Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II

Article abstract of DOI:10.1016/j.ijbiomac.2018.04.131

Selective carbonic anhydrase (CA) inhibitors have gained a lot of importance owing to the implication of specific isoforms of CA in certain diseases like glaucoma, leukemia, cystic fibrosis, and epilepsy. A novel class of sulfonylurea derivatives was synthesized from corresponding sulfonyl chlorides and amines. Compounds with different pendant moieties in the sulfonylurea derivatives show significant interactions with human carbonic anhydrase II (CAII). In vitro evaluation of the sulfonylurea derivatives revealed that three compounds possess admirable inhibitory activity against CAII. Compounds containing methyl (G2), isopropyl (G4) and o-tosyl (G5) groups displayed IC₅₀ (109-137 μm) for CAII. Fluorescence binding and cytotoxicity studies revealed that these compounds are showing good binding affinity (18-34 μM) to CAII and non- toxic to human cells. Further, molecular docking studies of G2, G4 and G5 with CAII showed that these compounds fit nicely in the active site of CAII. Molecular dynamics simulation studies of these compounds complexed with CAII showed that essential interactions were maintained up to 50 ns of simulation. These results indicate the promising nature of the sulfonylurea scaffold towards CAII inhibition and opens scope of hit to-lead optimization for discovery of effective drugs against CAII-associated disorders.

Full text of DOI:10.1016/j.ijbiomac.2018.04.131

Accepted Manuscript
Implication of sulfonylurea derivatives as prospective inhibitors
of human carbonic anhydrase II
Danish Idrees, Murtuza Hadianawala, Amarjyoti Das Mahapatra,
Bhaskar Datta, Sonam Roy, Shahzaib Ahamad, Parvez Khan, Md.
Imtiyaz Hassan
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DOI:
Reference:
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doi:10.1016/j.ijbiomac.2018.04.131
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24 April 2018
Please cite this article as: Danish Idrees, Murtuza Hadianawala, Amarjyoti Das Mahapatra,
Bhaskar Datta, Sonam Roy, Shahzaib Ahamad, Parvez Khan, Md. Imtiyaz Hassan ,
Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic
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ACCEPTED MANUSCRIPT
Running head: Sulfonylurea derivatives as CAII Inhibitors
Implication of sulfonylurea derivatives as prospective inhibitors of
human carbonic anhydrase II
Danish Idrees1, Murtuza Hadianawala2, Amarjyoti Das Mahapatra2, Bhaskar Datta2,*, Sonam
Roy1, Shahzaib Ahamad3, Parvez Khan1 and Md. Imtiyaz Hassan1

1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar,
New Delhi 110025, India.
2Department of Chemistry, Indian Institute of Technology, Palaj, Gandhinagar, Gujarat -
382355, India.
3Department of Biotechnology, College of Engineering & Technology, IFTM University,
Lodhipur-Rajput, Delhi Road, Moradabad, India.
* All correspondence should be addressed to,
Md. Imtiyaz Hassan, Ph.D.
E-mail: mihassan@jmi.ac.in
Bhaskar Datta, Ph.D.
E-mail: bdatta@iitgn.ac.in
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ABSTRACT
Selective carbonic anhydrase (CA) inhibitors have gained a lot of importance owing to the
implication of specific isoforms of CA in certain diseases like glaucoma, leukemia, cystic
fibrosis, and epilepsy. A novel class of sulfonylurea derivatives was synthesized from
corresponding sulfonyl chlorides and amines. Compounds with different pendant moieties in the
sulfonylurea derivatives show significant interactions with human carbonic anhydrase II (CAII).
In vitro evaluation of the sulfonylurea derivatives revealed three compounds possessing
admirable inhibitory activity against CAII. Compounds containing methyl (G2), isopropyl (G4)
and o-tosyl (G5) groups displayed IC50 (109-137 μm) for CAII. Fluorescence binding and
cytotoxicity studies revealed that these compounds are showing good binding affinity (18-34
µM) to CAII and non- toxic to cells. Further, Molecular docking studies of G2, G4 and G5 with
CAII showed these compounds fit nicely in the active site of CAII. Molecular dynamics
simulation studies of these compounds complexed with CAII also showed essential interactions
which were maintained up to 50 ns of simulation. These results indicate the promising nature of
the sulfonylurea scaffold towards CAII inhibition and opens scope of hit to-lead optimization for
discovery of effective drugs against CAII-associated disorders.
