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Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors

of Human P-glycoprotein

Article in Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) · May 2020
DOI: 10.2174/1871520620666200516144403

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RESEARCH ARTICLE
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Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors of

Human P-glycoprotein The journal for in-depth reviews and high quality research
papers on Anti-Cancer Agents

Saira Farman1, Aneela Javed2, Arshia3, Khalid M. Khan3,5, Abdul Nasir4, Asif Ullah Khan1,
Muhammad A. Lodhi1, Humaira Gul6, Faisal Khan1, Muhammad Asad1 and Zahida Parveen1,*

1
Department of Biochemistry, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan; 2Atta-Ur-Rehman School of
Anti-Cancer Agents in Medicinal Chemistry

Applied Biosciences, National University of Science and Technology (NUST), Sector H-12, Islamabad, Pakistan; 3H.E.J. Research
Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan;
4
Synthetic Protein Engineering Lab (SPEL), Department of Molecular Science and Technology, Ajou University, Suwon 443-749,
South Korea; 5Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin
Faisal University, P.O. Box 31441, Dammam, Saudi Arabia; 6Department of Botany, Abdul Wali Khan University, Mardan, Khyber
Pakhtunkhwa, Pakistan

Abstract: Background: Human P-glycoprotein (P-gp) is a transmembrane protein that belongs to the ATP-
Binding Cassette (ABC) transporters family. Physiologically, it exports toxins out of the cell, however, its over-
expression leads to the phenomena of Multidrug-Resistance (MDR) by exporting a diverse range of compounds,
which are structurally and chemically different from each other, thus creating a hurdle in the treatment of vari-
ous diseases including cancer. The current study was designed to screen benzophenone sulfonamide derivatives
as a class of inhibitors and potential anticancer agents for P-gp.

Methods: A total number of 15 compounds were evaluated. These compounds were screened in daunorubicin
efflux inhibition assays using CCRF-CEM Vcr1000 cell line that overexpressed human P-gp. Cytotoxicity assay
ARTICLE HISTORY was also performed for active compounds 11, 14, and 13. These scaffolds were then docked in the homology
model of human P-gp using mouse P-gp as a template (PDB ID: 4MIM) and the recently published Cryo Elec-
tron Microscopy (CEM) structure of human mouse chimeric P-gp to find their interactions with specified resi-
Received: July 13, 2019 dues in the binding pocket. Analysis was performed using Labview VI and Graph pad prism version 5.0.
Revised: February 24, 2020
Accepted: March 08, 2020
Results: Results revealed the potency of all these compounds in low nanomolar range whereas, compound 14
was found to be most active with IC50 value of 18.35nM±4.90 followed by 11 and 13 having IC50 values of
DOI:
10.2174/1871520620666200516144403 30.66nM±5.49 and 46.12nM±3.06, respectively. Moreover, IC50 values calculated for 14, 11 and 13 in cytotox-
icity assay were found to be 22.97µM±0.026, 583.1µM±0.027 and 117.8µM±0.062, respectively. Docking
results showed the interaction of these scaffolds in transmembrane helices (TM) where Tyr307, Tyr310, Tyr953,
Met986 and Gln946 were found to be the major interaction partners, thus they might play a significant role in
the transport of these scaffolds.
Conclusion: Benzophenone sulfonamide derivatives showed IC50 values in low nanomolar range comparable to
the standard inhibitor Verapamil, therefore they can be good inhibitors of P-gp and can serve as anticancer
agents. Also, they have shown interactions in the transmembrane region sharing the same binding region of
verapamil and zosuquidar.

Keywords: P-gp, benzophenone sulfonamide, docking, inhibition, ABCB1, MDR.

1. INTRODUCTION as well as its involvement in various physiological processes i.e,

cellular xenobiotic defense has grabbed exceptional attention.
ATP-Binding Cassette (ABC) transporter proteins form the Among the chemically diverse substances which are substrates of
largest family of transmembrane proteins. They are named after the P-gp are included antibiotics, protease inhibitors, chemotherapeu-
sequence identity in their Nucleotide-Binding Domain regions tics and immunosuppressants [4, 5].
(NBDs). In humans, there are 48 ABC proteins, which are mainly
exporters. These are further divided into seven subfamilies desig- To understand the broad specificity of substrates for P-gp, bind-
nated A-G. Among these, three are Multidrug Resistance (MDR) ing site identification of both the substrates and inhibitors is of
proteins that include ABCB1 (P-gp), ABCG2 (BCRP) and ABCC1 much importance. This is also a very crucial step towards investi-
(MRP1) [1, 2]. Great advancement has been shown in the past sev- gating the drug transport mechanism. Although several groups have
eral years in order to study the role of MDR transporter proteins [3]. contributed very well towards this, resulting in diverse experimental
P-gp, due to its expression in excretory organs and cell membranes data but the number and location of the drug-binding sites are still
to be discovered. Earlier, one of the groups suggested a single large
binding pocket [6, 7]. Loo and Clarke also characterized P-gp for its
*Address correspondence to this author at the Department of Biochemistry, interaction and transport-ability in their cross-linking experiments.
Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan; The different patterns obtained from such experiments led to the
Tel: +92 342 6223824; E-mail: [email protected] conclusion that P-gp could actually hold a variety of substrates in

