Biological evaluation of p-toluene sulphonylhydrazone as carbonic anhydrase IX inhibitors: An approach to fight hypoxia-induced tumors

Article abstract of DOI:10.1016/j.ijbiomac.2017.08.082

To find potential inhibitors of human carbonic anhydrase IX (CAIX), we have successfully deigned, synthesized and characterized three p-toluene sulphonylhydrazone derivatives (1–3). Molecular docking studies provided the structural basis of CAIX inhibition and a deeper insight into the protein-ligand interactions. p-Toluene sulphonylhydrazone derivatives show a well organized conformational compatibility with the active site of CAIX. The protein-ligand complex was stabilized by several non-covalent interactions offered by residues present in the active site cavity. The actual binding affinity of synthesized compounds with CAIX was experimentally measured by fluorescence and isothermal titration calorimetry (ITC). Results of both fluorescence binding and ITC measurements show the binding affinity of p-Toluene sulphonylhydrazone derivatives to the CAIX in the μM range. CAIX enzyme inhibition assay showed the IC₅₀ values in nM range. Though all the three compounds (1–3) showed a good binding with CAIX, compound 2 showed the best inhibition of CAIX activity. These compounds were non-toxic on normal cell lines (HEK-293) and significantly inhibit the proliferation of hypoxic cancer cells. All compounds induce apoptosis in the hypoxic cancer cells. These compounds may be further exploited as promising therapeutic agents to control the hypoxia-induced tumors.

Full text of DOI:10.1016/j.ijbiomac.2017.08.082

Accepted Manuscript
Title: Biological evaluation of p-toluene sulphonylhydrazone
as carbonic anhydrase IX inhibitors: an approach to fight
hypoxia-induced tumors
Authors: Aarfa Queen, Parvez Khan, Danish Idrees, Amir
Azam, Md. Imtaiyaz Hassan
PII:
S0141-8130(17)32590-4
DOI:
Reference:
http://dx.doi.org/10.1016/j.ijbiomac.2017.08.082
BIOMAC 8074
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
17-7-2017
10-8-2017
13-8-2017
Please cite this article as: Aarfa Queen, Parvez Khan, Danish Idrees,
Amir Azam, Md.Imtaiyaz Hassan, Biological evaluation of p-toluene
sulphonylhydrazone as carbonic anhydrase IX inhibitors: an approach
to fight hypoxia-induced tumors, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.082
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Biological evaluation of p-toluene sulphonylhydrazone as
carbonic anhydrase IX inhibitors: an approach to fight hypoxia-
induced tumors
Aarfa Queen1, Parvez Khan2, Danish Idrees2, Amir Azam1,* and Md. Imtaiyaz Hassan2,*
1Department of Chemistry, Jamia Millia Islamia, New Delhi-110025, India
2Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-
110025, India
Running head: CAIX Inhibitors
*To whom correspondence should be addressed
Dr. Md. Imtaiyaz Hassan
Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, New Delhi-110025, India
Tel.: +91-9990323217
E-mail: mihassan@jmi.ac.in
Prof. Amir Azam
Department of Chemistry
Jamia Millia Islamia, New Delhi-110025, India
Tel.: +91-11-26981717/3254; fax: 91-11-26980229;
E-mail: amir_sumbul@yahoo.co.in
Highlights
 Three p-Toluene sulphonylhydrazone derivatives are synthesized and tested for CAIX inhibition
assay.
 Compound 1 and 2 bind CAIX with high affinity.
 Both compound 1 and 2 inhibits CAIX enzyme activity with IC50 values in nM range.
 These compounds induce apoptosis in hypoxic HeLa and HT-29 cells.
1

ABSTRACT
To find potential inhibitors of human carbonic anhydrase IX (CAIX), we have successfully
deigned, synthesized and characterized three p-Toluene sulphonylhydrazone derivatives (1-
3). Molecular docking studies provided the structural basis of CAIX inhibition and a deeper
insight into the protein-ligand interactions. p-Toluene sulphonylhydrazone derivatives show a
well organized conformational compatibility with the active site of CAIX. This protein-ligand
complex was stabilized by several non-covalent interactions offered by residues present in the
active site cavity. The actual binding affinity of synthesized compounds with CAIX was
experimentally measured by fluorescence and isothermal titration calorimetry (ITC). Results
of both fluorescence binding and ITC measurements show the binding affinity of p-Toluene
sulphonylhydrazone derivatives to the CAIX in the µM range. CAIX enzyme inhibition assay
showed the IC50 values in nM range. Though all the three compounds (1-3) showed a good
binding with CAIX, compound 2 showed the best inhibition of CAIX activity. These
compounds were non-toxic on normal cell lines (HEK-293) and significantly inhibit the
proliferation of hypoxic cancer cells. All compounds induce apoptosis in the hypoxic cancer
cells. These compounds may be further exploited as promising therapeutic agents to control
the hypoxia-induced tumors.
Keywords: CAIX Inhibitors; Tumor Hypoxia; Enzyme inhibition
1. Introduction
Cancer is one of the leading causes of human death. A large number of drug therapies
have been employed to fight cancer but still an effective medication is required [1, 2]. In last
few decades, some potential drug targets of human cancer have been identified which
2

