Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-[1,2,3]triazolo[4,5d]pyrimidin 6(7H)yl)urea

Article abstract of DOI:10.1016/j.jmgm.2016.01.006

Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug design and discovery process. Here, we performed fluorescence-bind

Full text of DOI:10.1016/j.jmgm.2016.01.006

Accepted Manuscript
Title: Design and synthesis of a novel class of carbonic
anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-
3
H-[1,2,3]triazolo[4,5d]pyrimidin 6
(7H)yl)urea
Author: Shikha Kumari Danish Idrees Chandra Bhushan
Mishra Amresh Prakash Wahiduzzaman Faizan Ahmad Md.
Imtaiyaz Hassan Manisha Tiwari
PII:
S1093-3263(16)30006-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jmgm.2016.01.006
JMG 6650
To appear in:
Journal of Molecular Graphics and Modelling
Received date:
Revised date:
Accepted date:
30-10-2015
11-1-2016
16-1-2016
Please cite this article as: Shikha Kumari, Danish Idrees, Chandra Bhushan
Mishra, Amresh Prakash, Wahiduzzaman, Faizan Ahmad, Md.Imtaiyaz
Hassan, Manisha Tiwari, Design and synthesis of a novel class of
carbonic
anhydrase-IX
inhibitor
1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-
[1,2,3]triazolo[4,5d]pyrimidin 6 (7H)yl)urea, Journal of Molecular Graphics and
Modelling http://dx.doi.org/10.1016/j.jmgm.2016.01.006
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Design and synthesis of a novel class of carbonic anhydrase-
IX inhibitor 1-(3-(phenyl/4-fluorophenyl) -7-imino-3H-
[1,2,3] triazolo[4,5d]pyrimidin 6 (7H)yl)urea
a,†
b†
a†
b†
Shikha Kumari , Danish Idrees , Chandra Bhushan Mishra , Amresh Prakash ,
b
b
b,*
a,
Wahiduzzaman , Faizan Ahmad , Md. Imtaiyaz Hassan and Manisha Tiwari *
a
Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New
b
Delhi 110025, India
†These authors equally contributed
*
Corresponding Author
Md. Imtaiyaz Hassan, Ph.D.
Assistant Professor)
(
Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
E-mail: mihassan@jmi.ac.in
Dr. Manisha Tiwari
(
Assistant Professor)
Dr. B. R. Ambedkar Centre for Biomedical Research,
University of Delhi, Delhi 110007, India
E-mail: mtiwari07@gmail.com

Graphical abstract
Tyr7
Thr199
Thr200
His119
His96
His94
Trp209
Cartoon view of TPUI docked with CAIX

Highlights

Pyrimidine urea derived compound TPUI binds to CAIX with high affinity and
selectivity.



TPUI is preferentially docked in the active site cavity of CAIX.
Fluorescence binding study is consistent with docking and MD simulation studies.
TPUI appeared as novel class of CAIX inhibitor against hypoxia-induced cancer.
ABSTRACT
Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of
hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug
design and discovery process. Here, we performed fluorescence-binding studies and inhibition
assay combined with molecular docking and molecular dynamics (MD) simulation analyses to
determine the binding affinity of two synthesized triazolo-pyrimidine urea derived (TPUI and
TPUII) compounds with CAIX and CAII. Fluorescence binding results are showing that molecule
D
TPUI has an excellent binding-affinity for CAIX (k = 0.048 µM). The TPUII also exhibits an
appreciable binding affinity (k =7.52 µM) for CAIX. TPUI selectively inhibits CAIX as
D
compared to TPUII in the 4-NPA assay. Docking studies show that TPUI is spatially well-fitted in
the active site cavity of CAIX, and is involve in H-bond interactions with His94, His96, His119,
Thr199 and Thr200. MD simulation studies revealed that TPUI efficiently binds to CAIX and
essential active site residual interaction is consistent during the entire simulation of 40 ns. These
studies suggest that TPUI appeared as novel class of CAIX inhibitor, and may be used as a lead
molecule for the development of potent and selective CAIX inhibitor for the hypoxia-induced
cancer therapy.
Keywords: Hypoxia-induced cancer; carbonic anhydrase IX inhibitor; drug design and discovery;
triazolo-pyrimidine urea; MD simulation

1
. Introduction
Carbonic anhydrase (CAs) are zinc containing metalloenzymes which primarily regulate
physiological pH in human body [1]. CA catalyzes a rapid reversible conversion of carbon dioxide
+
) which regulates the
-, homeostasis, electrolyte
(
CO
2
) and water (H
2
O) into a proton (H ) and bicarbonate ion (HCO
3
physiological processes connected with the transport of CO
2
/ HCO
3
secretion in a varieties of tissues/organs, biosynthetic reactions, respiration, calcification,
tumorigenicity and bone reabsorption [2, 3]. In human there are 16 αCA isozymes have been
reported which exhibit variable tissue distribution and functions [4].
CAIX is an attractive target for cancer treatment because of its limited expression in the normal
tissues and predominant expression in varieties of tumour cells [5, 6]. CAIX is a hypoxia-inducible
protein that regulates cellular pH to encourage cancer cell survival and invasion in the hypoxic
microenvironments and serve as a potential biomarker in the poor prognosis of breast cancer
metastasis and survival [7-9]. Furthermore, extracellular location of this isozyme is favourable for
designing of a selective inhibitor which inhibits the membrane associated CAs without interacting
to other cytosolic and mitochondrial CAs [10-12]. Expression of CAIX is induced under hypoxic
conditions and their activity leads to pH imbalance in tumor tissues and thus provides favorable
environment for the survival of cancer cells [13, 14]. Moreover, it contributes to cancer progression
by stimulating cancer cell migration, adhesion and invasion. Now a day’s CAIX has been
identified as an important biomarker of hypoxia and its over-expression is often associated with a
poor responsiveness to the classical radio and chemotherapies [15, 16]. Therapeutic inhibition of
CAIX has been shown to decrease primary tumor growth and metastasis in various tumor models
[
17].