1875-5992/20 $65.00+.00 © 2020 Bentham Science Publishers

1740 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

its drug-binding pocket, which is enclosed in various domains of (3µM) and incubated at 37°C for 30min under continuous agitation.
the transmembrane region between the two halves through an in- Loaded cells were split into nine FACS tubes and then centrifuged
duced-fit mechanism [8, 9]. Later on, in order to further explore the (Eppendorf 5810 R) at 500g (6min, 4°C). The supernatant was re-
unusual behavior of P-gp, two binding sites were proposed and the moved and the cells were re-suspended in ice cold washing media
interactions among the P-gp substrates were described as coopera- (RPMI 1640 media containing 2% FCS, pH 7.4). Washing was
tive, competitive and non-competitive nature [10]. In addition, repeated and the pellets were kept on ice. Efflux media (2% FCS,
Parveen et. al., (2011) reported dual pseudo-symmetric drug trans- pH 7.4) was prepared and divided into nine eppendorf tubes (1mL
location pathways in P-gp by doing site-directed mutagenesis where each) and kept at 37°C. Eight concentrations (serial 1:3 dilution)
rhodamine123 prefers one site (site 1) and verapamil, vinblastine were evaluated for each inhibitor. Daunorubicin efflux was meas-
and propafenones go to the other site (site 2). This duality has also ured continuously for 5min at 37°C by Becton Dickinson FACS
been further elaborated by tyrosine mutations [11]. Calibur flow cytometer (Becton Dickinson, Islamabad, Pakistan).
In addition to binding site identification, another method that The excitation and emission wavelengths were 502nm and 588nm.
may help in reverting drug resistance property of P-gp is its modu- To generate the dose-response curve K (First order rate constant)
lation using low molecular weight compounds. Therefore, much values were plotted against inhibitor concentration and IC50 values
effort has been devoted to the development of inhibitors of P-gp were calculated using non-linear regression analysis in Graph Pad
transporter. The inhibition of transport by P-gp has also been em- Prism Version: 5.0.
ployed in numerous studies [12]. There are several drugs which
have reached clinical trials, however, due to toxicity reasons, their 3.2. Cytotoxicity Assay
use has been restricted [13-15]. Therefore, there is a need for new
The cytotoxicity assay was performed to evaluate the sensitivity
scaffolds that may better modulate P-gp and may also help in un-
of CCRF-CEM vcr1000 cells towards benzophenone sulfonamide
derstanding the molecular basis of drug-transporter interactions [16-
derivatives. CCRF-CEM vcr1000 cells were seeded at a density of
18]. In this regard, benzophenone sulfonamide derivatives can be
5000-10,000 cells per well in 96 well plate. Various concentrations
good alternatives.
of compounds were then added and incubated at 37°C, 5% CO2 for
Benzophenones are also known as diphenyl ketones. Besides 24 hours. Next day, 20µL of MTT reagent ((3-(4, 5-
their other biochemical properties, they have been observed to have dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added
antitumor activity [19]. Dihydroxy-4-methoxy benzophenone [20] at a concentration of 0.5mg/mL or 5mg/mL to each well and incu-
and 2-aminobenzophenone are derivatives of this class. Methoxy bated for 2-4 hours to allow MTT to get metabolized. After incuba-
at para position imparts a greater efficacy to the benzophenones tion, 100µL of DMSO (stop solution) was added to each well and
[21]. was allowed to stand for 10 min to dissolve the blue color. Absor-
Another class is of sulfonamide, which is further divided into bance was taken on ELISA at 450nm/520nm.
five subclasses. Various biologically active substances have a sul-
fonamide group [22-24]. Aromatic and hetero-aromatic sulfona- 3.3. In Silico Studies
mides have been found to inhibit the enzyme carbonic anhydrase, The in silico study comprised of homology modelling and mo-
thus showing antitumor activity [25]. The current study was de- lecular docking of a class of benzophenone sulfonamide deriva-
signed to screen benzophenone sulfonamide derivatives as inhibi- tives. The homology model of human P-gp was generated on the
tors for P-gp in daunorubicin efflux inhibition assays and the most basis of the crystal structure of mouse P-gp as a template (PDB ID:
active compounds in the cytotoxicity assay. Moreover, these com- 4MIM) because of its close similarity. Molecular docking was per-
pounds have been docked in the homology model of human P-gp formed both with the modelled human P-gp and the recently pub-
built on the template of mouse P-gp [26] and the recently published
lished CEM structure of human P-gp (PDB ID: 6FN4).
Cryo Electron Microscopy (CEM) structure of human P-gp [27] in
order to map the interactions of these compounds with residues of
3.4. Homology Modeling
human P-gp.
As the crystal structure of mouse P-gp was available, therefore,
2. MATERIALS AND METHODS homology model of human P-gp was generated using mouse P-gp
as a template (PDB ID: 4MIM). To build the model, the human P-
2.1. Inhibition Assays
gp sequence was taken from UniProt Database (Accession no:
2.1.1. Cell Line P08183), which was then aligned with mouse P-gp sequence using
BLASTp [30]. Both sequences showed 87% sequence similarity,
The resistant CCRF-CEM Vcr1000 cell line used in this study thus the mouse P-gp can be used as the best template to generate
was received from Prof. Dr. Volker Gekeler [28]. This cell line was homology model. A total of 100 models were generated using the
maintained in RPMI 1640 medium containing 10% FCS. Molecular Operating Environment (MOE). The best model was
2.1.2. Compounds selected for further studies.
A series of synthetic benzophenone sulfonamide derivatives (n 3.5. Molecular Docking
= 15) synthesized and recently reported was tested in this study
with respect to p-gp inhibitory potential. Structures of all these The modeled human P-gp was then energy minimized and pro-
synthetic compounds were elucidated by 1H-NMR, 13C-NMR, tonated to be used for molecular docking analysis. The drug-
EIMS and FAB spectroscopic techniques [29]. binding residues of the transmembrane region, which were already
reported in the literature were selected in the sequence section in
3. EXPERIMENTAL MOE to define a binding pocket for molecular docking [31]. The
structures of low molecular weight benzophenone sulfonamide
3.1. Inhibition of Daunorubicin Efflux derivatives (n=15) were drawn in Chemdraw. Compounds library
Cells were harvested at a density of 1x106 cells/mL/data point was generated by converting the structures to the 3D saved in .mol
and centrifuged at 500g (6min, 4°C). The supernatant was removed format and docked to P-gp with a selection of the total number of
by aspiration and the cells were re-suspended in loading media thirty conformations for each compound. Top poses were selected
(RPMI 1640 containing 10% FCS, pH7.8) containing daunorubicin mainly on docking score and molecular interactions.
Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 1741