includes carbonic anhydrase (CA) [3-7], an enzyme involved in the pH regulation. CA is a
zinc containing metallo-protein that efficiently catalyze the reversible hydration of carbon
dioxide [6]. Among the 14 isoforms of CA, CAIX is a transmembrane isoform having
extracellular-facing catalytic site [6, 8]. CAIX is known to possess highest H+ transfer rate
among all the isoforms [9].
CAIX is up-regulated in several cancers and plays significant role in tumor acidification
(tumor hypoxia), progression and metastasis [9, 10]. Hypoxia conditions upregulates the
expression of CAIX and therefore, its expression measure of hypoxia. High expression of
CAIX generally correlates with poor prognosis. Hence, it is an important prognostic indicator
in oncology [11, 12]. CAIX is present in around tumor which regulates pH by ion transport
across the plasma membrane and thus guard tumor cells from acidosis, thereby balancing the
intracellular accumulation of acidic metabolic products [9, 13]. The use of CAIX expression
is an attractive and promising candidate marker for systemic anticancer therapy [10, 14, 15].
The detection and characterization of hypoxic areas in the solid tumor is an important
topic of discussion in cancer research since hypoxia itself is an independent predictor of death
in various types of cancer [14]. Hypoxic cancerous cells do not respond to chemotherapies
and radiation therapies [2, 14, 16]. Thus, an approach for molecular imaging based on
synthesis of selective ligands or inhibitors of CAIX which is overexpressed at sites of
hypoxia is required. Such an approach could help to design and target selective potent
inhibitors of CAIX for clinical uses to fight against cancer and act as anti-hypoxia therapy
[15].
Sulfonamides, sulfamates, sulfamides, phenols and coumarins are some of the carbonic
anhydrase inhibitors [17-20]. Sulphonamide causes the inhibition of catalytic activity of CA,
by regulating acid-base balance and transport of ion in many tissues. CAIX sulphonamide
3

inhibitors gets accumulated in hypoxic cells only and thereby causing the reversal of
acidification by CAIX [21]. Acetazolamide is a potent CA inhibitor that inhibits in the
nanomolar range, ethoxolamide and topiramate are some other known CA inhibitors (Fig. 1)
[22]. Benzene sulfonamides are classical CA inhibitors bearing various substituents, which
bind to zinc ion in a deprotonated form (RSO2NH-) by exchanging a water molecule [23].
Recently, some other inhibitors such as thiourea derivatives [15, 24] and sulfonyl
semicarbazide derivatives have been reported [25, 26] (Fig. 1).
Despite a number of novel potent CA inhibitors have been synthesized, the selectivity of CA
isozyme is still a mystery [27]. Hydrazone derivatives are extensively studied pharmacophore
having wide range of biological activities [28, 29]. Some hydrazone derivatives of 2,6-
dimethylimidazo (2,1-b) (1,3,4)thiadiazole-5-carbohydrazide [30], hydrazinopyrimidine-5-
carbonitrile derivatives [31], coumarin substituted hydrazide–hydrazone derivatives [32] are
known to possess anticancer activities [33] .
In view of the above properties of the sulphonamide and hydrazone skeletons, we
have selected and synthesized three compounds in which the two above mentioned
pharmacophore (sulphonamide and hydrazone) were covalently linked to generate a single
hybrid molecule (Fig. 2). Selected compounds were synthesized and evaluated against the
human CAIX isozyme in anticipation that the hybrid molecules would show potent activity of
the two scaffolds. The in vitro results of the hybrids were further validated by molecular
docking, enzyme inhibition and binding studies. These compounds were further investigated
for their anticancer properties and used to analyze their apoptotic potential against human
cancer cell lines in normoxic as well as in hypoxic conditions.
2. Material and Methods
4