Inorganic anions, sulfonamides, sulfamates, sulfamides, phenols are important class of
pharmacophores that inhibit CAIX very efficiently by participating in Zn binding [18-20].
Acetazolamide 1 (Figure S1) is a potent CA inhibitor which inhibits CA isoform in a nonselective
manner through participating in Zn-binding [21]. Recently, Korkmaz et al. [22] reported a series
of phenyl thiourea derivatives as novel class of CA inhibitors. In this series derivative 2 (Figure
S1) inhibit CA with micro molar range activities. Coumarins appeared as new class of CAIs that
binds (in hydrolyzed form) at the entrance of the CA active site and does not interact with the Zn
metal. Thiocoumarine derivatives 3 (Figure S1) exhibits an excellent inhibitory activity for CAIX
without showing Zn metal binding in active site of CA [23]. Moreover, coumarin derivative 4
(
Figure S1) was also successfully inhibits CA isoform without displaying Zn-binding [24]. Thus,
coumarins show entirely new category of mechanism to inhibit CA enzyme. Numerous, potent
sulfonamide based CAIX inhibitors have already been identified in the last years that can
efficiently inhibit CAIX activity [22]. However, most of the clinically used CA-inhibitors exhibit
broad CA-inhibition and possesses poor isozyme subtype selectivity [25, 26]. Therefore, a
selective CAIX inhibitor is essentially required in cancer therapy without side effects related to
the inhibition of other CA isozymes [2, 27]. Novel chemical scaffolds which endowed with
improved activity as well as selectivity for CAIX are essentially required for the development of
successful anticancer agents.
Triazoles have emerged as an important class of heterocyclic compound to design potent
therapeutic drug molecules [28]. Numerous triazole derivatives have already showed promising
anticancer activity in various in vitro as well as in vivo models [29-31]. Recently, triazole-linked
O-glycosides (compound 5, Figure S1) have been used as a selective and potent CAIX inhibitor
[27]. Moreover, pyrimidine derivatives (compound 6, Figure S1) also displayed potent CA

inhibitory activity. [32, 33]. Hence, triazole coupled pyrimidine molecules would be a promising
scaffold for the development of potent and selective CAIX inhibitors towards the cancer therapy.
In this view, we have design and synthesized urea pendant triazolo-pyrimidine derivatives (TPUI
and TPUII) as a novel class of CAIX inhibitors (Figure S1, compound 7). The designed molecules
are endowed with essential pharmacophoric requirement such as hydrophobic domain (triazolo-
pyrimidine) as well as hydrogen bond donor (HBD)/ hydrogen bond acceptor (HBA) (Urea
fragment) domain (Figure S1). The designed compounds have a urea functional group instead of
the traditional sulfonamide group. This alteration might open a new door for a novel class of
pharmacophore as selective CAIX inhibitors. Binding affinities of TPUI and TPUII with CAIX as
well as CAII were assessed by spectrofluorimeter. In vitro CA inhibition studies of TPUI and
TPUII against CAIX as well as CAII were studied by the 4-NPA spectrophotometric assay.
Moreover, CAIX selective derivative TPUI was docked with human CAIX to evaluate its
interaction potential and sitting pattern in active site CAIX isoforms. Furthermore, MD simulation
studies were also performed to evaluate the extensive binding prototype of synthesized triazolo-
pyrimidine urea derivatives with CAIX.
2
. Materials and Methods
2
.1. Synthesis of TPUI and TPUII
All chemicals, reagents and solvents were procured from Sigma Aldrich (St. Louis, MO, USA),
S.D Fine Chemicals (India) and Merck (Darmstadt, Germany). The homogeneity and purity of the
compounds were checked by thin layer chromatography (TLC), performed on commercially
available silica gel (Kieselgel 60, F254) coated aluminium sheets (Merck) by using methanol:

chloroform (5:95) as solvent system. The visualization on TLC was done by both ultra-violet (UV)
light (λ= 254 nm) and iodine indicator. All compounds were purified by column chromatography
by using silica gel 100-200 mesh (Merck). Melting points were determined in open capillary tubes
in a Hicon melting point apparatus (Hicon, India). The nuclear magnetic resonance (NMR) spectra
were obtained on high resolution Jeol-400MHz NMR spectrophotometer (USA) in CDCl
3
and
DMSO-d using Tetramethylsilane (TMS) as the internal reference. Chemical shifts (δ) were
6
expressed in parts per million relative to TMS and the following abbreviations were used to
describe the peak patterns when appropriate: s, (singlet); d, (doublet); t, (triplet); m, (multiplet);
brs, (broad singlet); dd, (double doublet).The coupling constant (J) values are given in hertz (Hz).
Mass spectra (LRMS) were recorded on an Agilent 6310 Ion trap LC/MS and elemental analysis
(
C, H and N) was carried on Elementar analysensysteme. A detail of chemical analysis is provided
in the Supplementary Text for Chemistry Section.
2
.2. Cloning, Expression and Purification of CAIX and CAII
The 1131-bp coding region (38-414aa) of catalytic domain of CAIX gene was sub-cloned into
pET21c (Novagen) vector with the C-terminal His6 tag. Similarly, CAII gene of 801-bp was sub-
cloned into pET15d vector (Novagen) with N-terminal His6Xtag. Both proteins were expressed in
Escherichia coli BL21 (DE3) cells. CAIX was expressed at 16 °C for 14 hours after induction with
0
.5mM IPTG (Sigma). CAIX was expressed as insoluble protein and form inclusion bodies (IBs)
and pellets were dissolved in the suspension buffer (50 mM phosphate, pH 7.4, 300 mM NaCl, 1%
Triton-X and 1% N-lauroylsarcosine) to solubilize IBs. These solubilized IBs were sonicated and
centrifuged for 30 minutes at 12,000 rpm. This step was repeated thrice and finally supernatant
was applied on a Ni-NTA Sepharose column (GE Health-care). CAIX was eluted from the column