4. RESULTS values were found to be 785.7±24 for 1 and more than 1000nM for
2 making it an inactive compound. Structural descriptions of 1 and
4.1. Inhibition of Daunorubicin Efflux 2 revealed the presence of additional -OH group in 2.
In the first series of experiments, benzophenone sulfonamide Compounds 3, 4, 7, 9, and 14 shared the same parental structure
derivatives (n=15) were evaluated for their inhibitory potential that was nitrobenzene sulfonohydrazide. The IC50 values for 3, 4, 7
using daunorubicin efflux inhibition protocol. A total number of and 9 were greater than 1000nM hence they were inactive. While
eight different concentrations of each inhibitor was used to generate compound 14 turned out to be the most active compound that
a dose-response curve. Graphs were plotted using log inhibitor showed the least IC50 value of 18.35±4.90nM. When structural
concentration on X-axis and first-order rate constants (K) on Y- comparison was made, it revealed that all these five compounds
axis. Representative curves of three most active compounds are have -NO2 group at different positions, which influences the activ-
shown in Fig. (1a-c), where circles represent the data points and ity in a prominent way. This can be seen clearly in 7 that possesses
lines show a trend in the activity, which increased with the increase -NO2 at ortho position, thus being highly inactive. However, the
in concentration until it reached maxima. The best comparison was activity was resumed in 3 and 4 by introducing a -NO2 group at
made on the basis of IC50 values, which were calculated from dose- meta and para positions and they also had an additional dimethyl-
response curves of each compound using non-linear regression amino group in their structure. Moreover, compound 14 gained much
analysis in graph pad prism Version: 5.0 and are shown in Table 1 more activity (18.35nM) that had -NO2 group at meta position and
along with structures of compounds. additionally had para -Cl group, which was lacking in all others.
Fifteen benzophenone sulfonamide derivatives which were For compounds 5 and 6, IC50 values were found to be 248±
evaluated in the current study were categorized into subclasses on 49nM and 100.4±0.02nM, respectively, showing about 2.5-folds
the basis of their parental structures for clarity. The simplest struc- difference in the activity. There was a -OMe (methoxy group) instead
ture among these derivatives was of compound 8 that is a sulfono- of -NO2 in compound 5. There are two compounds in this library
hydrazide having IC50 value of 811.6±16.55nM. All the other four- (benzophenone sulfonamides) that possess naphthalene-sulfono-
teen compounds were derivatives of 8 with different modifications, hydrazide as their core structure that include 12 and 13. IC50 values
which showed a range of activities with different IC50 values indi- are 324.2±17.04 and 46.12±3.06nM, respectively, with a 7-fold
cating the variation due to the structural differences of these low difference in the activity. Compounds 12, 13, are naphthalene-2-
molecular weight compounds. Compounds 1 and 2 contain dimeth- sulfonohydrazide and naphthalene-1-sulfonohydrazide, respec-
ylbenzene-sulfonohydrazides as their parental structure. Their IC50 tively. Compounds 10 and 11 are chlorobenzene-sulfonohydrazides.

(a) (b)
First order rate constant (k)

First order rate constant (k)

0.010 0.015

0.008
0.010
0.006

0.004
0.005
0.002

0.000 0.000
0 1 2 3 4 5 0 2 4 6
Compound 14 conc. (m
mM) Compound 13 conc. (mM)

(c)
First order rate constant (k)

0.015

0.010

0.005

0.000
0 1 2 3 4 5
Compound 11 conc. (mM)

Fig. (1). Inhibition of daunorubicin transport by 11, 13 and 14. (a) Structure and dose-response curve of 11 (IUPAC: 3, 5-dichloro-N'-(diphenylmethylene)-2
hydroxybenzenesulfonohydrazide). (b) Structure and dose-response curve of compound 13 (IUPAC: 5-(dimethylamino)-N'-(diphenylmethylene) naphthalene-
1-sulfonohydrazide. (c) Structure and dose-response curve of compound 14 (IUPAC: 4-chloro-N'-(diphenylmethylene)-3 nitrobenzene sulfonohydrazide. The
graphs represent the inhibitory activity of 11, 13 and 14. Different concentrations (nM) of these scaffolds were used. The graphs were plotted using log inhibi-
tor concentration on X-axis and fractional inhibition of daunorubicin transport on Y-axis. Measurements were taken on FACS caliber Flow cytometer. Solid
lines represent hyperbolic dose-response curves, which were generated in graph pad prism using non-linear regression analysis, while circles represent the
individual data points, which are average of two independent experiments performed in duplicate. IC50 value was calculated from the curve as 50% occupancy
values and are represented as mean ± SEM (shown in Table 1). (A higher resolution / colour version of this figure is available in the electronic copy of the
article).
1742 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

Table 1. IC50 values for daunorubicin efflux inhibition by benzophenone sulfonamide derivatives. IC50 values were calculated from dose-response
curves all 15 compounds as 50% occupancy values and are represented as mean ± SEM. Experiments were performed in duplicate.