Keywords: Carbonic anhydrase, Sulfonyl urea derivatives, Enzyme inhibition, Structure based
drug design
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1. Introduction
Carbonic anhydrases (CA, EC.4.2.1.1) catalyze the reversible hydration of carbon dioxide (CO2)
and enable regulation of pH of living system [1-3]. These are metalloenzymes predominantly
possess zinc(II) in the active site cavity which is essential for their enzymatic activities [4, 5].
There are 16 isoforms of CA which are distributed to almost all organelle of human body and are
essential for varieties of cellular processes [2, 6]. Apart from their main cellular function,
inhibition of these isozymes is used to address a wide range of disease conditions including
glaucoma to cancer [7-11].
Sulphanilamide is one of the earliest reported and widely applicable CA inhibitors [12].
Elucidation of the sulphonamide group as a Zn(II) binding entity spurred the development of CA
inhibitors (CAIs) with a modular architecture. CAIs have been designed and developed with a
zinc binding group (ZBG), an aromatic scaffold and a capping group or tail segment [12, 13].
The aromatic scaffold and tail ensure better positioning of the ZBG [14]. CAIs possessing such
modular architectures have been implicated in the direct coordination to Zn(II) in the active site
or to Zn(II)-bound solvent molecules as part of their mechanism of action [8, 15]. While such a
design paradigm has resulted in large numbers of CAIs, poor isozyme selectivity has prevented
their wider clinical application [12, 16]. Several new classes of compounds such as coumarins
and phenols have been identified for their ability to bind at the entrance of the CA active site [17,
18] to manifest in the inhibition of CA [18, 19].
Attempts have been made to combine the strengths of the two approaches to CA inhibition,
namely, interaction with Zn(II) and active site entrance blockage [20]. However, such strategies
met with a little success [21]. For example, sulfonamide-containing CAIs have been designed
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with sugar-containing tail segments for targeting specific isozymes [22, 23]. Unfortunately, such
anomeric sulphonamides do not exhibit differences in Kis between the cytosolic CAII and the
membrane-associated isozyme CAIX [24]. In this context, several bioisosters of sulphonamides
such as sulfamates and sulfamides have also been explored as CAIs [25, 26]. While many of
these have been identified as tight-binding inhibitors, isozyme-specificity as an innate trait has
remained elusive.
Interestingly, the sulfonylurea group is a bioisostere of sulphonamide which has received scant
attention for use in CAIs. We have recently reported sulfonylurea-derivatives as inhibitors of
CAII [27]. CAII is a ubiquitous isozyme of CA and creates challenges for selective targeting of
tumor-specific isozymes (CAIX and CAXII). We already reported the structural and biophysical
properties of different CAs [28-31]. Here, we report sulfonylurea-derivatives for their ability for
effective CAII inhibition in nm range. Three molecules (G2, G4 and G5) have been identified
with micromolar IC50s. Binding mode analysis by molecular docking reveals an interesting
association of these sulfonylurea-derivatives in the active site of the enzyme. Notably, hydrogen
bonding and hydrophobic interactions are observed with several key active site residues such as
His64, Asn67, Thr199 and Thr200. The present study highlights sulfonylurea as a novel scaffold
for effective CAII inhibition and expands the scope for discovery of effective drugs against
epilepsy and glaucoma.