with elution buffer (50 mM phosphate, pH 7.4, 300 mM NaCl, 1% N-lauroylsarcosine and 350
mM imidazole). CAIX was refolded by dialyzing against refolding buffer (50 mM phosphate
o
buffer pH 7.4 and 150 mM NaCl) for 36 hrs at 4 C with five successive buffer changes. We also
performed enzyme assay of refolded CAIX (34). Figure S2 A and B are showing the SDS-PAGE
and Western blot, respectively of purified CAIX.
CAII was expressed at 37 °C for 3 hours after induction with 0.25 mM IPTG (Sigma) as soluble
protein, harvested cells were suspended in buffer (50 mM phosphate, pH 7.4, 300 mM NaCl, 1%
Triton-X and 0.1mg/ml lysozyme). Cell lysate was sonicated and centrifuged to remove cell debris.
Supernatant was applied to Ni-NTA Sepharose column (GE Health-care) and eluted with elution
buffer (50 mM phosphate, pH 7.4, 300 mM NaCl, and 300 mM imidazole). The eluent was
collected, concentrated and loaded to gel-filtration chromatography (superdex200, GE Healthcare
Bio-Sciences) in 50 mM phosphate, pH 7.4 and 150 mM NaCl buffer (35). Both proteins were
analyzed by SDS-PAGE and identified by Western blotting with anti-histidine antibodies. Figure
S2 C and D are showing the SDS-PAGE and Western blot, respectively of purified CAII.
2
.3. Fluorescence spectra measurements
Fluorescence measurement was performed in the Jasco spectrofluorimeter (Model FP-6200) using
mm quartz cuvette. Protein concentration of 3.4 μM and 2.8 μM were used for CAII and CAIX,
5
respectively. Compounds (TPUI and TPUII) were used in a concentration 1-10 μM. The protein
solution was excited at 292 nm, and emission spectra were recorded between 300 - 400 nm. Both
compounds were dissolved in DMSO and then diluted to 1 mg/ml in the 50 mM phosphate buffer
pH 7.4 and 150 mM NaCl. The protein solution was titrated with increasing concentration of
ligands and a final spectrum was collected after subtracting intensity of the buffer/ligand from each

spectrum. The decrease in fluorescence intensity is due to the addition of ligand has been used to
calculate the binding affinity by Stern-Volmer equations with slight modification [34].
log(Fo-F)/F = logK
a
+ nlog[Q ]
(1)
Where, F
is the binding constant and n is the number of binding sites, Q represents quenching constant. For
the ligand-protein complex, the values for K and n can be derived from the intercept and slope.
o
is the fluorescence intensity of protein and F is the fluorescence intensity of ligand, K
a
a
2
.4. Carbonic anhydrase inhibition assay
Carbonic anhydrase inhibition was measured by previously reported method [35] based on the
principle that p-nitrophenol acetate (4-NPA) is hydrolyzed by CA to form p-nitophenol which
was measured in Jasco UV/visible spectrophotometer (Jasco V-660, Model B 028661152)
equipped with peltier-type temperature controller (ETCS). Absorption was measured at 415 nm
using 1 cm path length cuvettes. Reaction mixture contained 50 mM Tris buffer (pH 7.5), 3 mM
substrate p-nitrophenol acetate, different concentration of test compounds (0.5 mM) and protein
concentration was used 5 µM - 10 µM. All experiments were carried out at least three times at 25
o
o
±
.1 C. After 3 min incubation at 25 C reading was measured.
2
.5. Molecular docking
Atomic coordinates of CAIX and CAII were taken from the Protein Data Bank (www.rcsb.org)
36, 37]. Steepest descent method from Gromacs 4.5.5 was used to optimize the coordinates and
[
both proteins were saved in saved in .pdbqt format to carry docking analysis. AutoDock 4.2 with
standard protocol was used to dock the TPUI and TPUII with both isozymes. The Lamarckian

genetic algorithm (LGA) was applied to deal with protein-ligands interactions [38]. The most
favourable free binding energy and docking orientations lying within the range of 2.0 Å in root-
mean square deviation (rmsd) tolerance were used to cluster the molecule and ranked accordingly.
PyMOL was used to visualize molecular interactions. All calculations were carried out on
workstation machines running Linux x 86 as operating systems.
2.6. Molecular dynamics simulation
MD simulations were performed with Gromacs 4.5.5 suite of programs, using CHARMm force-
field and spc216 water molecules. The trajectory files were analysed using GROMACS utilities:
g_gyrate, g_rmsd and g_rmsf to obtain the radius of gyration (Rg), RMSD and root mean square
fluctuation (RMSF). The coordinates of CAIX (PDB ID: 3IAI) was energy minimized with
-1
steepest descent up to a tolerance of 100 kj mol to remove bad contacts. Two independent MD
simulations were carried out with standard protocol in water for CAIX and CAIX-TPUI complex
at 300K, respectively to evaluate the structural changes, binding affinity and stability in course of
simulation. SwissParam server is used to generate the topology file for ligand [39]. The SHAKE
algorithm was used to restrain the covalent bonds containing hydrogen atoms at their equilibrium
distances and periodic boundary conditions were applied. Particle Mesh Ewald (PME) method was
used for computing the long-range electrostatic interactions. A 1.4 nm cut-off for van der Waal
interactions and a 1.0 nm cut-off for Coulombs interaction were adopted in the simulation,
respectively. Analytical graphs were plotted using Xmgrace, VMD and PyMol were used for
structure and molecular interaction analysis [40].