Comp. No. Structures I.U.P.A.C. Name IC50 ± SEMa [nM]

4-Chloro-N'-(diphenylmethylene)-
O H 2,5dimethylbenzenesulfonohydrazide
785.7±24
1 N
S N
O
Cl
OH

4-Chloro-N'-((4-hydroxyphenyl)(phenyl)methylene)-
>1000
2 2,5dimethylbenzenesulfonohydrazide
O H
N
S N
O
Cl

((4(Dimethylamino)phenyl)(phenyl)methylene)-3-
3 nitrobenzenesulfonohydrazide >1000
O O H
N
- N+ S N
O
O

N'-((4-(Dimethylamino)phenyl)(phenyl)methylene)-4-
>1000
4 O H nitrobenzenesulfonohydrazide
N
S N
O
-
O
N+
O

N'-((4-(Dimethylamino)phenyl)(phenyl)methylene)-4- 248 ± 49
5 H
O methoxybenzenesulfonohydrazide
N
S N
O
O

N'-((4-(Dimethylamino)phenyl)(phenyl)methylene)
6 100.4±1.01
H benzenesulfonohydrazide
O N
S N
O

O H
N
7 S N N'-(Diphenylmethylene)-2-nitrobenzenesulfonohydrazide >1000
O
O-
N+
O
(Table 1) contd….
Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 1743

Comp. No. Structures I.U.P.A.C. Name IC50 ± SEMa [nM]

8 H O N'-(Diphenylmethylene)methanesulfonohydrazide 811.6±16.55
N
N S
O

O H
N
9 S N N'-(Diphenylmethylene)-4-nitrobenzenesulfonohydrazide >1000
O
-O
N+
O

O H
10 N 2,4-Dichloro-N'-(diphenylmethylene)benzenesulfonohydrazide >1000
S N
O
Cl Cl

30.66±5.49

O H
N 3,5-Dichloro-N'-(diphenylmethylene)-2-
11 Cl S N hydroxybenzenesulfonohydrazide
O
OH
Cl

12 O H N'-(Diphenylmethylene)naphthalene-2-sulfonohydrazide 324.2±17.04
N
S N
O

O H
N 5-(Dimethylamino)-N'-(diphenylmethylene)naphthalene-1-
13 S N 46.12±3.06
sulfonohydrazide
O

O O H
14 N 4-Chloro-N'-(diphenylmethylene)-3-nitrobenzenesulfonohydrazide 18.35±4.90
-O N+ S N
O
Cl

O H
15 N N'-(Diphenylmethylene)-4-methoxybenzenesulfonohydrazide 312.1±5.58
S N
O
O
SEMa means Standard Error of the Mean; NA.b means Not Active.

The IC50 value of 12 is greater than 1000nM, which makes it an groups are present at ortho and para positions in compound 10,
inactive compound while that for 13 is 30.66±5.49nM. Chlorine while compound 11 has -OH group at ortho and -Cl at meta position.
1744 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

Fig. (2). Cytotoxicity assay of active compounds 11, 13 and 14. The composite graph was plotted using log inhibitor concentration on X-axis and absorbance
on Y-axis. Solid lines represent compound 11 (blue), 13 (red) and 14 (yellow). Circles, squares and triangles represent the individual data points of the respec-
tive curves. These curves were generated in Graph Pad Prism 6.using non-linear regression analysis. Data were fit to sigmoidal dose–response curves, which
are average of two independent experiments performed in duplicate. Different concentrations (0.09 to 20µM) of these scaffolds were used. Measurements were
taken on ELISA. IC50 values were calculated from these curves as 50% occupancy values and are represented as mean ± S.E. (A higher resolution / colour
version of this figure is available in the electronic copy of the article).

