
S, 9.34.
Compound P3: Yield 82% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.62 (d, J = 8.3 Hz, 2H, ArH), 7.34–6.98 (m, 12H, ArH),
5.05 (d, J = 9.3 Hz, 1H, –NH–), 4.87 (s, 2H, –OCH2), 4.25 (dt,
J1 = 9.3 Hz, J2 = 6.0 Hz, 1H, –CH–), 3.04–3.02 (dd, 3J1 = 6.0 Hz,
4J2 = 1.7 Hz, 2H, –CH2–), 2.39 (s, 3H, –CH3) ppm. 13C NMR
(100 MHz, CDCl3): d 170.8, 143.7, 136.8, 134.9, 134.8, 129.8,
129.6, 128.7, 128.6, 127.4, 127.3, 67.5, 56.7, 39.6, 21.7 ppm. Anal.
Calcd [C23H23NO4S]: C, 67.46; H, 5.66; N, 3.42; S, 7.83. Found: C,
66.74; H, 5.19; N, 3.62; S, 7.44.
4.2. Syntheses of N-tosyl a-amino acid derivatives G(1–4) and P
(1–4)
Syntheses of G1 and P1: To a stirred solution of glycine (0.75 g,
10.0 mmol) or -phenylalanine (1.65 g, 10.0 mmol) in 20 mL of 1 M
L
NaOH solution at 25 °C, p-toluenesulfonyl chloride (1.91 g,
10.0 mmol) was added. After overnight stirring at room tempera-
ture, the solid residue was filtered off and the aqueous reaction
portion was acidified with 1 M HCl. The obtained solid was filtered
and purified by column chromatography on silica with hexane and
EtOAc (1:2) to obtain compounds G1 and P1 as white solids.
(Yields, 81% and 76%, respectively.)
Compound G4: Yield 78% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.75 (d, J = 8.3 Hz, 2H, ArH), 7.30 (d, J = 8.3 Hz, 2H, ArH),
5.73 (broad singlet, 1H, –NH–), 3.71 (s, 2H, –CH2–), 3.47–3.45 (m,
2H, aliphatic ring hydrogens), 3.20–3.17 (m, 2H, aliphatic ring
hydrogens), 2.41 (s, 3H, –CH3), 1.64–1.44 (m, 6H, aliphatic ring
hydrogens) ppm. 13C NMR (100 MHz, CDCl3): d 164.8, 143.7,
136.2, 129.8, 127.4, 45.5, 43.6, 26.2, 25.4, 24.3, 21.7 ppm. Anal.
Calcd [C14H20N2O3S]: C, 56.73; H, 6.80; N, 9.45; S, 10.82. Found:
C, 56.21; H, 6.29; N, 9.69; S, 10.36.
Compound G1: Yield 81% as white solid. 1H NMR (400 MHz,
DMSO-d6): d 12.66 (broad singlet, 1H, –COOH), 7.93 (t, J = 6.1 Hz,
1H, –NH–), 7.67 (d, J = 8.4 Hz, 2H, ArH), 7.37 (d, J = 8.3 Hz, 2H,
ArH), 3.54 (d, J = 6.1 Hz, 2H, –CH2–), 2.37 (s, 3H, –CH3) ppm. 13C
NMR (100 MHz, CDCl3): d 171.1, 143.8, 136.3, 129.9, 127.3, 40.3,
21.6 ppm. Anal. Calcd [C9H11NO4S]: C, 47.15; H, 4.84; N, 6.11; S,
13.99. Found: C, 48.36; H, 5.11; N, 5.93; S, 14.27.
Compound P4: Yield 80% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.65 (d, J = 8.3 Hz, 2H, ArH), 7.26–7.11 (m, 7H, ArH),
5.77 (d, J = 9.4 Hz, 1H, –NH–), 4.33 (ddd, J1 = 9.4 Hz, J2 = 8.5 Hz,
J3 = 6.1 Hz, 1H, –CH–), 3.20–2.58 (m, 6H, aliphatic hydrogens),
2.38 (s, 3H, –CH3), 1.42–0.76 (m, 6H, aliphatic hydrogens) ppm.
13C NMR (100 MHz, CDCl3): d 168.5, 143.5, 137.1, 135.8, 129.8,
129.6, 128.6, 127.5, 127.3, 53.4, 46.3, 43.1, 41.2, 25.5, 25.0, 24.1,
21.6 ppm. Anal. Calcd [C21H26N2O3S]: C, 65.26; H, 6.78; N, 7.25;
S, 8.30. Found: C, 65.91; H, 6.23; N, 7.68; S, 7.87.