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2. Materials and methods
2.1. Synthesis of sulfonylurea
Benzenesulfonyl chloride (0.5 g, 2.8 mmol) was treated with pyridine (0.4 mL, 5.0 mmol) and
allowed to stir for 5 min. The resultant solution was transferred to a mixture of sodium cyanate
(0.25 g, 3.9 mmol) in acetonitrile (5 mL) and allowed to stir for about 4 hour at room
temperature. To the resultant mixture aniline (0.364 mL, 4 mmol) was added and the stirred for
about 1 hr at room temperature. The resulting reaction mixture was poured on crushed ice and
acidified with dil HCl (pH 5 - 6). Aqueous layer was extracted with ethyl acetate three times and
combined extract was washed with brine and dried over anhydrous Na2SO4. Solvent was
evaporated under vacuum and resulting residue was further purified by column chromatography
to afford a white solid compound G7 (125 mg, 20%).
1H NMR (CDCl3, 500 MHz): δ 8.47 (brs, 1H, NH), 7.95 (d, 2H, J = 7.5 Hz), 7.65 (t, 1H, J = 7.5
Hz), 7.54 (t, 2H, J = 7.5 Hz), 7.4 (d, 2H, J = 8 Hz), 7.32 (t, 2H, J = 7.5 Hz), 7.14 (t, 1H, J = 7.5
Hz); 13C NMR (CDCl3, 125 MHz): 148.57, 139.51, 136.55, 134.06, 129.58, 129.18, 126.89,
124.94, 120.40; MS (ESI): 277.093 [M+H]+, 299.07 [M+Na]+
2.2. Synthesis of thiosulfonylurea-derivatives
To (970.6 mg, 4.6 mmol) p-Chlorobenzenesulfonyl chloride, 10 ml aqueous ammonium
hydroxide was added and refluxed for 1 h. Reaction mixture was then quenched with water and
acidified with dil HCl (pH 5 - 6). The resulting product was filtered, dissolved in DCM and dried
over anhydrous Na2SO4. Solvent was evaporated under vacuum and resulting residue was used
for the next step without further purification. To a solution of the sulfonamide (1.00 mmol) in
DMF, KOtBu (1.20 mmol) was added and stirred for 15 min. To this solution phenylisocyanate
(0.6 mL, 5.5 mmol) was added and heated at 80 °C for 1 h. Reaction mixture was quenched with
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water and acidified with dil HCl (pH 5-6). Product was isolated by filtration and was further
purified using flash chromatography to yield title compound G8 (1.1 g, 74%).
1H NMR (CDCl3, 500 MHz): δ 9.65 (brs, 1H, NH), 8.19 (brs, 1H, NH), 7.88 (d, 2H, J = 8.5
Hz), 7.57 (d, 2H, J = 8.5 Hz), 7.46 (d, 2H, J = 8.0 Hz), 7.39 (t, 2H, J = 8.0 Hz), 7.29 (t, 1H, J
= 8.0 Hz); 13C NMR(CDCl3, 125 MHz): 176.49, 141.31, 137.15, 136.88, 130.07, 129.18,
128.55, 127.41, 124.29; MS (ESI): 327.00 [M+H]+
2.3. Carbonic anhydrase inhibition assay
The inhibitory action of synthesized compounds was measured by esterase assay against CAII
using earlier described methods (28, 30). The esterase activity of CAII was measured by
following the change in absorbance at 400 nm of p-nitrophenyl acetate(4-NPA) to 4-
nitrophenolate ion over a period of 3 min at 25°C using a spectrophotometer Jasco UV/visible
spectrophotometer (Jasco V-660, Model B 028661152). Reaction mixture contained 50 mM
Tris buffer (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 3 mM substrate p-nitrophenol acetate.
1µM of protein concentration was used and different concentrations of synthesized compounds
were used in a total volume of 500µl. A reference measurement was obtained by preparing the
same reagents in cuvette without enzyme solution. The synthesized compounds were tested in
triplicate at each concentration used. Control enzyme activity in the absence of a drug was taken
as 100%. Acetazolamide (AZM) was used as a reference compound. The IC50 values for each
synthesized compounds were calculated by using GraphPad Prism (Version 5.0) graphing
software.