2.1. Materials used
N-lauryl sarcosine, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
and other reagents were purchased from Sigma Aldrich (St. Louis, MO). Ni-NTA column and
gel filtration column (Superdex-200) were purchased from GE healthcare (GE Healthcare
Life Sciences, Uppsala, Sweden). HeLa (human cervical cancer cells), human colon
carcinoma cells (HT-29), and human embryonic kidney cells (HEK-293) cells were procured
from National Centre for Cell Sciences (NCCS), Pune, India. FITC-Annexin-V detection kit
was purchased from BD-Pharmingen, BD Biosciences (USA). Dulbecco minimal essential
medium (DMEM), RPMI-1640 and Ham’s F-12 nutrients mix cell culture medium and fetal
bovine serum (FBS) was purchased from Gibco life sciences. All reagents used were of
molecular biology grade. All the reagents, chemicals and solvents were purchased from
Sigma–Aldrich (St. Louis, MO, USA), S.D Fine Chemicals (India) and Merck (Darmstadt,
Germany). The purity of the compounds was tested by thin layer chromatography (TLC).
2.2. Synthesis of inhibitors
The selected compounds were synthesized by earlier reported method [34, 35]. The
synthetic pathway leading to target compounds (1–3) is depicted in Scheme 1 (see
supplementary data). Briefly, p-Toluene sulfonyl chloride was stirred in excess of hydrazine
hydrate until a white precipitate of p-toluene sulfonyl hydrazide was formed. p-Toluene
sulfonyl hydrazide reacted with different derivatives of aldehydes in ethanol to get
hydrazones which was crystallised to give final products. All the melting points of the
synthesized compounds were determined in pyrex capillaries using a basic melting point
apparatus and were not corrected. Pre-coated aluminum sheets (silica gel 60 F254, Merck
Germany) were used for thin-layer chromatography (TLC) and spots were visualized under
UV light. Elemental analysis was carried out on CHNS Elementar (Vario EL-III) and the
5

results were within  0.3% of the theoretical values. IR spectra were recorded on Bruker FT-
1
13
IR spectrophotometer on a Zn Se crystal as KBr discs. H NMR and C NMR spectra were
recorded on Bruker Spectrosp in DPX 400 MHz using DMSO-d6 as a solvent and
tetramethylsilane (TMS) as an internal standard. Splitting patterns are designated as follows;
s, singlet; d, doublet; m, multiplet. Chemical shift values are given in ppm. The FAB mass
spectra of the compounds were recorded on JEOL SX 102/DA-6000 mass spectrometer using
Argon/Xenon 6KV, 10 mA) as the FAB gas and m-nitro benzyl alcohol (NBA) was used as
the matrix.
2.2.1.General procedure for the synthesis of p-Toluenesulfonyl hydrazide
A slight excess of hydrazine hydrate (2 mL) was added to a stirring solution of p-toluene
sulfonyl chloride (2 gm, 10.49 mmol) at 25°C. The reaction mixture was stirred at room
temperature for overnight. Solid precipitates were filtered and washed with water, to remove
hydrazine hydrochloride. White hydrazide obtained was recrystallized from ethanol to give
pure product.
2.2.2.General procedure for the synthesis of p-Toluenesulfonyl hydrazones (1-3)
To a stirring solution of p-Toluene sulfonyl hydrazide (0.25 g, mmol) in ethanol one
equivalent of different substituted aldehyde was added. The reaction mixture was stirred at
room temperature for 12h to get precipitate of hydrazone which was filtered dried and
recrystallized from ethanol.
2.3. Molecular docking
The atomic co-ordinate of CAIX was taken from protein data bank (www.rcsb.org,
PDBID: 3IAI), and optimized using steepest descent method from Gromacs 4.5.5. The 2D
6

and 3D structures of all the synthesized compounds were drawn with the help of Chembio
Draw ultra. Further calculations and file preparations are done according to our published
protocol [15, 36]. After preparing the coordinate files of CAIX and respective compound, it
was subjected to docking using AutoDock 4 package [37]. The interaction between CAIX
and each compound were analyzed using the Lamarckian genetic algorithm (LGA). The
binding energy was calculated using van der Waals, electrostatic interactions and hydrogen
bonding. Finally docked complexes of CAIX were further optimized, validated and explored
using “Receptor–Ligand Interactions” modules of Discover Studio 4.0 [38].
2.4. Expression and purification of proteins
CAIX and CAII were successfully expressed in E. coli (BL21 strain) and
subsequently purified [15, 39, 40]. In brief, for CAIX the pellet obtained from the culture was
dissolved in cell-lysis buffer (50mM Tris, 250mM NaCl, 0.1 mM PMSF and 1% Triton-100)
and inclusion bodies were prepared. Further, inclusion bodies were dissolved in sarcosine
buffer (50mM Tris, 1.5% N-laurosyl sarcosine, pH 8.0) and were centrifuged for 25 min at
12,000 rpm and the supernatant was collected. From this supernatant, CAIX protein was
purified using Ni-NTA affinity chromatography. In case of CAII, expression vector
containing gene of CAII, expressed in E. coli (BL21 strain). After cell lysis, filtered
supernatant was loaded on Ni-NTA affinity column, pre-equilibrated with Tris-HCl buffer
(pH 7.5, 500 mM NaCl and 5% Glycerol). The eluted protein was concentrated and further
purified by gel-filtration chromatography (Superdex 200 pg connected to the Akta purifier,
GE Healthcare). The purity of eluted CAIX and CAII was then checked by running SDS-
PAGE.
2.5. Fluorescence measurements
7