The last compound of this series is 15, which has -OMe attached to 84.3% of residues in the most favored region, 11.5% in the allowed
benzene sulfonohydrazide and its IC50 value is 312.1 ± 5.58nM. The and 4.3% in the outlier region. Further validation of the model was
above results showed that compound 14 is the most active among the done by Ucl-Doe Errat2 software and the overall quality of the
benzophenone sulfonamides followed by 11 and 13. model was 91.086%. The results are shown in Table 2. Therefore,
the model was used for further docking studies.
4.2. Analysis of Cytotoxicity Assay
The reported residues of literature were selected to describe a
The above three active compounds 11, 13 and 14 were further binding pocket for these fifteen compounds. Energy minimization
evaluated for their cytotoxic properties using MTT ((3-(4,5- was done using LigX, (minimization tool) of MOE. All fifteen ben-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye. P-gp zophenone-sulfonamide derivatives were then docked into the mod-
overexpressing cell line CCRF-CEM Vcr-1000 was incubated with eled human P-gp using MOE. Different conformations of each
different concentrations of these active compounds overnight and docked compound showed their molecular interactions with the
further stained with MTT. A composite dose-response curve for residues in the Transmembrane region (TM) of human P-gp model.
compounds 11, 13 and 14 was generated with log inhibitor concen- All of these compounds have been described on the basis of the
tration on X-axis and absorbance on Y-axis as shown in Fig. (2). residues with which they interact in different poses. Met68 and
IC50 values calculated using non-linear regression analysis were Met949 are common to all the compounds. Additionally, compound 1
found to be 22.97µM±0.026, 583.1µM±0.027 and 117.8µM±0.062 interacted with Leu975 and Ser979. Compound 2 had two new
for 14, 11 and 13, respectively. It can be noticed that these com- interactions with Tyr953 and Met986 along with those mentioned
pounds were specifically active in a low nanomolar range in daun- for 1. Compound 3 had been found to interact with Leu332, Phe335,
orubicin efflux inhibition study. However, in connection to the Phe728, Tyr953 and Ser979 while compound 4 interacted with
cytotoxicity assay, it kills 50% of the cells at a very high concentra- Tyr310, Phe728, Tyr953, Leu975, Ser979 and Met986.
tion clearly indicating that it is bearable at physiological conditions.
Interactions for compound 5 were found with Phe335, Phe942
4.3. In Silico Analysis of Benzophenone Sulfonamide Derivatives and Ser979, however, compound 6 interacted with Tyr310, Leu332,
Phe335, Tyr953 and Leu975. Compound 7, which is the parent
4.3.1. Templates Used for Molecular Docking
structure, showed interactions with Leu67 and Tyr953. Compound
Two templates were used in the current study for molecular 8 showed Tyr953 and Cys956 as directly interacting partners. A
docking of a class of benzophenone sulfonamide derivatives in total of three affinities were shown by compound 9 including
order to map the interactions of these scaffolds in the binding Leu332. Compounds 10 and 15 showed two common residues
pocket of human P-gp. The crystal structure of mouse P-gp (PDB: Leu332 and Tyr953 alongwithPhe971, Phe728, Leu332, Cys956
4M1M) [26] was mostly used as a template for human P-gp there- and Phe978 in compound 10. Compound 12 had been found to
fore, benzophenone sulfonamides were initially docked into the interact with Met949, Cys956, Tyr953 and Phe978.
homology model based on mouse template. Secondly, the recently Compound 13 was the third most active compound of benzo-
published CEM structure of human-mouse chimeric-P-gp was used phenone sulfonamide derivatives. Docking results of this ligand
directly to dock the same class of compounds [27].

4.4. Molecular Docking Analysis of Mouse-Based Human P-gp Table 2 Quality parameters of human P-gp homology model based
Homology Model on mouse template.

The structure of mouse P-gp (PDB: 4M1M) can provide great Ramachandran Plot Homology Model Based on Mouse-Template
help towards building a homology model for human P-gp due to its
87% sequence identity and 94% similarity with human P-gp. There- Favoured region 84.3%
fore, in the present study, crystal structure of mouse Pgp was used Allowed region 11.5%
as a template to build the homology model of human P-gp. To vali-
Outlier region 4.3%
date the model, Ramachandran plot was generated, which showed
Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 1745

Fig. (3). Interaction diagram (2D) of modeled human P-gp with docked compound 11. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).

Fig. (4). Interaction diagram (2D) of human-mouse chimeric P-gp with docked compound 11. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).
1746 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

Fig. (5). Interaction diagram (2D) of modeled human P-gp with docked compound 13. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).

Fig. (6). Interaction diagram (2D) of human-mouse chimeric P-gp with docked compound 13. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).
Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 1747

(a) (b)
Phe Phe Met Tyr
372 726 958 853
Val
Met 904
949 Ser
Leu
Met 902 Tyr
67
- 68 951
Cl O

N + Cl
Leu
O Gln
O 905
545 O
+
Leu N S N
352 N N
Leu O
N S O 905 Met
H O-
H 75
Phe O
976 Leu Met
Phe
Tyr 352 25
903
953

Val
Met 976
989 Phe
Val Phe 901
Met 71 Phe 228
68 335

(c)
Leu
875
Val
Phe 904
908
Met Ile
943 329
Cl
Phe
335 O -
+ O Phe
N S N 328
N
H O O
Phe
Tyr 992 Leu
953 332
Met
75
Met
68
Gln Val
946 72

Leu
67

polar sidechain acceptor solvent residue arene-arene

acidic sidechain donor metal complex arene-cation
basic backbone acceptor solvent contact
greasy backbone donor metal contact
proximity ligand receptor
contour exposure contact

Fig. (7). Interaction diagram (2D) of modeled human P-gp with docked compound 14. (A higher resolution / colour version of this figure is available in the
electronic copy of the article).
(a) (b)
Met
Leu 65 Ile
64 340 Val
952 Leu
332
Met Met
67
Phe 68 Leu Phe
37 975 714
Gln
596 Phe
- 336
O H O O
O
O N
+ Phe +
S N - 205 N S N
O O
N N O
H
Tyr Tyr Cl Ile
Phe Cl 736
560 953 232
Tyr
953
Met Phe Phe
949 653 Phe
Ser Leu 543
Met Met 71
993 335 Phe
906 956 Ser
978 929