2.4. Fluorescence binding study
The binding study of compounds (G2, G4 and G5) with CAII protein was performed by
monitoring the changes in fluorescence intensity of protein. All fluorescence measurement was
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performed in the Jasco spectrofluorimeter (Model FP-6200) using 5 mm quartz cuvette
(excitation at 292 nm, emission at 300–400 nm). Protein concentration of 1 µM CAII and
compounds were used in a concentration 1–10 µM. Compounds were dissolved in DMSO and
then diluted to 1 mg/ml in the 50 mM Tris pH 8.0 and 150 mM NaCl. The protein solution was
titrated with increasing concentration of compounds. The decrease in fluorescence intensity is
due to the increasing concentration of compounds has been used to calculate the binding affinity
by Stern–Volmer equations with slight modification.
log (Fo –F)/F = logKa + nlog[Q]
(1)
Where Fo is the fluorescence intensity of protein and F is the fluorescence intensity of ligand, Ka
is the binding constant and n is the number of binding sites, Q represents quenching constant. For
the ligand–protein complex, the values for Ka and n can be derived from the intercept and slope.
2.5. Cell culture and cytotoxicity assay
HEK293 cell lines were routinely maintained and cultured in a 5% CO2 humidified incubator at
37°C, in DMEM supplemented with 10% heat inactivated fetal bovine serum (Gibco), 1%
penicillin, streptomycin solution (Gibco). Cells were routinely maintained and trypsinized not
more than 30 passages. To see the cytotoxicity of synthesized compounds on cell viability and
proliferation, standard MTT assay was used as described earlier [32, 33]. Briefly, cells were
seeded (8000-9000/well) in a 96-well plate and allowed to grow overnight. On the next day, cells
were treated with increasing concentrations (5–200 μM) of studied compounds in a final volume
of 200 µl for 24 and 48 h at 37º C in a humidified CO2 incubator. At the end of the treeatment, 20
µl of MTT solution and 100 µl of DMEM (from 5mg/ml stock solution in PBS) was added to
each well and incubated for 4-5 h at 37ºC in a humidified CO2 incubator. Finally the supernatant
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was aspirated and the colored formazan crystal produced from MTT was dissolved in 150µl of
DMSO. The absorbance was measured at 570 nm with the help of multiplate ELSIA reader
(BioRad). The percentage of cell viability was calculated and plotted as a function of
concentration of compound.
2.6. Docking studies
Molecular docking approaches have been used to know the binding mode of ligand and receptor
in drug discovery. We have used Autodock (version 4.2), an automated tool to perform docking
which was installed on Windows 7 OS (on HP Proliant DL180 G6 workstation) [34-36]. We
have used crystal structure of human carbonic anhydrase II (CAII) (PDB-ID: 1CA2). 2D
chemical structures of the ligand was drawn and converted into 3D structure by using Chemdraw
version 12 and utilized MMP2 and MM2 force-field to energy minimize the compounds. After
energy minimization compound G2, G4 and G5 required to convert into docking input file
format (i.e. .pdb to .pdbqt), ligands and protein files converted into .pdbqt. The ligands was
docked into the cavity site of CAII protein, when we employ the auto-grid box dimensions were
set to be 126 x 126 x 126 Å of XYZ axes and default grid point spacing of 0.375 Å was
established by using the Auto-Grid module. To predict the bounded conformation based on the
empirical force filed Lamerckian Genetic Algorithm (LGA) runs was performed [37]. After
completion of docking run all the compounds .dlg file were analyzed to know the binding pattern
with protein. We applied blind docking approach for the docking of 1CA2, further we also used
CASTp server to predict the binding site of protein. The autodock4 predicted the binding exactly
at the binding site as predicted by castp (CASTp server widely used server know the protein
binding pockets) [38]. PyMOL software was used to visualize the protein ligand binding site
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[39]. Finally, docking results provide good binding free energy that forms stable complexes
between CAII and compound G2, G4 and G5 by forming hydrogen bonding.