The binding study of each synthesized compounds with that CAIX protein was carried
by monitoring changes in fluorescence intensity of protein. The stock solutions of
synthesized compounds were made in the DMSO, and diluted to 1 µM/µl working
concentration in the 50 mM phosphate buffer. All fluorescence experiments were carried out
on Jasco spectroflourimeter (FP-6200) using a 5 mm quartz cuvette (excitation at 280 nm,
emission at 300-400 nm). To obtain final spectra, each time corresponding blank is
subtracted. Each experiment was performed in triplicates. A decrease in fluorescence
intensity with increasing concentration of compound was utilized as a measure for the
calculation of binding constant (Ka), number of binding sites present in protein (n) using the
modified Stern-Volmer equation:
log (Fo-F)/F = log Ka + nlog[L]
(1)
where, Fo = fluorescence intensity of native protein, F = fluorescence intensity of
protein in the presence of ligand, Ka = Binding constant, n = number of binding sites, L =
concentration of ligand. The values for binding constant (Ka) and number of binding sites (n)
were derived from the intercept and slope, respectively.
2.6. Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) measurements were studied using a VP-ITC
microcalorimeter from MicroCal, Inc (GE, MicroCal, USA) according to our published
protocol [41]. Briefly, protein was extensively dialyzed against 50 mM phosphate buffer and
from the stock solution of ligand working ligand solutions was prepared in last dialyzing
buffer. As stock solutions of compounds was prepared in DMSO, equal amount of DMSO,
according to the amount added in preparing ligand solution was added to the protein solution
(up to 1.5 % v/v) in order to prevent signal stability problems during ITC measurements.
8

Blank experiments were carried out to obtain the heat of dilution of each compound. For
analysis, heat of dilution of each compound with that of buffer was taken and subtracted from
the heat of dilution obtained from the compound-protein titrations. The binding data was
analysed using MicroCal Origin 7.0 software provided with the instrument.
2.7. CA inhibitory assay
CAIX and CAII enzyme inhibition assays were performed using reported method [7, 15].
This procedure is based on spectrophotometric determination of p-nitrophenol which is a
yellow colored compound that is formed as hydrolysis product of p-nitrophenyl acetate (4-
NPA) catalyzed by CA. The absorbance was then measured at 400nm by UV
spectrophotometer V-660. The IC50 values for each test compounds were calculated by using
Graph Pad Prism (Version 6.0) software.
2.8. Cell viability assay
The studied cell lines (HeLa and HT-29) were maximally passaged for 3 months.
Cells were incubated at 37 ºC with 5 % CO2 (for normoxic conditions), 1 % O2 and 5 % CO2
balanced with N2 or grown in the presence of deferoxamine (to mimic hypoxic conditions) as
reported [42, 43]. We have selected HeLa and HT-29 cells because it has been reported that
these cells naturally express CAIX [43]. To determine the cytotoxic and anticancerous
activities of compounds, MTT assay was carried out as described [36, 41]. Briefly, 10×103
viable cells were seeded in a 96 well plates and incubated with increasing concentrations of
compounds (5 µM-100 µM). After 48 h of treatment, mixture of medium and compound were
removed, cells were washed with phosphate buffer saline (PBS) twice. Approximately, 20 µl
MTT (from 5 mg/ml stock) and 100 µl DMEM was added to each well and incubated for 4-5
h at 37 ºC, in a CO2 incubator. Finally, the residual MTT and medium was aspirated and the
9

crystals of formazan were dissolves by adding 100 µl DMSO in each well. The micro-titer
plates were then agitated for 15-20 minutes on an orbital plate shaker and absorbance at 570
nm was taken on a titerplate reader (BioRad). Absorbance value obtained was translated into
percentage viability in comparison to the control cells.
2.9. Cell apoptosis assay
To determine cell apoptosis, annexin-V staining was used [44, 45]. HeLa cells were
treated with IC50 concentration of each compound for 24 h at 37ºC, and the control cells were
treated with the media only. After 24 h, nearly 2x106 cells were trypsinized and washed two
times with 5-6 ml of PBS by centrifugation; at 1800 rpm for 4 min. Staining of Annexin-V
was done by using FITC-Annexin-V kit by following the manufacturer’s instructions (BD-
Biosciences, USA). For each sample nearly, 10,000 events were taken by flow cytometry BD
FACScanto and data were analyzed with help of flowJo software. DAPI was used to stain
nuclei of the cells [41]. Fluorescence images were taken on Nikon-EclipseTS100 microscope.
3. Results
3.1. Synthesis
All compounds were characterized by IR, 1H NMR, 13C NMR and mass spectrometry
(see supplementary data). The purity of the compounds was confirmed by elemental analysis
and data was found in accordance with ± 0.3 %.
3.1.1.N'-[(1E)-(2-hydroxyphenyl) methylene]-4-methylbenzenesulfonohydrazide (1)
10