3
. Results and discussion
3
.1. Chemistry
Synthesis of TPUI and TPUII has been performed according to scheme 1 [41, 42] (Supplementary
Text). Briefly, aniline/p-fluoroaniline was converted into respective azides by treating sodium
nitrate as well as sodium azide in HCl at RT. Equimolar ratio of azides 3-4 and cyanoacetamide
has been refluxed in ethanol to get amide derivatives 5 and 6. Further, dehydration of amide
derivatives has been done with POCl
3
in acetonitrile to achieved cyanotriazole derivatives 7 and
8
. Extension of amino functional group of triazole 7 and 8 has been completed with refluxing
triethyl orthoformate in acetic anhydride. Cyclized triazolo-pyrimidine urea derivatives 11(TPUI)
and 12 (TPUII) were synthesized by reacting semicarbazide with Schiff base intermediate 9 and
1
0 in ethanol by using triethylamine as basic catalyst. Synthesized compounds were successfully
characterized by NMR, mass spectrometry and elemental analysis.
3
.2. Florescence binding study
The fluorescence emission spectra of CAIX solution in the absence and presence of different
concentrations of compounds (TPU-I and TPU-II) in PBS at 25°C are shown in Fig. 1A and 1C.
The maximum emission peak was observed at 343 nm (excitation wavelength was 292 nm). Upon
incubation of different concentrations (1-10 µM) of compounds with CAIX the maximum
fluorescence peak intensity was reduced significantly indicating that CAIX-compound complexes
were formed. Change in the intrinsic fluorescence intensity of CAIX was observed when
compounds bind to CAIX. The values fluorescence intensity at 343 nm was plotted as a function
of [ligand] to calculate the binding affinity (Figure 1B and 1D.).

A similar fluorescence quenching results were observed for CAII with same compounds but their
binding affinities are comparatively low (Figure 2). The binding affinity of TPUI for CAII and
CAIX are 111.223 µM and 0.048641 µM (Fig. 1B and 2B). TPUII showed binding affinity for
CAII and CAIX are 240.98 µM and 7.52 µM (Fig. 1D and 2D). Results clearly indicate that TPUI
exhibited greater affinity for CAIX over CAII as compared to TPUII. It is analyzed that
introduction of fluorine atom at para-position on phenyl ring of triazolopyrimidine loss affinity for
CAIX as compared to phenyl ring containing triazolopyrimidine.
3
.3. Carbonic anhydrase inhibition
Inhibition activities of TPUI, TPUII and AZM were measured against CAII and CAIX by
previously reported 4-NPA assay [35]. Inhibition studies results showed that the TPUI possessed
1
6% and 53% inhibition activities against CAII and CAIX, respectively. Moreover, TPUII
possessed 43% and 54% inhibition activities against CAII and CAIX, respectively. Standard
inhibitor AZM showed 76% inhibition for CAII and 63% inhibition for CAIX. Our study clearly
indicates that TPUI is selectively inhibits CAIX (53%) over CAII (16%) as compared to AZM
which did not displayed impressive selective inhibition profile for both CAIX (76%) and CAII
(
63%). These inhibition results are also consistent with florescence binding studies where TPUI
showed good affinity towards CAIX as compared to CAII.
3
.4. Molecular docking
Docking analysis of TPUI was carried with both isozymes CAII and CAIX to elucidate interaction
pattern of synthesized inhibitors. Result showed that TPUI is nicely bounded into the active site
of CAIX with minimum binding energy (ΔG) -10.89 kcal/mol (K = 0.032 μM) (Table 1). Although,
i

2
+
TPUI does not bind with Zn metal in active site but the best binding mode of TPUI in the active
site of CAIX is stabilized with four H-bond interaction with His 94, His119, Thr199 and Thr200
(Figure 3A). Active site residues Leu98, Val121, Val143, Leu198 and Trp209 are involved in
hydrophobic interaction and Trp5 is stabilized phenyl moiety of triazolo-pyrimidine with π-π
interaction (Figure 3B). Structure of TPU scaffold is provided in the Figure 3C.
Molecular docking result with CAII shows binding affinity -9.47 kcal/mol (K = 87.32 μM) for
i
TPUI (Figure 4A). At the active site of CAII, TPUI shows H-bond interaction with Tyr7, Asn67,
His96, Thr200, Asn244, whereas residues Ala65, Phe66, Trp97, Leu98 and Val121, Val143,
Leu198 and Trp209 are involved in hydrophobic interaction (Figure 4B). The phenyl moiety of
TPUI is stabilized with π-π interaction of Phe95 and Phe131. Active site topology of CAII is highly
conserved with CAIX [2, 36]. However, molecular docking result shows a quite different
orientation of TPUI at the active site of CAII (Table 2) and anchored near the outer hydrophobic
core of active site and is largely stabilized with Ala65, Phe66, Trp97, Leu98 and Val121, Val143,
Leu198 and Trp209.
Structural analysis shows that CAIX active site is located in a large conical cavity which spans
2
+
from the surface to the centre of isozyme and zinc ion (Zn ) is coordinated with imidazole ring
of three histidine molecules (His-64, His-94 and His-119), at the bottom of cavity [36]. And active
site cavity is well partitioned in lower and upper hydrophobic domain and core region is rich in
hydrophilic amino acid residues [27, 36]. Whereas, CAII active site is comprised with two distinct
domain (i) upper layer is lined with hydrophobic residues: Ile91, Val121, Phe131, Val135, Leu141,
Val143, Leu198, Pro202, Leu204, Val207 and Trp 209 (ii) lower domain is facilitated with
hydrophilic residues Tyr7, Asn62, His64, Asn67, Thr199 and Thr200 [43] (Figure 3 and 4). This