Val Met
992 992

(c) (d)
Leu Phe
Leu
64 983
Met 224
949
Leu
332 Met Asn
Gln
68 842
Met 725
67
O
Phe Phe Ile
71 536 340 + -
Phe
N O
728 O
Phe N
752
O Phe N S Cl
O 143 H
O
- N+ S O
O
N N
H Ile
340 Phe
Cl 503
H Ala
957 Tyr Val
Leu Phe 996
Met Tyr 307
905 343 Phe 336 380
Phe
583 335
Tyr
953 Gln
Phe
978 990

polar sidechain acceptor solvent residue arene-arene

acidic sidechain donor metal complex arene-cation
basic backbone acceptor solvent contact
greasy backbone donor metal contact
proximity ligand receptor
contour exposure contact

Fig. (8). Interaction diagram (2D) of human-mouse chimeric P-gp with docked compound 14. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).
1748 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

Table 3. List of benzophenone sulfonamide derivatives with their LogP values and docking scores in modeled human P-gp and human-mouse
chimeric P-gp structure.

Compound LogP Modeled Human P-gp Human-Mouse Chimeric P-gp

No. Docking Score Docking Score

1 4.69 -9.30 -8.76

2 4.83 -10.05 -10.94
3 3.39 -10.10 -9.98
4 3.39 -10.93 -11.95
5 3.49 -10.31 -10.48
6 3.48 -10.50 -10.55
7 3.33 -12.35 -11.99
8 1.99 -10.78 -10.26
9 3.33 -12.19 -9.93
10 4.72 -13.23 -10.74
11 4.43 -13.22 -10.63
12 4.57 -11.23 -9.75
13 4.64 -14.10 -10.80
14 3.98 -12.21 -11.64
15 3.43 -12.88 -9.99

revealed Phe978, Leu975, Met68, Tyr953 and Val982 involved in total of eight interactions were found. Tyr307 along with Gln725
the binding. Compound 11 was the second most active compound and Tyr953 together with Gln946 were found to be in interaction.
of benzophenone sulfonamide derivatives, which interacted with six Phe71 and Phe336 alongwith methionines Met67 and 68 partici-
residues Leu332, Gln946, Phe971, Leu975, Met949 and Tyr953. pated in ligand binding.
Compound 14 was the most active of benzophenone sulfonamide
When compound 9 was docked into the human-mouse chimeric
derivatives having one -NO2 and one -Cl attached to its benzene
P-gp structure, different docking poses were obtained. The most
ring. Docking poses showed Met75, Tyr953, Ser952 and Met949
noticeable residues were Phe336, Phe983, Met67, Tyr953 and
involved in interactions.
Gln946. Molecular docking of compound 10 in human-mouse chi-
meric P-gp revealed Tyr953, Tyr310, Gln725, Phe336 and Phe983.
4.5. Molecular Docking Analysis using Human-Mouse Chimeric
Compound 12 in the binding cavity of human-mouse chimeric P-gp
P-gp Structure
resulted in a single interaction with Tyr310. Docking of compound
In another series, the CEM structure of human-mouse chimeric 15 provided different conformations showing Phe71, Phe336,
P-gp (PDB: 6FN4) published recently by Alam et al., (2018) [28] Phe983, Gln946 and Met986 and Gln946.
was used to further validate the interactions of benzophenone sul- Compound 13 showed possible interactions with Phe336,
fonamide derivatives in the binding pocket. This chimeric structure Leu64, Met68 and Tyr310. Docking of compound 11 revealed pos-
was actually a hybrid made up of human and mouse P-gp (ABCB1- sible interactions with three residues Met67, Tyr310 and Phe336.
HM). This structure was used directly for the molecular docking of Docking of the most active compound 14 established seven promi-
benzophenone sulfonamide derivatives. The group reported zosu- nent interactions with Phe336, Phe983, Tyr953, Met68, Gln725,
quidar binding pocket, which includes Trp232, Ala233, Phe305, Asn842 and Leu975 (Figs. 3-8).
Ile306, Phe336, Gln725, Glu875, Leu879, Gln946, Tyr950, Tyr953,
Phe983, Met986 and Val991. 5. DISCUSSION
Compound 1 was observed to interact with six residues of the
A research of 4 decades since the discovery of P-gp revealed
human-mouse chimeric P-gp. His60, Gln195, Gln 946, Ser344,
that its over-expression leads to multidrug resistance in different
Glu875 and Met986. Interactions of compound 2 in the binding
types of diseases including cancer [32]. As a result, cancerous cells
cavity of human-mouse chimeric P-gp were found with His60,
became resistant to a huge list of various available anticancer drugs
Thr945, Tyr310, Glu946, Ser344 and Met986, Glu875 and Phe942.
[33]. The P-gp inhibitors, which are identified till now and used in
Compound 3 showed five interactions with binding site residues
numerous MDR diseases in clinical procedures, have unluckily very
His60, Met67, Gln946, Tyr950 and Met986. Compound 4 in differ-
limited success [34, 35]. Besides these facts, researchers all over the
ent docking poses was found to interact with His60, Gln195,
world are still trying to design and screen inhibitors that could tar-
Glu875, Gln946 and Met986. Molecular docking interactions of
get P-gp and reverse resistance [36]. Various first generation and
compound 5 in the binding pocket of human P-gp were found with
second generation inhibitors have been made and tested, resulting
glutamines, Gln195, Gln347, Gln946, Glu875 and His60.
in limited success therefore, the third-generation P-gp inhibitors
Different conformations obtained from docking of compound 6 like elacridar, tariquidar and zosuquidar are in clinical trials in order
in the structure of human-mouse chimeric P-gp revealed Leu64, to fulfill the drug delivery to the tissues [37]. There is another cate-
Met986, Glu875 and Gln946 residues. Docking poses of compound gory of inhibitors like Nobiletin, which has been proposed to be
7 showed interaction with two of the most important tyrosines re- used in combination with other drugs for the treatment of MDR
ported in literature i.e., Tyr310 and Tyr953. The other two residues [38]. Klepsch et al., (2011) used the propafenone type of inhibitors
His60, Gln725 and Gln946 were also found. A well-interacted pat- for P-gp inhibition [39]. In another dual inhibition type of study, a
tern of compound 8 with the surrounding residues was observed. A series of fifteen propafenone analogues were evaluated and they
Benzophenone Sulfonamide Derivatives as Interacting Partners and Inhibitors Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 1749