2.7. MD Simulations
The general methodology of MD simulations was carried out on CAII and complex with
compound G2, G4 and G5 using GROMSCS-Version 4.6.5 (on HP Proliant DL580 G7
workstation). The topology of CAII was produced on the basis of GROMOS96 53A6 force-field
[40]. Due to the lack of suitable force field parameters for drug molecules in the GROMACS
software package, PRODRG server [41] is a program for generation of molecular topologies and
coordinate files. After creating the topology, coordinate file of compound G2, G4 and G5 were
immersed into SPC/E (model) water box of cubic shape. In this mode, counter ions was added
for entire system neutralized. In order to neutralized system and energetically minimized via two
methods steepest descent and conjugate gradient algorithms (CGA) converged criterion within of
0.005 kcal mol-1. To increase the reliability of MD simulations apply restrain both the drug and
protein structure before the equilibration phase. Once equilibration phase was done separately in
constant temperature (300 K), constant volume ensemble (NVT) as well as constant temperature
(300K), constant pressure (NPT) conditions, appropriate time scale for each 100 ps. By keeping
the native binding coordinate condition of ligand and protein restrained. Nose Hoover thermostat
and Parrinello-Rahman barostat (1 bar) were maintained temperature and pressure ensemble,
respectively. Finally, the MD simulation production with 300 K temperature and each of the
independent trajectories used 50 ns (LINCS Algorithm). Each complex behavior, at time step of
2fs was used in water environment. The knowledge of analyzed the MD trajectories Root Mean
Square Deviation (RMSD), radius of gyration (Rg), root means square fluctuations (RMSF) and
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secondary structure content were developed by Do_dssp program. In this study, we performed 50
ns (50,000 ps) MD simulations of WT CAII, compound G2, G4 and G5. The totaling of 200 ns
of trajectories analysis was calculated for the protein backbone with respect to the initial
conformation.
3. Results and Discussion
3.1.Synthesis and characterization of sulfonylurea and thiosulfonylurea-derivatives
Sulfonylurea derivatives are widely used in anti-diabetic medicines. These have been
traditionally prepared by the treatment of sulphonamide with appropriate isocyanate in presence
of base [42]. We have recently reported a general method for synthesis of sulfonylurea
derivatives from sulfonyl chlorides and amines [43]. The same method was used for synthesis of
several compounds in this work (Scheme 1 and Table 1).
In each case the sulfonyl chloride was first treated with pyridine followed by addition of sodium
cyanate and reaction with the suitable amine. However, the single-pot reaction was ineffective
for the synthesis of thiosulfonylurea derivatives. These were synthesized by the conventional
approach of first converting the sulfonyl chloride into sulphonamide followed by reaction with
isothiocyanate. The yield of the single-pot method ranged from modest to comparable in contrast
to the conventional approach (Scheme 2 and Table 2). Nevertheless, the hazardous nature of the
conventional approach as well as the wider scope afforded by the direct use of sulfonyl chlorides
and amines maintains the attractive nature of the single-pot method. The final compounds were
obtained as solids and were purified and characterized using standard techniques. Stock solutions
of the sulfonylurea and thiosulfonylurea-derivatives were prepared in DMSO and used for
1
biological assays. The synthesized compounds were characterized by H NMR, 13C NMR and
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mass spectroscopy. The purity of the target compounds was evaluated by elemental analysis (see
supplementary data).
Molecule #
G1
R
-CH3
R’
G2
-CH3
G3
G6
-CH3
-Cl
G7
-H
Scheme 1. Synthesis of sulfonylureas G1-3, 6 and 7
Compound
G4
R
R’
-H
G5
G8
G9
-H
-Cl
-H
-CH3
-H
-H
Scheme 2. Synthesis of sulfonylureas G4, G5, G8 and G9
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3.2.CAII inhibition assay
The inhibition studies of synthesized compounds against CAII were carried out using the
esterase activity assay of CA, with p-nitrophenyl acetate (4-NPA) as a substrate which is
colorless. The inhibitory activity of compounds against CAII is shown in Table 3. Compounds
G2, G4 and G5 show good inhibition of CAII. All these compounds have phenyl rings at the two
ends of the sulfonylurea moiety with various alkyl substitutions. The importance of the phenyl
groups is most evident from a comparison of performance with compounds G1 and G3. A less
bulky isobutyl (G1) or an aromatic but bulkier benzothiazole (G3) are unable to provide suitable
bonding contacts with the enzyme site. Within the molecules with phenyl rings at the terminal
ends of sulfonylurea the combination of methyl and phenyl (G2), 4-Isopropyl and phenyl (G4)
and o-methyl and phenyl group (G5) led to the most profound inhibition of CAII with compound
G2 having IC50 = 137.41 μm, compound G4 having IC50 = 109.34 μm and compound G5 have
IC50 = 125.62 μm. The good inhibition by compounds G2, G4 and G5 is indicative of the
important interactions effected by the phenyl rings with the alkyl substitutions providing suitable
interaction with the enzyme active site. Interestingly, use of the thiosulfonylurea compounds G8
and G9 do not lead to any significant differences in CAII inhibition as compared to their
sulfonylurea counterparts G6 and G7. These results point to the structural scaffold provided by
phenyl substituted sulfonylurea as a promising new class of selective CAII inhibitors.
