Molecular mimics of classic p-glycoprotein inhibitors as multidrug resistance suppressors and their synergistic effect on paclitaxel

Article abstract of DOI:10.1371/journal.pone.0168938

P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells and considered as a main component of multidrug resistance (MDR) to chemotherapies. In this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide

Full text of DOI:10.1371/journal.pone.0168938

RESEARCH ARTICLE
Molecular Mimics of Classic P-Glycoprotein
Inhibitors as Multidrug Resistance
Suppressors and Their Synergistic Effect on
Paclitaxel
Moustafa E. El-Araby1,2*, Abdelsattar M. Omar1,3, Maan T. Khayat1, Hanan A. Assiri4,
Ahmed M. Al-Abd5,6
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi
Arabia, 2 Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University, Cairo,
Egypt, 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt,
4 Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia,
5 Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah,
Saudi Arabia, 6 Department of Pharmacology, Medical Division, National Research Centre, Cairo, Egypt
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* madaoud@kau.edu.sa
OPEN ACCESS
Abstract
Citation: El-Araby ME, Omar AM, Khayat MT, Assiri
HA, Al-Abd AM (2017) Molecular Mimics of Classic
P-Glycoprotein Inhibitors as Multidrug Resistance
Suppressors and Their Synergistic Effect on
Paclitaxel. PLoS ONE 12(1): e0168938.
P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells
and considered as a main component of multidrug resistance (MDR) to chemotherapies. In
this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide deriva-
tives) were synthesized aiming to develop a molecular framework that effectively sup-
presses MDR. When tested for their influence on Pgp activity, four compounds coded Cur1-
01, Cur1-12V, Curox-1 and Curox-3 significantly decreased remaining ATP concentration
indicating Pgp substrate site blocking. On the other hand, Cur-3 and Cur-10 significantly
increased remaining ATP concentration, which is indicative of Pgp ATPase inhibition. The
cytotoxicity of synthesized compounds was examined against Pgp expressing/highly resis-
tant colorectal cancer cell lines (LS-174T). Compounds Cur-1 and Cur-3 showed consider-
able cytotoxicity with IC50 values of 7.6 and 8.9 μM, respectively. Equitoxic combination (at
IC50 concentrations) of PTX and Cur-3 greatly diminished resistant cell clone from 45.7% to
2.5%, albeit with some drop in potency from IC50 of 7.9 nM to IC50 of 23.8 nM. On the other
hand, combination of PTX and the non-cytotoxic Cur1-12V (10 μM) significantly decreased
the IC50 of PTX to 3.8 nM as well as the resistant fraction to 16.2%. The combination test
was confirmed using the same protocol but on another resistant CRC cell line (HCT-116) as
we obtained similar results. Both Cur-3 and Cur1-12V (10 μM) significantly increased the
cellular entrapment of Pgp probe (doxorubicin) elevating its intracellular concentration from
1.9 pmole/cell to 3.0 and 2.9 pmole/cell, respectively.
doi:10.1371/journal.pone.0168938
Editor: Ashraf B. Abdel-Naim, Ain Shams
University, EGYPT
Received: September 24, 2016
Accepted: December 8, 2016
Published: January 9, 2017
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This Project was funded by the Deanship
of Scientific Research (DSR), at King Abdulaziz
University, Jeddah, Saudi Arabia, under grant no.
G-416-166-36. The authors, therefore,
acknowledge with thanks DSR for technical and
financial support, http://dsr.kau.edu.sa/Default.
aspx?Site_ID=305&Lng=EN. The funders had no
role in study design, data collection and analysis,
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P-GP Inhibitors and Paclitaxel Synergism
decision to publish, or preparation of the
manuscript.
Introduction
Resistance to chemotherapy has been standing as the major obstacle to declare a clear victory
in the fight with the “Emperor of All Maladies” [1, 2]. There are several mechanisms by which
tumor cells develop resistance towards cytotoxic drugs aiming to stay alive and continue their
malignant pattern of growth and proliferation [3, 4]. Multidrug resistance through efflux of
chemotherapeutic agents outside cancer cells is recognized as a major cancer resistance mech-
anism that is responsible for failure of treatment after initial rapid improvement [5]. When
patients are placed in a chemotherapy regimen, cancer cells strike back by over expression of
ATP-dependent Binding Cassette (ABC) transporter proteins. ABC is a superfamily of mem-
brane-bound transporters that are able to uptake xenobiotics and chemical substances unidi-
rectional from inside to outside the cell [6]. The earliest discovered and most studied ABC
transporter protein is the P-glycoprotein (Pgp); it is also known as multidrug resistance pro-
tein 1 (MDR-1), ATP-binding cassette sub-family B member 1 (ABCB1) or cluster of differen-
tiation 243 (CD243). Pgp was first identified by Victor Ling and coworkers in 1970s as a
protein responsible for multidrug permeability in Chinese hamster ovary cells [7, 8]. After
cloning of Pgp cDNA [9], its wild gene alleles (as well as mutant gene alleles) were found to be
amplified in tumor cells as a response to chemotherapeutic agents leading to development and
spread of MDR events within tumor cells [10].
Competing Interests: The authors have declared
that no competing interests exist.