Compound 1 was succesfully synthesized and confirmed by IR, 1H NMR, 13C NMR
13
analysis. Observations from the results of IR, 1H NMR, C NMR are described as (White
o
solid, Ethanol) Yield: 78 %; mp: 149-151 C; Anal. calc. for C14H14N2O3S: C 57.9, H 4.9, N
9.6, O 16.5, S 11.0 %; found: C 58.1, H 4.9, N 9.5, O 16.5, S 11.2 %; IR νmax (cm-1): 3183
1
(N-H), 2926 (Ar-H), 1616 (C=N), 1495 (C=C), 1330, 1168 (SO2) ; H NMR (DMSO-d6) δ:
11.43(s, 1H, -NH), 10.18 (s, 1H, -OH), 8.16 (s, 1H, -CH=N), 7.75 (d, 2H, J=8.4 Hz, Ar-H),
7.41 (d, 2H, J=8.4 Hz, Ar-H), 7.23-7.19 (m, 1H, Ar-H), 6.85-6.79 (m, 2H, Ar-H), 2.34 (s,
13
3H-CH3); C NMR (DMSO- d6)  (ppm): 156.47, 146.19, 143.59, 135.83, 131.40, 129.72,
127.50, 127.13, 119.39, 119.00, 116.16, 20.95; ESI-MS (m/z): 291.1 (M++1).
3.1.2. N'-[(1E)-(4-methoxyphenyl)methylene]-4-methylbenzenesulfonohydrazide (2)
Compound 2 was succesfully synthesized and confirmed by IR, 1H NMR, 13C NMR
13
analysis. Observations from the results of IR, 1H NMR, C NMR are described as Light
o
orange needles, 20% DCM: Hexane) Yield: 83%; mp: 102-104 C; Anal. calc. for
C15H16N2O3S: C 59.2, H 5.3, N 9.2, O 15.8, S 10.5 %; found: C 58.9, H 5.2, N 9.4, O
16.1, S 10.24 %; IR νmax (cm-1): 3222 (N-H), 2842 (Ar-H), 1575 (C=N), 1520 (C=C),
1326, 1162 (SO2); 1H NMR (DMSO- d6) δ: 11.21 (s, 1H, -NH), 7.84 (s, 1H, -CH=N), 7.75 (d,
2H, J=8.4Hz, Ar-H), 7.49 (d, 2H, J=8.8Hz, Ar-H), 7.39 (d, 1H, J=8.8 Hz, Ar-H), 6.93(d,
13
J=8.4 Hz, 2H, Ar-H), 2.34 (s, 3H, -CH3); C NMR (DMSO- d6)  (ppm): 160.74, 147.00,
143.29, 136.19, 129.56, 128.32, 127.21, 126.25, 114.22. ESI-MS (m/z); 305.0 (M++1).
3.1.3. N'-[(1E)-(3,4-dimethoxyphenyl)methylene]-4-methylbenzenesulfonohydrazide (3)
Compound 3 was succesfully synthesized and confirmed by IR, 1H NMR, 13C NMR
13
analysis. Observations from the results of IR, 1H NMR, C NMR are described as (Yellow
o
needles, 10% DCM: Hexane) Yield: 80 %; mp: 131-132 C; Anal. calc. for C16H18N2O4S: C
11

57.5, H 5.4, N 8.4, O 19.1, S 9.6 %; found: C 57.3, H 5.2, N 8.3, O 19.2, S 9.5 %; IR νmax
1
(cm-1): 3198 (N-H), 2987, 2838 (Ar-H), 1583 (C=N), 1521 (C=C), 1320, 1168 (SO2); H
NMR (DMSO- d6) δ: 11.20 (s, 1H, -NH), 7.82 (s, 1H, -CH=N), 7.76 (s, 1H, Ar-H), 7.48 (s,
1H, Ar-H), 7.39 (s, 1H, Ar-H), 7.37 (s, 1H, Ar-H), 7.12-7.05 (m, 1H, Ar-H), 6.95 (s, 1H, Ar-
13
H), 6.92 (s, 1H, Ar-H), 2.34 (s, 3H, -CH3); C NMR (DMSO- d6)  (ppm): 151.11, 149.39,
147.67, 143.82, 136.65, 130.04, 127.75, 126.92, 121.40, 112.01, 109.16: ); ESI-MS (m/z):
334.9.0 (M+ + 1).
3.2. Molecular docking
To find out the interactions between CAIX and synthesized compounds, molecular
docking was carried out. Docking studies helps to estimate the binding energies, interacting
residues, intermolecular distance between the atoms of ligands and interacting residues of
CAIX. By following the criteria of binding energy, type and extent of interactions exists, the
best-docked CAIX-ligand complexes were selected. CAIX shows a substantial binding with
1, 2 and 3 compounds (Table 1). Docking suggesting that the compound 1 and 2 bind into the
active site cavity of CAIX (Fig. 3 & 4).
All the three compounds bind with CAIX significantly (Fig. 3 & 4). Compound 1
forms a complex with CAIX by offering four hydrogen bonds (Fig. 4B), present between
Gln92, Leu198, Thr199 and Thr200 at 3.0, 3.1, 3.0 and 2.7 Å, respectively. Compound 2
forms six hydrogen bonds with His94, Thr199 (2) and Thr200 (3) residues of CAIX (Fig.
4C), average distances of these hydrogen bonds are 3.3, 2.8, 3.0, 3.0, 3.0 and 2.9 Å,
respectively suggesting a strong binding. Compound 3 on the other hand, offering only two
hydrogen bonds, exists between Arg102 (2.2 Å) and Ser227 (3.0 Å) (Fig. 4D). Analysis of
docked structure shows that CAIX offers numerous van der Waals, covalent and electrostatic
interactions to all the three compounds (Fig. 3A).
12