3.3.Fluorescence binding studies
The fluorescence emission spectra of CAII solutions in the absence and presence of different
concentrations of compounds in Tris at 25◦C are recorded. The maximum emission peak of CAII
was observed at 343 nm (excitation wavelength was 292 nm). Upon incubation of different
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concentrations (1–10 µM) of compounds with CAII the maximum fluorescence peak intensity
was reduced significantly indicating that CAII formed complexes with compounds. The change
in the intrinsic fluorescence intensity of CAII was observed when compounds bind to them. The
values fluorescence intensity at 343 nm was plotted as a function of [ligand] to calculate the
binding affinity of compounds against CAII (Figure 1). The fluorescence quenching results were
observed for CAII with compounds but the binding affinities of compounds (G4 and G5) are
comparatively high than compound (G2). The binding affinity of compounds for CAII is 34.20
µM, 18.57 µM, and 19.21 µM (Table 4).
3.4.Cytotoxicity studies
Three selected compounds (G2, G4 and G5) were evaluated on HEK-293 cell lines by MTT
assay. These compounds were screened in the concentration range of 0-200 µM, and treatments
were given for 24 and 48 h. The results shown that compound G2, G4 and G5 does not elicit any
towards HEK-293 cell lines (Figure 2). Cytotoxicity of these compounds for HEK-293 cells
were also studied at their respective IC50 (from enzyme inhibition assay) value for 72 h, we
observed that more than 90% embryonic kidney cells were viable even after 72 h of incubation
(data not shown). Results from cytotoxicity studies clearly indicate that the studied compounds
are non-toxic to HEK-293 cells.
3.5.Molecular docking studies
Molecular docking studies are widely used for predicting the binding study and inhibition
constant (Ki) of compounds. In this study, CAII was docked with Compounds G2, G4 and G5 to
elucidate their interaction patterns. Result suggested that the compounds G2, G4 and G5 are
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nicely bounded into the active site of CAII with lowest binding energy reported in Table 4. The
G2 in the active site of CAII is stabilized with six H-bond interaction with Asn62, His64, and
Asn67 (Figure 3A). The G4 in the active site of CAII is stabilized with three H-bond
interactions with Asn62, His64, Gln92 and Thr200 (Figure 3B). Furthermore, G5 is stabilized by
six H-bond interactions with His64, Asn67 Gln92 and Thr200 (Figure 3C).
Finally docking study concluded that the compounds G2, G4 and G5 have showed good binding
energy value of
and
(ΔG in kcal/mol), respectively (see Table 4). The
inhibitory constant, binding energy for CAII and these compounds may be considered as
candidate in development of selective CAII inhibitor.