Compound P1: Yield 76% as white solid. 1H NMR (400 MHz,
DMSO-d6): d 12.70 (broad singlet, 1H, –COOH), 8.17 (d, J = 8.8 Hz,
1H, –NH–), 7.45 (d, J = 8.2 Hz, 2H, ArH), 7.24–7.10 (m, 7H, ArH),
3.84 (ddd, J1 = 8.8 Hz, J2 = 8.8 Hz, J3 = 5.8 Hz, 1H, –CH–), 2.92 (dd,
2J1 = 13.7, 3J2 = 5.7 Hz, 1H, –CHaHb–), 2.70 (dd, 2J1 = 13.7,
3J2 = 8.8 Hz, 1H, –CHaHb–), 2.34 (s, 3H, –CH3) ppm. 13C NMR
(100 MHz, CDCl3): d 174.7, 143.8, 136.6, 135.0, 129.8, 129.7,
128.7, 127.4, 127.2, 56.5, 39.0, 21.7 ppm. Anal. Calcd [C16H17NO4S]:
C, 60.17; H, 5.37; N, 4.39; S, 10.04. Found: C, 59.36; H, 5.11; N, 4.83;
S, 9.27.
4.3. Purification of carbonic anhydrase isozymes from human
erythrocytes by affinity chromatography
Syntheses of G(2–4) and P(2–4): General experimental proce-
dure is as follows: Glycine or
L
-phenyl alanine derivative (as HCl
Erythrocytes were purified from fresh human blood obtained
from the Blood Centre of the Research Hospital at Atatürk Univer-
sity. The blood samples were centrifuged at 1500 rpm for 15 min
and the plasma and buffy coat were removed. The red cells were
isolated and washed twice with 0.9% NaCl, and hemolyzed with
1.5 volumes of ice-cold water. The ghost and intact cells were
removed by centrifugation at 20,000 rpm for 30 min at 4 °C. The
pH of the hemolysate was adjusted to 8.7 with solid Tris.9a
Firstly, benzoyl chloride was stirred for four hours at room
temperature in CH2Cl2 cellulose. After the spacer arm cellulose
added as a benzyl group and finally diazotized sulfanilamide
clamped to the para position of benzyl group as ligand. The
hemolysate was applied to the prepared Cellulose-benzyl-
sulfanilamide affinity column equilibrated with 25 mM Tris–
HCl/0.1 M Na2SO4 (pH 8.7). The affinity gel was washed with
25 mM Tris–HCl/22 mM Na2SO4 (pH 8.7). The human carbonic
anhydrase (hCA I and hCA II) isozymes were eluted with 1 M
NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M
NaClO4 (pH 5.6), respectively. All procedures were performed at
4 °C.11
salt) (10 mmol) and Na2CO3 (2.1 g, 20 mmol) were dissolved in
anhydrous dichloromethane (50 mL) and cooled in an ice bath
before tosyl chloride (1.9 g, 10 mmol) was added. The resultant
reaction mixture was stirred overnight at room temperature. After
the reaction, more solvent was added and the residue was washed
sequentially with 0.1 M HCl, saturated NaHCO3 and brine. Organic
portion was dried with Na2SO4 and concentrated in vacuo to give
crude product. The product was purified by column chromatogra-
phy on silica with hexane and EtOAc (1:1).
Compound G2: Yield 78% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.74 (d, J = 8.3 Hz, 2H, ArH), 7.31 (d, J = 8.3 Hz, 2H, ArH),
5.04 (t, J = 5.1 Hz, 1H, –NH–), 3.78 (d, J = 5.1 Hz, 2H, –CH2–), 3.64
(s, 3H, –OCH3), 2.42 (s, 3H, –CH3) ppm. 13C NMR (100 MHz, CDCl3):
d 169.4, 144.0, 136.3, 129.9, 127.4, 52.7, 44.2, 21.7 ppm. Anal.
Calcd. [C10H13NO4S]: C, 49.37; H, 5.39; N, 5.76; S, 13.18. Found:
C, 50.63; H, 5.09; N, 5.36; S, 12.87.
Compound P2: Yield 81% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.63 (d, J = 8.3 Hz, 2H, ArH), 7.25–7.20 (m, 5H, ArH),
7.08–7.05 (m, 2H, ArH), 5.03 (d, J = 9.1 Hz, 1H, –NH–), 4.21 (dt,
J1 = 9.1 Hz, J2 = 6.0 Hz, 1H, –CH–), 3.49 (s, 3H, –OCH3), 3.03 (d,
J = 6.0 Hz, 2H, -CH2-), 2.40 (s, 3H, –CH3) ppm. 13C NMR (100 MHz,
CDCl3): d 171.4, 143.8, 136.8, 135.1, 129.7, 129.5, 128.7, 127.4,
127.3, 56.7, 52.5, 39.5, 21,6 ppm. Anal. Calcd [C17H19NO4S]: C,
61.24; H, 5.74; N, 4.20; S, 9.62. Found: C, 60.67; H, 5.39; N, 4.46;
S, 9.17.