3.3 Fluorescence binding studies
To estimate the actual binding affinity of p-Toluene sulfonylhydrazone derivatives,
fluorescence and ITC measurements were performed. Binding constants of all three
compounds toward purified CAIX were determined from fluorescence-monitored compound-
CAIX titration by using modified Stern-Volmer equation (equation 1). The best quenching on
addition of increasing amount of compounds were observed in case of compound 1 and 2
(Fig. 5A). For compound 3 a relatively lower intensity of quenching was observed in
comparison to compound 1 and 2 (Fig. 5A). Binding affinities of these compounds to CAIX
were estimated by calculating binding constant with the help of modified Stern-Volmer plot
and found Ka = 6.9 × 107 M-1, Ka = 4.2 × 108 M-1 and Ka = 7.4 × 103 M-1 for compounds 1, 2
and 3, respectively. These results are suggesting that compound 2 is the best molecule which
may be further exploited for inhibition of CAIX.
3.4. ITC measurements
To ascertain binding affinity and nature of interaction of these compounds with
CAIX, we performed ITC measurement. The upper panel of Fig. 6 displays raw data with
negative heat pulsations indicating exothermic binding. After subtracting the dilutions of each
compound and protein, the final ITC isotherm and fitted titration data of CAIX with each
compound is shown in lower panel of Fig. 6. The results shown (Fig. 6) were obtained from
single-site model of fitting. Different thermodynamic parameter associated with binding of
each synthesized compound with CAIX (association constant and ΔH, enthalpy change) is
shown in Table 2. ITC Results also confirms that although three compounds shows binding
with CAIX, but binding affinity of compound 1 and 2 is relatively higher (as depicted from
their dissociation constant values) and can be used as potential inhibitors against CAIX.
13

3.5. Enzyme inhibition assay
Enzyme inhibition potential of compounds 1, 2, 3 against CAIX and CAII was
assayed by measuring the esterase activity of CA using nitrophenol acetate (4-NPA) as a
substrate. For enzyme inhibition studies, acetazolamide was used as a positive control. We
found that compounds 1, 2 and 3 are showing appreciable inhibition of CAIX activity with an
IC50 value of 78 nM, 56 nM and 10.89 μM, respectively. Whereas these compounds show
less inhibition against CAII with following IC50 values, 99 μM, 90 μM and 160 μM,
respectively (Table 2). It can be easily observed from IC50 values of these compounds that
compound 1 and 2 inhibits CAIX more specifically than CAII.
3.6. Cell viability assay
To see the effect of synthesized compounds on the proliferation of HT-29 and HeLa
cells, MTT assay was carried out. Treatment of all compounds inhibit the viability of HeLa
and HT-29 cell lines in a dose-dependent (5–100 μM) manner (Fig. 7). IC50 values of each
compound after 48 h treatment are given in Table 2. Cell viability studies on hypoxic as well
as normoxic HT-29 and HeLa cells were found to be dose dependent effect of compounds (1-
3) on the cell viabilities. Interestingly, it was found that all compounds reduce the cell
viability in both conditions, but inhibition is more prominent in case of hypoxic cells as
compared to normoxic cells (Fig. 7). It was found that compound 1 and 2 inhibits the
proliferation of HT-29 and HeLa cells (both in hypoxic as well as normoxic conditions) to a
greater extent than compound 3 (Fig. 7).
3.7. Apoptosis assay
To further elaborate our observations regarding the functional influence of present CAIX
inhibitors in terms of apoptotic potential, HeLa and HT-29 cells were treated with the
14