3.6.MD Simulation
MD simulation analysis of CAII and ligand-bound with compounds G2, G4 and G5 are
indicated in black, red, green and blue, respectively. The average RMSD values computing
know the protein stability in-terms of backbone RMSD vs time (ps) of 50 ns each. The
observed RMSD of only CAII increases till ~5 ns fluctuate ~0.1 nm and then achieved the
equilibrium, 15 to 30 ns quite disrupt the RMSD and remains stable throughout the
simulation. Results suggest that the CAII remain stable during the MD simulation, the average
RMSD calculated 0.16 nm. WT and all other compound comparatively achieved equilibrium
approximately ~5 ns of the MD simulation. Maximum fluctuation occurs with G5, after
equilibration RMSD sharply increased at ~ 12 ns and stable till 30 ns. However, another
fluctuation increase at 30 ns then stabilized protein throughout the simulation and the average
RMSD is 0.30. From Figure 4 the average RMSD value of WT and all the complexes are
reported in Table 5. These results suggested that the G4 have least average RMSD value 0.21
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as compare to all other compounds. Similar results found in docking study that provide lowest
binding energy -7.56 stabilize the protein complex with G4.
The Rg is measurement of compactness of CAII and complexed with compounds G2, G4 and
G5 represented in Figure 5. The Rg value of WT comparatively to all the compound are less
than WT, that suggesting compounds provides significant compactness and rigidity to the
protein and thus increasing the complex stability. The Average Rg values are mention in
Table 5.
Structural mobility analysis plays a key role in MD simulation which is analyzed through
g_rmsf module of gromacs. The flexibility of protein structure from Figure 6 illustrate the
RMSF of residues was plotted in the presence of compounds G2, G4 and G5. We observed
that the G2 at residues 8-10 (loops region) increases fluctuation ~5 nm and residues 18-20,
125-130 (α-helices region) fluctuation bars reaches ~ 3.0 nm and 200-204 (loops region)
fluctuation bars ~3.0 nm. Similarly, compound G5 shown at residues 22-24 (α-helices region)
fluctuation bars 3.5, residues 100-105 (loops region), 171-173 (??-sheets region) fluctuation
bars ~2.5 nm and 235-240 (loops region) ~3.0 nm. Finally, compound G4 displays the least
fluctuations as compare to other compounds, the residues 53-58 (loops region) only ~2.0 nm.
The RMSF plot clearly indicates that the G4 stabilize CAII as compared to other compounds,
similar result found in RMSD.
Protein functionality is affected by tertiary as well as secondary structure. Investigation of
secondary structure provide an idea about the conformation changes of whole protein
structures in each time scale from the entire MD trajectory (Figure 7A-D). Secondary
structure changes in the presence of different compounds are described in Table 6. Major
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protein component of secondary structure like ??-sheets content and helices increases in the
presence of G4. Moreover, in the case of G2 and G5 helices decreases. In the case of
compound G4 a considerable increase was observed as compared to other compounds, and
suggesting maximum stability to protein.
Conclusions
A new series of compounds incorporating the sulfonylurea and sulfonylthiourea moieties have
been prepared and investigated for their interaction with human CAII. Several molecules in this
class were identified as having CAII inhibitory activity. In particular, the biological evaluation of
these compounds reveals good inhibitory activity of compounds G2, G4 and G5. These
compounds were also found to exhibit good binding affinity for CAII. The present molecules
represent a promising structural motif that can be further explored for enhancing isoform
selective inhibition of CA. In particular, the currently reported molecules may constitute
interesting candidates for the development of novel antiepileptic strategies.
Acknowledgement: DI thanks to the University Grants Commission for the award of fellowship.
This work is supported by Council of Scientific and Industrial Research, India (Grant No.
37(1665)/15/EMR-II). B.D. is grateful to Center for Biomedical Engineering, IIT Gandhinagar
for financial support of this work.