analysis shows that hydrophilic core residues CAII are highly conserved with CAIX. However,
the residual difference at the outer linings of CAII active site, specially the hydrophobic residues,
facilitated the binding of TPUI towards the outer surface of CAII. Moreover, the compressed outer
rim of CAII active site, probably hitched the binding of triazolopyrimidine urea and major
interaction is observed at surface. The surface view of both isozymes docked with TPUI is shown
in (Figure 5A and 5B).
Result shows that interaction and environment at the binding site of CAIX is more favourable for
TPUI. Moreover, docking result clearly indicates that TPUI binds in CAIX and CAII with totally
different manner (Table 1). TPUI participates in hydrogen bonding interaction with His94, His119,
Thr199 and Thr200 in active site of CAIX and exhibits hydrogen bonding interaction with Tyr7,
Asn67, His96, Thr200, Asn244 in active site of CAII (Figure 3, 4 and Table 1). Moreover,
orientation of TPUI in CAIX and CAII was absorbed totally different. Additionally, docking
studies show that TPU has emerged as novel class of selective CAIX inhibitor which did not
+
interact with Zn metal but exhibited excellent binding pattern in active site of CAIX.
3
.5. MD simulations
Docking procedures are used to predict the affinity between the ligand and protein through the
identification of correct conformation of ligands in the binding pocket of any protein [44-48].
However, MD simulation provides a dynamic picture of protein ligand interactions [49-55]. In the
present study, best binding conformation of TPUI into the binding site of CAIX, having highest
docking score was taken for further analysis. We performed 40 ns MD simulation and found that
TPUI remain inside the binding pocket of CAIX. Dynamic stability of CAIX and its complex with
α
TPUI, over the simulation of 40 ns was analyzed using backbone RMSD of C atoms. RMSD

result shows a stable conformation is maintained throughout the simulation time of 40 ns. Plot of
time versus RMSD is shown in Figure 6A.
Trajectory for CAIX achieved a stable equilibrium in 5 ns, with the initial drift of 0.05 nm. A small
transition in trajectory is observed after 20 ns which is repeated up to 30 ns with drift of 0.05 nm.
However, trajectory attains the stability after 30 ns and a continuous equilibrium is maintained up
to end of simulation time. RMSD plot of CAIX-TPUI complex shows a significant drift of ~ 0.1
nm at 15 ns, indicates the conformational change in CAIX, due to spatial fitting of TPUI in the
active site. The small but continuous rise in trajectory represented the various interactions with
ligand. The trajectory for isozyme became stable after 25 ns with drop down of 0.08 nm and
further, it maintained stable conformation throughout the simulation time of 40 ns. The less change
in RMSD of backbone atoms strongly suggests the stable dynamic behaviour of CAIX-TPUI
complex.
Furthermore, the Rg analysis is carried to measure the compactness of isozyme. The average value
of Rg, calculated from trajectory is shown in Figure 6B. Plot for CAIX, clearly indicates that a
steady Rg value 1.77 nm is maintained over the course of 40 ns. The average Rg value of 1.70 nm
is found for CAIX-TPUI complex showed the stable isozyme-inhibitor interaction. The relative
decrease in Rg (0.07 nm), lead to increase in compactness of isozyme, suggests that binding of
TPUI may enhance the stability of isozyme. The stable back bone RMSD and decrease in Rg score
from native structure strongly indicate that association of TPUI with CAIX is retained during the
entire simulation time.
The average position fluctuation of residues in a dynamic system is analysed with RMSF of all Cα
atoms. Figure 6C clearly shows residues belong to stable secondary structure conformation (α/β

sheet) have stable fluctuation. The residues having higher fluctuations are belonging to loops.
Result shows binding of TPUI lead to increase fluctuation of active site residues. This observation
is consistent with RMSD and Rg data, and result revelled H-bond interaction with His94, His96,
His119, Thr199 and Thr200 plays important role in activity of CA inhibitors. Upper and lower
hydrophobic domains are essential to anchor inhibitor at the active site of isozyme. Moreover,
amino acid differences in the hydrophobic domain of CAIX and CAII are significantly important
for the selective binding TPUI and TPUII with isozyme CAIX. Result showed that hydrophobic
domain may be good target for the design of isozyme selective inhibitor. To gain insight into the
effect of active site residues involve in interaction with inhibitor, we carried solvent accessible
surface area of the CAIX (Figure 6D) and result is found to consistent with RMSF. MD simulation
is precisely used to evaluate the molecular docking results.
4
. Conclusions
The rational drug design for selective inhibition of CA isozymes is a difficult target, due to
conservation of active site structure and topology within the CA enzyme family. However, the
selective inhibition of CAIX is essentially required for cancer therapy without side effect
associated with CAII inhibition. Here we have successfully designed and synthesized TPUI and
TPUII as potent and selective CAIX inhibitors. Both TPUI and II appeared as potent CAIX
inhibitor in silico as well as in vitro studies. However, TPUI displayed greater inhibition activity
and selectivity for CAIX isozyme. These studies successfully provided an endeavour for the
rational design of triazolopyrimidine urea as a novel class of CAIX inhibitor. This molecule may
be explored as lead in development of selective CAIX inhibitor for the therapy of various cancers
in future.

Conflict of interest
Authors declare no conflict of interest regarding any financial and personal relationships with other
people or organizations that could inappropriately influence (bias) this work.
Acknowledgements
SK, DI, AP and CMB are thankful to University Grants Commission (UGC), New Delhi, India,
for their fellowship. MT is thankful to University of Delhi for providing financial support. Authors
sincerely thank Jamia Millia Islamia for providing high speed server in the Central Instrumentation
Facility. Harvard University-plasmid facility is acknowledged providing the CAVA gene. FA and
MIH are thankful to the Department of Science and Technology (Government of India) and Indian
Council for Medical Research (ICMR) for financial support. Authors thank Department of Science
and Technology, Government of India for FIST support (SR/FST/LSI-541/2012).