respective IC50 concentration of each compound for 24 h. Double staining was used to see
early apoptosis (annexin-V staining) and late apoptosis (PE-staining/7-AAD). It was found
that treatment of cells with compound 1, 2 and 3 induces apoptosis in both the cancer cell
lines (Fig. 8). Induction of apoptosis was also studied in both hypoxic as well as normoxic
cells. Consistent to the cell viability studies as described in previous section, here also it was
observed that though induction of apoptosis takes place in both experimental conditions
(hypoxia/normoxia), but more number of cells are found to be positive for annexin-V and PE-
staining when treatment is given in hypoxic conditions (see lower row of Fig. 8A). Another
important observation from these results is that out of three studied inhibitors (1, 2 and 3), if
we compare efficacy, then compound 2 is most effective as it induces apoptosis in more
number of cells, which was followed by 1 whereas 3 is least effective. In case of normoxic
HT-29 cells compounds 1, 2 and 3 induces apoptosis in 4.79 %, 6.49 % and 6.28 % of cells,
but in hypoxic condition the percentage of apoptotic cells increases to 37.09 %, 74.1 % and
15.49 %, respectively. Similarly, compounds 1, 2 and 3 treatment to normoxic HeLa cells
prompts apoptosis in 7.86 %, 24.15 % and 7.15 % of cells, whereas in hypoxic conditions
51.39 %, 92.84 % and 19.2 % of cells found to be apoptotic, respectively. Results of DAPI-
staining suggest that treatment of HeLa cells with compound 1, 2 and 3, induces nuclear
morphological changes or nuclear fragmentation also (Fig. 8B). It can be easily observed
from Fig. 8B that compound 1 and 2 induces nuclear fragmentation in more number of cells
(as shown by red color open head arrows) than compound 3. It also supports the
anticancerous behavior of these inhibitors effectively.
4. Discussion
Cancer is a worldwide substantial health problem. Management and prevention of
cancer is necessary because of social and financial burden to the society. Despite appreciative
15

understanding of the diverse molecular corridors involved in progression of different types of
cancer, the use of specifically targeted bio macromolecules has failed to deliver significant
developments in the survival of cancer patients. Numerous reports have shown the
association of various signaling pathways in the progression of different cancers [41, 46, 47].
In the field of cancer biology, now day’s tumor microenvironment becomes an important
hallmark that has been targeted for drug discovery [1, 48]. Uses of structural information of
cancer targets to design potent inhibitors using molecular docking and simulation are
extensively used to fight against different types of cancer [49-60].
CAIX becomes a prognostic indicator and important drug target. As it gets up-regulated
in numerous cancers and plays a substantial role in acidification of tumor (tumor hypoxia),
progression and metastasis [9, 10]. Therefore expression of CAIX is a measure of hypoxia
and correlates with poor prognosis [11, 12]. High expression of CAIX is an attractive and
promising candidate marker for systemic anticancer therapy and drug development [10, 14,
15, 61]. In this study we report the use of p-Toluene sulfonylhydrazone derivatives as
potential inhibitors of CAIX.
Initially binding affinity of synthesized molecules with CAIX has been studied by
molecular docking that suggests a relatively strong binding affinity. The compound-CAIX
docked complex was stabilized by hydrogen bonding as well as π-π interactions (Fig. 3 & 4).
Results obtained from docking were further confirmed by the fluorescence binding and ITC
measurements, showing that compound 1 & 2 binds with the CAIX with high affinity (in μM
range). To further ascertain the functional significance of p-Toluene sulfonylhydrazones
derivatives, enzyme inhibition assay was performed which clearly indicated a significant
inhibition potential towards CAIX (Table 2).
Structure analysis of compound 1 is suggesting that the hydroxy (-OH) group is more
reactive and electron donating in comparison to methoxy (-OCH3) group due to steric
16

repulsion of the methyl group on the lone pair of oxygen. The methoxy derivative of p-
toluene sulfonylhydrazone 2 possesses maximum enzyme inhibition activity against CAIX.
The electron releasing methoxy group provides moderate basicity as compared to 1 which is
more basic due to the presence of hydroxy group. In compound 3, two methoxy groups lead
to p-Toluene sulphonylhydrazone derivatives least basic as compared to compound 1 and 2.
Interestingly it does not bind as strongly with CAIX as compound 1 and 2. Thus, the basicity
of the compounds may influence the interaction of these compounds with CAIX. Studied
compounds efficiently bind to the CAIX and appreciably inhibit the enzymatic activity of
CAIX and thus they may be explored for the development of unique pharmacophore.
Cancer cells multiply in an uncontrolled fashion and are generally resilient to apoptosis
in response to several chemotherapeutic drugs that has been developed in the last decades [2,
16]. Our results indicated that p-Toluene sulfonylhydrazone have the ability to sensitize
hypoxic cancer cells toward apoptosis (Fig. 8). It is an important aspect in drug development
that the synthesized drug molecule should be nontoxic or minimally toxic; our results also
suggested no toxicity of synthesized molecules in the studied concentration range towards
normal HEK-293 cells.
5. Conclusion
In summary, studies presented here shown that synthesized tosylhydrazine derivatives
are binding to the CAIX with a significant binding affinity and resulting in inhibition of its
enzyme activity which many be associated with its anticancerous activity. Further chemical
modifications may be helpful to improve their binding affinity and specificity and exploited
further for an effective hypoxia-induced cancer management. This study offers first evidence
that p-Toluene sulfonylhydrazone derivatives bind CAIX and showed anticancer activity on
different cell lines by inducing apoptosis.
17