Table 4. Comparison of binding pocket residues for benzophenone sulfonamide derivatives and standard known inhibitors in modeled human P-
gp structure and human-mouse chimeric P-gp.

1. Verapamil Leu65, Met69, Phe72, Thr199 , Ser222 , Ile306, Tyr307 , Phe336, Leu339, Ile340, Ala342,
Phe343 , Phe728 , Ala841, Ile868, Gly872 , Phe942, Thr945, Tyr953, Phe957 , Leu975, Phe978,
Val981, Val982, Phe983, Gly984, Ala985, Met986

2. Zosuquidar Trp232, Ala233, Ile306, Phe336, Phe303, Gln725, Glu946, Phe983, Tyr950, Tyr953, Leu879,
Gln990, Val991, Glu875, Met986

3. Interacting residues in modeled human P-gp Met68, Met75, Tyr310, Leu332, Phe335, Phe728, Phe942, Gln946, Met949, Ser952, Tyr953,
Cys956, Phe971, Leu975, Phe978, Ser979, Val982, Met986

4. Interacting residues in human-mouse chimeric P-gp His60, Tyr307, Tyr310, Phe336, Gln725, Met986, Gln946, Glu875, Tyr953

5. Common interacting residues Tyr307, Tyr310, Tyr953, Met986, Gln946.

concluded that in case of P-gp, the logP values of compounds are actions as a side chain acceptor with all the three active scaffolds
highly influenced as compared to BCRP [40]. (14, 13, and 11).
There are certain properties which must be considered for When these results were compared with the literature, it had
molecules to be drug-like e.g. logP values and molecular weight. been noticed that several groups reported the importance of tyrosine
Practically, most of the chemical substances which are routinely residues for P-gp-substrate interactions. A study demonstrated the
evaluated in clinical trials have much greater values of these two importance of domain interface tyrosine residues for the interaction
properties. Therefore, compounds having low lipophilicity and also of small molecules with P-glycoprotein. One of the tyrosines Y953
molecular weight (logP< 5; molecular weight < 500 Da) are re- (TM11) had been observed to form a hydrogen bond with rho-
quired for screening as inhibitors of human P-gp. damine123 and propafenone analogs [42]. This is consistent with
our docking results as tyrosine Y953 greatly interacted with the
In the present study, we evaluated a library of fifteen synthetic
benzophenone sulfonamide class of scaffolds.
compounds of benzophenone sulfonamide derivatives. These are
small compounds with low molecular weights ranging from 274- Pace et al. (2001) reported the role of tyrosines in active site
429g/mol. The logP values range from 1.99-4.83, as shown in Table 3. forming hydrophobic, hydrogen-bonding, π-π, and π-cation interac-
Both these values are lower than verapamil, which is the standard tions, thus providing stability to protein [43]. In our study also,
inhibitor having a molecular weight of 454.61g/mol and logP value important interactions were noticed with the tyrosine Tyr307,
of 5.04 due to which they are suitable as drug-like compounds for Tyr310 and Tyr953. The broad substrate specificity of P-gp makes
P-gp inhibition. Therefore, benzophenone sulfonamide derivatives it clear that there is no unique pharmacophore to describe the mo-
were screened in cell culture inhibition assays using CCRF-vcr1000 lecular features of a chemical entity to be recognized by P-gp.
over-expressed cell line to test their inhibitory potential on P-gp. Therefore, studies have reported multiple pharmacophores for P-gp
Three of these fifteen compounds including 14 (18.35±4.90nM), 13 even though they have in common, a certain degree of hydropho-
(46.12±3.06nM), and 11 (30.66±5.49nM) were found to be more bicity and the presence of hydrogen bond acceptors (ether, car-
active on the basis of IC50 values. In the future, these compounds bonyl, hydroxyl, tertiary amino groups). The presence of many
can be used as anticancer agents in reversing multidrug resistance, nonpolar residues facing the central cavity of P-gp, such as pheny-
thereby they might help in cancer treatment by targeting P-gp. lalanine, valine, leucine and isoleucine, as well as of residues able
to act as hydrogen bond donors, such as tyrosine and glutamine, are
In silico studies have been very helpful since the last 20 years consistent with the common features of multiple P-gp pharma-
describing the chemical substances whether they would bind with cophores. Seelig (1998) reported two P-gp pharmacophores based
P-gp or not. Properties like aromaticity, nitrogenecity and lipophil- on a comparison of 100 chemical compounds recognized as P-gp
icity can be better explained in this way [41]. Lipophilicity has also substrates [44].
been found related to the inhibitory activity. The benzophenone
sulfonamide derivatives were then evaluated in silico to find their Klepsch et al., (2011) used propafenone type of inhibitors for P-
gp inhibition. The homology model of human P-gp was built using
interaction with P-gp. These compounds were docked into the hu-
two templates, one of mouse (PDB: 3G5U) and another of Sav1866
man P-gp model by selecting the pocket, which is already reported
(PDB: 2HYD). For binding site identification, 5 propafenone ana-
for verapamil in the literature (Table 4). Interactions were found
logues i.e, GPV005, GPV186, GPV019, GPV062 and GPV366
with a number of residues including Met68, Met75, Tyr310,
were docked into the homology model of human P-gp. Y307 and
Leu332, Phe335, Phe728, Phe942, Gln946, Met949, Ser952,
Y310, according to the group, were found to have a critical role. In
Tyr953, Cys956, Phe971, Leu975, Phe978, Ser979, Val982 and
the case of 2HYD-P-gp, the RMSD clustering process showed fre-
Met986. Results showed that they interacted in the transmembrane quent interactions with the four residues Y307, Y310 (TM helix 5),
regions of P-gp, which is the drug binding site. Similarly, docking L724 (TM helix 7) and T769 (TM helix 8). Similarly, with 3G5U-
results of benzophenone sulfonamides with the human-mouse chi- pgp, one of the residues which showed greater interactions was
meric P-gp structure showed interactions with His60, Tyr307, Y310. The group proposed that if Y307 and Y310 would mutate
Tyr310, Phe336, Gln725, Met986, Gln946, Glu875 and Tyr953. with phenylalanine, 10-fold difference in activity of GPV005 and
Residues found common both in the modeled human P-gp and hu- GPV062 would be vanished [39]. Our results also revealed tyrosine
man-mouse chimeric structure were Tyr307, Tyr310, Tyr953, Y307 and Y310 as important interacting residues of the binding
Met986, Gln946 and Met 949. cavity, thereby being consistent with them.
These residues directly interacted with our active compounds Ma et al., (2015) performed a molecular docking study of no-
14, 13 and 11. When the type of interactions was considered, it had biletin into the homology modeled structure of P-gp. Results
been noticed that Tyr953 made polar H-bond interactions with showed the involvement of methoxyl group containing rings of
compounds 14 and 13, however, the same residues also had arene- nobiletin with the residues of the binding cavity. Predominantly,
arene interaction with compound 11. In addition to tyrosines hydrophobic interactions were observed with the residues of the
(Tyr307, Tyr310, Tyr953) Met949 also underwent prominent inter- binding cavity including Phe303, Tyr307, Tyr310, Leu332, Phe335,
1750 Anti-Cancer Agents in Medicinal Chemistry, 2020, Vol. 20, No. 14 Farman et al.