4.4. Esterase activity assay
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 °C using a spec-
trophotometer (CHEBIOS UV–VIS) according to the method
described by Verpoorte et al.13a The enzymatic reaction, in a total
volume of 3.0 mL, contained 1.4 mL 0.05 M Tris–SO4 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
Compound G3: Yield 86% as white solid. 1H NMR (400 MHz,
CDCl3): d 7.73 (d, J = 8.3 Hz, 2H, ArH), 7.37–7.23 (m, 7H, ArH),
5.05 (s, 2H, –OCH2–), 5.02 (broad singlet, 1H, –NH–), 3.82 (d,
J = 5.5 Hz, 2H, –NCH2–), 2.42 (s, 3H, –CH3) ppm. 13C NMR
(100 MHz, CDCl3): d 168.8, 144.0, 136.3, 134.8, 129.9, 129.8,
128.8, 128.4, 127.4, 67.7, 44.3, 21.7 ppm. Anal. Calcd [C16H17NO4S]:
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I. Fidan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
inhibitory effects of resveratrol, catechin, silymarin, dobutamine
and curcumin were examined. All compounds were tested in
triplicate at each concentration used. Different inhibitor
concentrations were used. hCA-I enzyme activities were
measured for G1–4 and P1–4 at cuvette concentrations. Control
cuvette activity in the absence of inhibitor was taken as 100%.
For each inhibitor an Activity (%)-[Inhibitor] graphs were drawn.
KI values were calculated from by using the Cheng–Prusoff
equation.13
method of smooth Particle Mesh Ewald were used for Coulomb
interactions.
Acknowledgments
This study was financed by TUBITAK (The Scientific and
Technological Research Council of Turkey) (Project no: 114Z731)
for Murat Senturk; and by Ondokuz Mayis University Scientific
Research Projects Council (Project no: 2013/PYO.ZRT.1901.13.004)
for Deniz Ekinci.
4.5. Protein determination
References and notes
Protein during the purification steps was determined spec-
trophotometrically at 595 nm according to the Bradford method,
using bovine serum albumin as the standard.16
1. (a) Ekinci, D.; Cavdar, H.; Durdagi, S.; Talaz, O.; Senturk, M.; Supuran, C. T. Eur. J.
Med. Chem. 2012, 49, 68; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
2. Hilvo, M.; Baranauskiene, L.; Salzano, A. M.; Scaloni, A.; Matulis, D.; Innocenti,
A.; Scozzafava, A.; Monti, S. M.; Di Fiore, A.; De Simone, G.; Lindfors, M.; Jänis,
J.; Valjakka, J.; Pastoreková, S.; Pastorek, J.; Kulomaa, M. S.; Nordlund, H. R.;
Supuran, C. T.; Parkkila, S. J. Biol. Chem. 2008, 283, 27799.
3. (a) Ekinci, D.; Cavdar, H.; Talaz, O.; Senturk, M.; Supuran, C. T. Bioorg. Med.
Chem. 2010, 18, 3559; (b) Alp, C.; Ekinci, D.; Gultekin, M. S.; Senturk, M.; Sahin,
E.; Kufrevioglu, O. I. Bioorg. Med. Chem. 2010, 18, 4468.
4. (a) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 1583; (b) Akin Kazancioglu, E.; Guney, M.; Senturk, M.; Supuran, C. T.
J. Enzyme Inhib. Med. Chem. 2012, 27, 880; (c) Ekinci, D.; Senturk, M.;
Kufrevioglu, O. I. Expert Opin. Ther. Pat. 1831, 2011, 21.
4.6. SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzymes. It was carried out in 10% and 3% acry-
lamide for the running and the stacking gel, respectively, contain-
ing 0.1% SDS according to Laemmli procedure. A 20 lg sample was
applied to the electrophoresis medium. Gels were stained for 1.5 h
in 0.1% Coommassie Brilliant Blue R-250 in 50% methanol and 10%
acetic acid, then destained with several changes of the same sol-
vent without the dye.17
5. (a) Ekinci, D.; al-Rashida, M.; Abbas, G.; Senturk, M.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2012, 27, 744; (b) Alp, C.; Ozsoy, S.; AlcanAlp, N.; Erdem, D.;
Gultekin, M. S.; Kufrevioglu, O. I.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2012, 27, 818; (c) Ekinci, D.; Kurbanoglu, N. I.; Salamci, E.; Senturk,
M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 845.
6. (a) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (b) Supuran, C. T.
Carbonic Anhydrases: Catalytic Mechanism, Distribution and Physiological
Roles. In Carbonic Anhydrase-Its Inhibitors and Activators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC: Boca Raton (FL), USA, 2004; pp 1–24.
7. Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2004, 19, 199.
8. (a) Alp, C.; Ozsoy, S.; Alcan Alp, N.; Erdem, D.; Gultekin, M. S.; Kufrevioglu, O. I.;
Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 818; (b)
Durdagi, S.; Senturk, M.; Ekinci, D.; Balaydin, H. T.; Goksu, S.; Kufrevioglu, O. I.;
Innocenti, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 1381;
(c) Balaydin, H. T.; Durdagi, S.; Ekinci, D.; Senturk, M.; Goksu, S.; Menzek, A. J.
Enzyme Inhib. Med. Chem. 2012, 27, 467.
9. (a) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.,
et al. J. Am. Chem. Soc. 2006, 128, 8329; (b) Weber, A.; Casini, A.; Heine, A.;
Kuhn, D.; Supuran, C. T.; Scozzafava, A., et al. J. Med. Chem. 2004, 47, 550.
10. Demirdag, R.; Comakli, V.; Senturk, M.; Ekinci, D.; Kufrevioglu, O. I.; Supuran, C.
T. Bioorg. Med. Chem. 2013, 21, 1522.
4.7. Molecular docking simulations
Glide docking protocols18 were applied for the prediction of
positions of used compounds into the active sites of CA I and CA
II enzymes. Active site of the enzyme was defined from the co-crys-
tallized ligands from PDB files. Crystal structures of hCA I, II and IX
are retrieved from Protein Data Bank server, (PDB codes: 2FW4,
5AML and 3IAI, respectively19–21). Throughout the docking simula-
tions both partial flexibility and full flexibility around the active
site residues are performed by Glide XP.
4.8. Molecular dynamic simulations
11. Ceyhun, S. B.; Senturk, M.; Erdogan, O.; Kufrevioglu, O. I. Pestic. Biochem.
Physiol. 2010, 97, 177.
12. (a) Cakmak, R.; Durdagi, S.; Ekinci, D.; Senturk, M.; Topal, G. Bioorg. Med. Chem.
Lett. 2011, 21, 5398; (b) Gulcin, I.; Beydemir, S.; Coban, A.; Ekinci, D. Fresen.
Environ. Bull. 2008, 17, 1283.
13. (a) Verpoorte, J. A.; Mehta, S.; Edsall, J. T. J. Biol. Chem. 1967, 242, 4221; (b)
Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
14. Nair, S. K.; Ludwig, P. A.; Christianson, D. W. J. Am. Chem. Soc. 1994, 116, 3659.
15. Venuti, M. C.; Alvarez, R.; Bruno, J. J.; Strosberg, A. M.; Gu, L.; Chiang, H.-S.;
Massey, I. J.; Chu, N.; Fried, J. H. J. Med. Chem. 1988, 31, 2145.
In order to investigate and track the behavior of used inhibitors
into the active site of the CAII, atomistic molecular dynamic (MD)
simulation was utilized. For this aim three systems, including con-
trol (apo form), G4-bound and mafenide-bound structures were
constructed based on top-docking poses. CAII was selected as a tar-
get for all systems and three classical MD simulations were inde-
pendently performed using Desmond code.22 All complexes were
solvated by TIP3P water model and then naturalized by adding
Na+ and ClÀ ions. The thickness of water layer was set to 10 Å.
Before the MD simulations the systems were minimized with a
maximum iteration of 2000 steps. Then, the systems were
submitted in 1 ns and 10 ns MD simulations for equilibration and
production MD runs for each systems. Temperature and pressure
were assigned on 300 K and 1.01325 bar, respectively using
Isothermal–isobaric (NPT) ensemble. Nose–hoover chain23 and
Martyna–Tobias–Klein24 were implemented thermostat and baro-
stat methods, respectively. Cut-off radios of 9 Å and long-range
16. Bradford, M. Anal. Biochem. 1976, 72, 248.
17. Laemmli, D. K. Nature 1970, 227, 680.
18. Friesner, R. A.; Banks, J. L.; Murphy, R. B., et al. J. Med. Chem. 2004, 47, 1739.
19. Temperini, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2006, 16,
5152.
20. Ivanova, J.; Leitans, J.; Tanc, M.; Kazaks, A.; Zalubovskis, R.; Supuran, C. T.; Tars,
K. Chem. Commun. (Camb) 2015, 7108.
21. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De
Simone, G. Proc. Natl. Acad. Sci. 2009, 106, 16233.
22. Desmond Molecular Dynamics System, Version 2.2; D.E. Shaw Research: New
York, NY, 2009.
23. Hünenberger, P. H. Adv. Polym. Sci. 2005, 173, 105.
24. Martyna, G. J.; Tobias, D. J.; Klein, M. L. J. Chem. Phys. 1994, 101, 4177.
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Please cite this article in press as: Fidan, I;. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.009