Acknowledgements
This work is supported by the Council of Scientific and Industrial Research, India (grant no.
37(1665)/15/EMR-II). AQ is thankful to the University Grant Commission (UGC) India for
financial support. We sincerely acknowledge Prof. Abdul Waheed, Saint Louis University for
providing the gene of CAII & CAIX. Authors thank to the Department of Science and
Technology, Government of India for the FIST support (FIST program No. SR/FST/LSI-
541/2012).
Disclosure of Conflicts of Interest
Authors have declared that there is no any conflict of interest.
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23

Figure Legends
Fig. 1: Structure of some known carbonic anhydrase inhibitors.
Fig. 2: Strategy for designing of carbonic anhydrase IX inhibitors.
Fig. 3: Molecular docking of synthesized compounds with CAIX: (A) 2D schematic
diagram showing interactions of compound 1, 2 and 3 to the CAIX. Residues involved
in hydrogen bonding, charge or polar interactions, van der Waals interactions are
represented in different color indicated in inset. (B) Surface view of CAIX complexed
with compound 1, 2 and 3.
Fig. 4: Interaction analysis of CAIX-inhibitor complex: (A) Cartoon model of CAIX
complexed with all three synthesized compounds. Docked poses of (B) compound
1 (C) compound 2 and (D) compound 3 with CAIX.
Fig. 5: Fluorescence binding studies of p-toluene sulfonylhydrazone derivatives with
CAIX. (A) Fluorescence spectra of CAIX (5-8 µM) with increasing concentrations
of each compound (0-100 µM). Excitation wavelength was fixed to 280 nm and
emission was recorded in the range 300-400 nm. (B) Modified Stern-Volmer plot
showing quenching of CAIX by each compound used to calculate binding affinity
(Ka).
Fig. 6: ITC measurement showing titration of p-toluene sulfonylhydrazone analogue
with CAIX. (Top) Raw data plot of heat produced against time for the titration of
800-1200 µM 1, 2 and 3 into 16-20 µM CAIX. (Bottom) Corresponding binding
isotherm obtained after integration of peak area and normalization to yield a plot of
molar enthalpy change against inhibitor-CAIX ratio. The one-site fit curve is
displayed as a thin red color line.
Fig. 7: Cell viability assay. (A) Effect of CAIX inhibitors on the viability of human cancer
cells (HeLa) and human normal cell (HEK-293) lines: Cells were treated with
24

increasing concentrations of each compound (0-100 μM) for 48 h. In case of
hypoxia-induced cells, we first exposed the cells to hypoxic conditions for 24 h,
and then treatment is given. Cell viabilities to that of the control were shown here.
Each data point shown is the mean ± SD from n=3. (B) Effect of CAIX inhibitors
on the viability of human cancer cells (HeLa) and human normal cell (HEK-293)
lines: Cells were treated with increasing concentrations of each compound (0-100
μM) for 48 h. Cell viabilities to that of the control were shown here. Each data
point shown is the mean ± SD from n=3
Fig. 8: Cell apoptosis studies. (A) Annexin-V staining of HT-29 and HeLa cells; cells were
treated with IC50 concentration of each inhibitor for 24 h and subsequently stained
with FITC-Annexin-V/7-AAD-staining. Apoptosis was quantified by flow
cytometry. Representative flow images showing FITC-Annexin-V/7-AAD labeled
cells, which directly corresponds to the percentage of apoptotic cells. (B)
Representative images of HeLa cells stained with DAPI after the treatment of each
compound, apoptotic nuclei were marked by red color arrow heads.
25

SO2NH2
S
H2NO2S
S
SO2NH2
O
N
N
O
O
N
O
HN
O
O
O
O
Topiramate
Ethoxolamide
Aceazolamide
R
R
H
S
O
H
H
N
N
N
S
O
N
N
C
O
N
O
H
H
H
O
H2NO2S
Thiourea derivatives
Sulfonyl semicarbazide derivatives
Figure 1
Figure 2
26

A.
Residue Interaction
Electrostatic
van der Waals
Covalent bond
Water
Compound 1
Compound 2
Compound 3
Metal
Residue Interaction
Electrostatic
Residue Interaction
Electrostatic
van der Waals
Covalent bond
Water
van der Waals
Covalent bond
Water
Metal
Metal
B.
Figure 3
B.
THR200
THR199
GLN92
LEU198
Compound 1
A.
C.
THR200
GLN92
Compound 2
LEU198
THR199
CAIX-Compound 1-3
Complex
D.
SER227
180°
ARG102
Figure 4
Compound 3
27

28

Compound 1
Compound 2
Compound 3
Figure 6
29