Leu336 and Leu339. Further, the methoxy and carbonyl groups of [4] Schinkel, A.H.; Jonker, J.W. Mammalian drug efflux transporters
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Not applicable. Freissmuth, M.; Ecker, G.F.; Chiba, P. Molecular dissection of dual
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Not applicable. tance. Nat. Rev. Cancer, 2005, 5(4), 275-284.
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review: Clinical relevance of drug drug and herb drug interactions
The data that support the findings of this study are available mediated by the ABC transporter ABCB1 (MDR1, P-glycoprotein).
from the corresponding author upon reasonable request. Oncologist, 2007, 12(8), 927-941.
http://dx.doi.org/10.1634/theoncologist.12-8-927 PMID: 17766652
[14] Khaleel, S.A.; Al-Abd, A.M.; Ali, A.A.; Abdel-Naim, A.B. Didox
FUNDING and resveratrol sensitize colorectal cancer cells to doxorubicin via
This study is funded by the Higher Education Commission activating apoptosis and ameliorating P-glycoprotein activity. Sci.
(HEC) Pakistan under the Project No: NRPU 3589. Rep., 2016, 6, 36855.
http://dx.doi.org/10.1038/srep36855 PMID: 27841296
[15] Qiu, Q.; Liu, B.; Cui, J.; Li, Z.; Deng, X.; Qiang, H.; Li, J.; Liao,
CONFLICT OF INTEREST C.; Zhang, B.; Shi, W.; Pan, M.; Huang, W.; Qian, H. Design, syn-
The author declares no conflict of interest, financial or other- thesis, and pharmacological characterization of N-(4-(2 (6,7-
Dimethoxy-3,4-dihydroisoquinolin-
wise. 2(1H)yl)ethyl)phenyl)quinazolin-4-amine derivatives: Novel in-
hibitors reversing P-glycoprotein-mediated multidrug resistance. J.
ACKNOWLEDGEMENTS Med. Chem., 2017, 60(8), 3289-3302.
http://dx.doi.org/10.1021/acs.jmedchem.6b01787 PMID: 28355069
Prof Dr. Peter Chiba (Medical University of Vienna, Austria) is [16] Xue, G.M.; Xia, Y.Z.; Wang, Z.M.; Li, L.N.; Luo, J.G.; Kong, L.Y.
highly acknowledged for providing plasmids and scientific discus- neo-Clerodane diterpenoids from Scutellaria barbata mediated in-
sions. hibition of P-glycoprotein in MCF-7/ADR cells. Eur. J. Med.
Chem., 2016, 121, 238-249.
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