References
1
.
Alterio V, Di Fiore A, D'Ambrosio K, Supuran CT, De Simone G. Multiple binding
modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15
different isoforms? Chem Rev 2012,112:4421-4468.
2
3
.
.
Imtaiyaz Hassan M, Shajee B, Waheed A, Ahmad F, Sly WS. Structure, function and
applications of carbonic anhydrase isozymes. Bioorg Med Chem 2013,21:1570-1582.
Reibring CG, El Shahawy M, Hallberg K, Kannius-Janson M, Nilsson J, Parkkila S, et al.
Expression patterns and subcellular localization of carbonic anhydrases are
developmentally regulated during tooth formation. PLoS One 2014,9:e96007.
4
5
6
7
8
9
1
1
.
D'Ambrosio K, Carradori S, Monti SM, Buonanno M, Secci D, Vullo D, et al. Out of the
active site binding pocket for carbonic anhydrase inhibitors. Chem Commun (Camb)
2
015,51:302-305.
.
Salmon AJ, Williams ML, Wu QK, Morizzi J, Gregg D, Charman SA, et al. Metallocene-
based inhibitors of cancer-associated carbonic anhydrase enzymes IX and XII. J Med
Chem 2012,55:5506-5517.
.
Doyen J, Parks SK, Marcie S, Pouyssegur J, Chiche J. Knock-down of hypoxia-induced
carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular
acidosis. Front Oncol 2012,2:199.
.
Nocentini A, Carta F, Ceruso M, Bartolucci G, Supuran CT. Click-tailed coumarins with
potent and selective inhibitory action against the tumor-associated carbonic anhydrases
IX and XII. Bioorg Med Chem 2015.
.
Syrjanen L, Luukkaala T, Leppilampi M, Kallioinen M, Pastorekova S, Pastorek J, et al.
Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin
neoplasms. APMIS 2014,122:880-889.
.
Hynninen P, Vaskivuo L, Saarnio J, Haapasalo H, Kivela J, Pastorekova S, et al.
Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours.
Histopathology 2006,49:594-602.
0.
1.
Dudutiene V, Matuliene J, Smirnov A, Timm DD, Zubriene A, Baranauskiene L, et al.
Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX. J
Med Chem 2014,57:9435-9446.
Gluszok S, Frederick R, Foulon C, Klupsch F, Supuran CT, Vullo D, et al. Design, solid-
phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as
carbonic anhydrase IX inhibitors. Bioorg Med Chem 2010,18:7392-7401.

1
2.
Morris JC, Chiche J, Grellier C, Lopez M, Bornaghi LF, Maresca A, et al. Targeting
hypoxic tumor cell viability with carbohydrate-based carbonic anhydrase IX and XII
inhibitors. J Med Chem 2011,54:6905-6918.
1
3.
4.
Supuran CT. Development of small molecule carbonic anhydrase IX inhibitors. BJU Int
2
008,101 Suppl 4:39-40.
1
Idrees D, Prakash A, Haque MA, Islam A, Ahmad F, Hassan MI. Spectroscopic and MD
simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA
induced by urea. J Biomol Struct Dyn 2015:1-37.
15.
16.
17.
18.
Vullo D, Franchi M, Gallori E, Pastorek J, Scozzafava A, Pastorekova S, et al. Carbonic
anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and
heterocyclic sulfonamides. Bioorg Med Chem Lett 2003,13:1005-1009.
Wang L, Yang C, Lu W, Liu L, Gao R, Liao S, et al. Discovery of new potent inhibitors
for carbonic anhydrase IX by structure-based virtual screening. Bioorg Med Chem Lett
2
013,23:3496-3499.
Thobani A, Fishman GA. The use of carbonic anhydrase inhibitors in the retreatment of
cystic macular lesions in retinitis pigmentosa and X-linked retinoschisis. Retina
2
011,31:312-315.
Altintop MD, Ozdemir A, Kucukoglu K, Turan-Zitouni G, Nadaroglu H, Kaplancikli ZA.
Synthesis and evaluation of new thiadiazole derivatives as potential inhibitors of human
carbonic anhydrase isozymes (hCA-I and hCA-II). J Enzyme Inhib Med Chem
2
015,30:32-37.
1
9.
SitaRam, Ceruso M, Khloya P, Supuran CT, Sharma PK. 4-Functionalized 1,3-
diarylpyrazoles bearing 6-aminosulfonylbenzothiazole moiety as potent inhibitors of
carbonic anhydrase isoforms hCA I, II, IX and XII. Bioorg Med Chem 2014,22:6945-
6
952.
2
2
2
2
0.
1.
2.
3.
SitaRam, Celik G, Khloya P, Vullo D, Supuran CT, Sharma PK. Benzenesulfonamide
bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic
anhydrase isoforms hCA IX and hCA XII. Bioorg Med Chem 2014,22:1873-1882.
Pichake J, Kharkar PS, Ceruso M, Supuran CT, Toraskar MP. Carbonic anhydrase
inhibitors: design, synthesis, and biological evaluation of novel sulfonyl semicarbazide
derivatives. ACS Med Chem Lett 2014,5:793-796.
Korkmaz N, Obaidi OA, Senturk M, Astley D, Ekinci D, Supuran CT. Synthesis and
biological activity of novel thiourea derivatives as carbonic anhydrase inhibitors. J
Enzyme Inhib Med Chem 2015,30:75-80.
Maresca A, Temperini C, Pochet L, Masereel B, Scozzafava A, Supuran CT. Deciphering
the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J
Med Chem 2010,53:335-344.