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Table 1: List of synthesized sulfonylureas G1 – G3, G6 and G7
Compound
G1
R
R’
Yield (%)
52
59
G2
41
25
G3
G6
Cl
H
20
G7
Table 2: List of synthesized sulfonylureas of G4, G5, G8 and G9
Compound
R/R’
Isopropyl / H
H / CH3
Cl / H
X
O
O
S
Yield (%)
81
52
74
77
G4
G5
G8
G9
H / H
S
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Table 3: In vitro carbonic anhydrase inhibitory activity
Compound
Structure of compound
Name of Structures
N-(isobutylcarbamoyl)-4-
methylbenzenesulfonamide
IC50 (µM)
339.60
G1
270.41
880.74
170.34
4-methyl-N-
(phenylcarbamoyl)benzenesulfo
namide
G2
G3
N-(benzo[d]thiazol-2-
ylcarbamoyl)-4-
methylbenzenesulfonamide
4-isopropyl-N-
(phenylcarbamoyl)benzenesulfo
namide
G4
G5
225.62
439.18
2-methyl-N-
(phenylcarbamoyl)benzenesulfo
namide
4-chloro-N-
(phenylcarbamoyl)benzenesulfo
namide
G6
G7
G8
G9
391.83
421.59
N-
(phenylcarbamoyl)benzenesulfo
namide
4-chloro-N-
(phenylcarbamothioyl)benzenes
ulfonamide
383.63
N-
(phenylcarbamothioyl)benzenes
ulfonamide
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Table 4: Binding parameters of each complex
Docked complex Binding free
Electrostatic
Energy
(kcal/mol)
Estimated
Inhibition
Constant
Ki (µM)
3.09
vdW + Hbond
+ desolv
Energy
*Binding
affinity
CAII-
energy (ΔG
Compounds
in kcal/mol)
(kcal/mol)
G2
G4
-7.32
-7.56
-0.19
-0.11
-8.52
34.20 µM
18.57 µM
4.05
-8.44
-7.51
G5
-6.69
-0.07
12.55
19.21 µM
*calculated from fluorescence binding study
Table 5: The average RMSD and Rg of WT-CAII and complex with CAII-compound G2, G4 and
G5 during the 50 ns MD simulation.
Compounds No.
Average RMSD
Average Rg
WT-CAII
G2
0.16±0.01
0.29±0.01
0.21±0.01
0.30±0.01
1.74±0.01
1.67±0.01
1.67±0.01
1.68±0.01
G4
G5
Table 6: The secondary structure plot representing structural content WT-CAII and complex
compound G2, G4 and G5 at 300 K of 50 ns MD simulation.
Secondary structure
compounds No.
structure
β-sheet
Coil
β-bridge
Bend
Turn
α-helix
WT-CAII
136.3
75.8
78.3
7.2
39.9
20.5
32.7
G2
G4
G5
135.7
136.6
140.3
79.3
76.0
82.6
70.9
72.7
71.6
7.3
7.2
6.9
43.8
45.4
40.8
25.6
26.4
23.4
23.3
29.8
27.3
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Figure Legends
Figure 1: Fluorescence emission spectra of (A) CAII with G4, (B) plot of log (Fo− F)/F vs log
[Q] for quenching of CAII by G4.
Figure 2: Cell viability assay. (A) Effect of CAII inhibitors on the viability of human normal
cell (HEK-293) lines: Cells were treated with increasing concentrations of each compound (0-
200 μM) for 48 h.
Figure 3: Surface representation of CAII docked with all three compounds G2, G4 and G5 in the
same active site pocket and hydrophobic core of active site is colored with element. (A) Cartoon
view of compound G2 docked with CAII, active site residue interactions with compound G2.
Residues are shown with stick and compound G2 is shown with ball and stick model, hydrogen
bonds are shown as broken lines (black). (B) Cartoon view of compound G4 docked with CAII.
Interactions of active site residue with compound G4. Residues are shown with stick and
compound G4 is shown with ball and stick model. (C) Cartoon view of compound G5 docked
with CAII. Interactions of site residue with compound G5. Hydrogen bonds are shown as broken
lines (black).
Figure 4: RMSD plot of CAII trajectories analysis produced by Gromacs; CAII (black),
compound G2 (red), compound G4 (green) and compound G5 (blue).
Figure 5: Rg plot of MD trajectories produced by Gromacs; CAII (black), compound G2 (red),
compound G4 (green) and compound G5 (blue).
Figure 6: RMSF plot, trajectories produced by Gromacs; CAII (black), compound G2 (red),
compound G4 (green) and compound G5 (blue).
Figure 7: DSSP program constitute secondary structure plot representing structural content (A)
CAII, (B) complex CAII-compound G2, (C) complex CAII-compound G4, (D) complex CAII-
compound G5 at 300 K of 50 ns MD simulation.
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