2
2
4.
5.
Maresca A, Temperini C, Vu H, Pham NB, Poulsen SA, Scozzafava A, et al. Non-zinc
mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide
inhibitors. J Am Chem Soc 2009,131:3057-3062.
Carradori S, De Monte C, D'Ascenzio M, Secci D, Celik G, Ceruso M, et al. Salen and
tetrahydrosalen derivatives act as effective inhibitors of the tumor-associated carbonic
anhydrase XII--a new scaffold for designing isoform-selective inhibitors. Bioorg Med
Chem Lett 2013,23:6759-6763.
26.
27.
28.
29.
30.
Cecchi A, Taylor SD, Liu Y, Hill B, Vullo D, Scozzafava A, et al. Carbonic anhydrase
inhibitors: inhibition of the human isozymes I, II, VA, and IX with a library of substituted
difluoromethanesulfonamides. Bioorg Med Chem Lett 2005,15:5192-5196.
Prakash A, Kumar K, Islam A, Hassan MI, Ahmad F. Receptor Chemoprint Derived
Pharmacophore Model for Development of CAIX Inhibitors. J Carcinog Mutagen
2
013,S8:1-9.
Wilkinson BL, Bornaghi LF, Houston TA, Innocenti A, Supuran CT, Poulsen SA. A
novel class of carbonic anhydrase inhibitors: glycoconjugate benzene sulfonamides
prepared by "click-tailing". J Med Chem 2006,49:6539-6548.
Akula NV, Kumar S, Singh V, Tiwari M. Homology modeling and QSAR analysis of
1
,3,4-thiadiazole and 1,3,4-triazole derivatives as carbonic anhydrase inhibitors. Indian J
Biochem Biophys 2010,47:234-242.
Almajan GL, Barbuceanu SF, Innocenti A, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Inhibition of the cytosolic and tumor-associated carbonic anhydrase
isozymes I, II and IX with some 1,3,4-oxadiazole- and 1,2,4-triazole-thiols. J Enzyme
Inhib Med Chem 2008,23:101-107.
3
3
1.
2.
Grandane A, Tanc M, Zalubovskis R, Supuran CT. Synthesis of 6-tetrazolyl-substituted
sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated
carbonic anhydrase isoforms IX and XII. Bioorg Med Chem 2014,22:1522-1528.
Riafrecha LE, Rodriguez OM, Vullo D, Supuran CT, Colinas PA. Attachment of
carbohydrates to methoxyaryl moieties leads to highly selective inhibitors of the cancer
associated carbonic anhydrase isoforms IX and XII. Bioorg Med Chem 2014,22:5308-
5
314.
3
3.
4.
Wilkinson BL, Bornaghi LF, Houston TA, Innocenti A, Vullo D, Supuran CT, et al.
Carbonic anhydrase inhibitors: inhibition of isozymes I, II, and IX with triazole-linked O-
glycosides of benzene sulfonamides. J Med Chem 2007,50:1651-1657.
3
Wang YQ, Zhang HM, Zhang GC, Tao WH, Fei ZH, Liu ZT. Spectroscopic studies on
the interaction between silicotungstic acid and bovine serum albumin. J Pharm Biomed
Anal 2007,43:1869-1875.

3
3
5.
6.
Pocker Y, Stone JT. The catalytic versatility of erythrocyte carbonic anhydrase. 3.
Kinetic studies of the enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry
1
967,6:668-678.
Alterio V, Hilvo M, Di Fiore A, Supuran CT, Pan P, Parkkila S, et al. Crystal structure of
the catalytic domain of the tumor-associated human carbonic anhydrase IX. Proc Natl
Acad Sci U S A 2009,106:16233-16238.
3
3
3
4
4
7.
8.
9.
0.
1.
Vidgren J, Liljas A, Walker NP. Refined structure of the acetazolamide complex of
human carbonic anhydrase II at 1.9 A. Int J Biol Macromol 1990,12:342-344.
Fuhrmann J, Rurainski A, Lenhof HP, Neumann D. A new Lamarckian genetic algorithm
for flexible ligand-receptor docking. J Comput Chem 2010,31:1911-1918.
Zoete V, Cuendet MA, Grosdidier A, Michielin O. SwissParam: a fast force field
generation tool for small organic molecules. J Comput Chem 2011,32:2359-2368.
Lill MA, Danielson ML. Computer-aided drug design platform using PyMOL. J Comput
Aided Mol Des 2011,25:13-19.
Chen X, Shi D. Synthesis and biological activity of 3-[(6-chloropyridin-3-yl)methyl]-6-
substituted-6,7-dihydro-3H-1,2,3-triazolo[4,5-d]-pyrimidin-7-imines J Heterocylic Chem
2
008,45:1493-1497.
4
2.
3.
Ried W, Laoutidis J. Synthese neuartiger triazoloanellierter 8-Azapurine Liebigs Annalen
der Chemie 1988,11:1107-1109.
4
Tanpure RP, Ren B, Peat TS, Bornaghi LF, Vullo D, Supuran CT, et al. Carbonic
anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic
halves of the carbonic anhydrase active site. J Med Chem 2015,58:1494-1501.
4
4.
5.
Thakur PK, Kumar J, Ray D, Anjum F, Hassan MI. Search of potential inhibitor against
New Delhi metallo-beta-lactamase 1 from a series of antibacterial natural compounds. J
Nat Sci Biol Med 2013,4:51-56.
4
Naz F, Shahbaaz M, Bisetty K, Islam A, Ahmad F, Hassan MI. Designing New Kinase
Inhibitor Derivatives as Therapeutics Against Common Complex Diseases: Structural
Basis of Microtubule Affinity-Regulating Kinase 4 (MARK4) Inhibition. OMICS
2
015,19:700-711.
4
4
6.
7.
Naz F, Shahbaaz M, Khan S, Bisetty K, Islam A, Ahmad F, et al. PKR-inhibitor binds
efficiently with human microtubule affinity-regulating kinase 4. J Mol Graph Model
2
015,62:245-252.
Naz H, Jameel E, Hoda N, Shandilya A, Khan P, Islam A, et al. Structure guided design
of potential inhibitors of human calcium-calmodulin dependent protein kinase IV
containing pyrimidine scaffold. Bioorganic & Medicinal Chemistry Letters 2016:(In
Press).

48.
49.
50.
51.
52.
53.
54.
55.
Aneja B, Irfan M, Hassan MI, Prakash A, Yadava U, Daniliuc CG, et al. Monocyclic
beta-lactam and unexpected oxazinone formation: synthesis, crystal structure, docking
studies and antibacterial evaluation. J Enzyme Inhib Med Chem 2015:1-19.
Hoda N, Naz H, Jameel E, Shandilya A, Dey S, Hassan MI, et al. Curcumin specifically
binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and
molecular dynamics simulation studies. J Biomol Struct Dyn 2015:1-13.
Anwer K, Sonani R, Madamwar D, Singh P, Khan F, Bisetty K, et al. Role of N-terminal
residues on folding and stability of C-phycoerythrin: simulation and urea-induced
denaturation studies. J Biomol Struct Dyn 2015,33:121-133.
Devika NT, Amresh P, Hassan MI, Ali BM. Molecular modeling and simulation of the
human eNOS reductase domain, an enzyme involved in the release of vascular nitric
oxide. J Mol Model 2014,20:2470.
Haque MA, Zaidi S, Ubaid-Ullah S, Prakash A, Hassan MI, Islam A, et al. In vitro and in
silico studies of urea-induced denaturation of yeast iso-1-cytochrome c and its deletants
at pH 6.0 and 25 degrees C. J Biomol Struct Dyn 2015,33:1493-1502.
Hassan MI, Kumar V, Somvanshi RK, Dey S, Singh TP, Yadav S. Structure-guided
design of peptidic ligand for human prostate specific antigen. J Pept Sci 2007,13:849-
8
55.
Khan FI, Shahbaaz M, Bisetty K, Waheed A, Sly WS, Ahmad F, et al. Large scale
analysis of the mutational landscape in beta-glucuronidase: A major player of
mucopolysaccharidosis type VII. Gene 2015.
Khan P, Parkash A, Islam A, Ahmad F, Hassan MI. Molecular basis of the structural
stability of hemochromatosis factor E: A combined molecular dynamic simulation and
GdmCl-induced denaturation study. Biopolymers 2015.

Figure-1: Fluorescence emission spectra of (A) CAIX with TPUI, (B) Plot of log (Fo-F)/F vs log
Q] for quenching of CAIX by TPU 1, (C) CAIX with TPUII, (D) Plot of log (Fo-F)/F
[
vs log [Q] for quenching of CAIX by TPUII.
Figure-2: Fluorescence emission spectra of (A) CAII with TPUI, (B) Plot of log (Fo-F)/F vs log
[
Q] for quenching of CAII by TPU 1, (C) CAII with TPUII, (D) Plot of log (Fo-F)/F
vs log [Q] for quenching of CAII by TPUII.
Figure-3: (A) Cartoon view of TPUI docked with CAIX. (B) Active site residue interactions with
TPUI. Residues are shown with stick and TPUI is shown with ball and stick model.
Hydrogen bonds are shown as broken lines (black). (C). Chemical structure of TPU
scaffold.
Figure-4: (A) Cartoon view of TPUI docked with CAII. (B) Active site residues interaction with
TPUI. Residues are shown with stick and TPUI is shown with ball and stick model.
Hydrogen bonds are shown as broken lines (black).
Figure-5: Surface representation of CAII (A) and CAIX, (B) docked with TPUI. Hydrophobic
core of active site is colored with element.
Figure-6: (A) Plot of time versus RMSD trajectory of isozyme CAIX (blue) and CAIX-TPUI
complex (red). (B) Time evolution of radius of gyration (Rg) values during 40,000 ps
(
40 ns) of MD simulation. The Rg plot for isozyme CAIX is shown with color black
and CAIX-TPUI complex is shown with color red. (C). RMSF plot for isozyme CAIX
blue) and CAIX-TPUI complex (red). (D) Accessible surfaces area (ASA) plot for
isozyme CAIX (blue) and CAIX-TPUI complex (red).
(

Figure 1.

Table 1: Docking score of TPUI and TPUII
CA IX
CA IX
Binding affinity (ΔG) Estimated K
i
Binding affinity (ΔG) Estimated K
i
TPU I
-10.89 kcal/mol
-10.26 kcal/mol
0.032 μM
1.67 μM
-9.47 kcal/mol
-8.53 kcal/mol
87.32 μM
192.6 μM
TPU II
Table 2: Molecular docking binding scores and binding interactions of compounds TPUI within
the CAIX and CAII active site (<5Å).
Residual interaction of PTU I with CAIX
H-bonds Hydrophobic
His 94, His119, Trp5, Leu98, Val121,
Thr199, Thr200 Val143, Leu198, Trp209 Thr200, Asn244
Residual interaction of PTU I with CAII
H-bonds Hydrophobic
Tyr7,Asn67,His96, Ala65, Phe66, Trp97,
Leu98, Val121, Val143,
Leu204,Val207, Trp209
Docking score (ΔG) : -10.89 kcal/mol
Docking score (ΔG) : -9.47 kcal/mol