The X-ray crystal structure of mouse Pgp protein (87% similarity to human Pgp), resolved
in 2009, was described as two trans-membrane domains each composed of six α-helices open-
ing inward during the relaxed state [11]. Each domain is linked in its intracellular face to a
nucleotide-binding domain (NBD) which is also known as the ATPase motif of the pump. The
surface between the two leaflets is wide and largely hydrophobic. Crystallographic data as well
as structure-binding relationship studies revealed a promiscuous substrate (inhibitor) binding
site inside the Pgp transmembrane part. It has great flexibility and also has larger than usual
numbers of specificity residues. Instead, it can be described as large and deep pocket sur-
rounded by clusters of hydrophobic residues. Therefore, it can accommodate less structurally
related compounds. This binding site was proved to recognize, bind and efflux more than 300
diverse organic compounds belonging to chemotherapeutic and non-chemotherapeutic classes
of drugs [12]. The Pgp efflux pump inhibitors may act through two different mechanisms: sub-
strate site blockage and ATPase inhibition. In recent years, Pgp allosteric inhibitors (ATPase
inhibitors) seemed more promising as several candidates (Tariquidar, Biricodar, Elacridar and
Zosuquidar) are advancing in clinical trials (Fig 1) [13]. There are some skepticism if any of
these compounds will eventually succeed to be the first-in-class MDR modulator to win
approval as adjuvant in cancer chemotherapy due to intolerable side effects, unpredictable
pharmacokinetics and drug-drug interactions [14]. Meanwhile, the direct pump inhibitors
that bind to substrate site are logically a primary molecular mechanism for development of
Pgp inhibitors. However, the studied compounds of this class, such as dexverapamil, caused
problems during clinical trials [15]. Fortunately, many kinase inhibitors, such as vemurafenib,
that were developed primarily as anticancer agents were found to directly block the ABCB1
(Pgp) as a secondary activity mechanism (Fig 1) [16].
There are many natural products that were characterized as MDR modulators through inhi-
bition of ABCB1 [17, 18]. Curcumin is a versatile natural molecule that is possessing multitude
of confirmed therapeutic activities but it is very safe and can be tolerated in high doses up to
10g/day [19, 20]. Regardless intensive research that confirmed the medicinal benefits of curcu-
min, the yellow condiment continues to intrigue more researchers to explore its activities on
recently validated therapeutic targets [21–23]. Curcumin ameliorates response of cancer
cells towards several cytotoxic drugs. Further investigations confirmed down-regulation of
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P-GP Inhibitors and Paclitaxel Synergism
Fig 1. Examples of Pgp inhibitors.
doi:10.1371/journal.pone.0168938.g001
MDR-1b gene and hence, Pgp expression by curcumin via interruption of PI3K/Akt/NFκB
pathway [24]. Due to high safety margin but low efficacy profile of curcumin, its analogues
coming from natural sources (curcuminoinds) or from synthetic sources, were investigated for
Pgp inhibitory activities. There are evidences that curcumin and its related compounds may
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P-GP Inhibitors and Paclitaxel Synergism
act also through competitive or allosteric (ATPase) inhibition of Pgp in resistant cancer cell
lines [25, 26].
The field of discovery of novel MDR modulators is anywhere but near closure [27]. In as
much as MDR poses such disastrous failures in chemotherapy and hence, mortality, efforts
must continue to discover improved MDR suppressors. Not separable, the impact of new
molecular leads on multi-drug resistance against cytotoxic drugs should be on the center of
the early in vitro studies. In this research, we are utilizing the structural architecture of curcu-
min to design compounds with confirmed Pgp inhibition activities and increased sensitivity of
resistant cancer cells towards paclitaxel (PTX).
Results
Design of Pgp Inhibitors
The design of our compounds was based on flexibility of the SAR of curcumin derivatives as
well as promiscuity of Pgp inhibition targets [25, 28, 29].
Curcumin molecule (diferulylmethane) is composed of two distinct parts: A) Two terminal
aromatic rings and B) Seven-atom linker with β-diketone in the center (Fig 2). The same main
features are shared between three Pgp inhibitors: curcumin, tetrahydrocurcumin and verapa-
mil (one of the earliest studied Pgp direct inhibitor). However, we acknowledge the differences
in hybridization pattern that should affect the molecular geometry. Many of the reported ana-
logues of curcumin are either arylvinylketones or bis-(arylvinyl)ketones. The curcumin ana-
logues that did not adhere to the 7-atom linker rule kept good to excellent Pgp inhibition
activities [30].
Chemical Synthesis
The synthesis of the oxazolones 3a-g followed standard Erlenmeyer chemistry for synthesis
(Fig 3)[31]. The acylglycines 1a-c were prepared in two steps by reaction of carboxylic acids
with ethylglycinate followed by saponification. The acylglycines were condensed with alde-
hydes 2a-d to furnish the oxa-analogues (4-oxazolones) 3a-g. Some oxazolones were reacted
with ammonium acetate in pyridine under anhydrous conditions to give the curcumin aza-
analogues 4b-g.
The N-phenylimidazolone 4a was prepared using the oxazolone 3a with aniline but the
reaction medium contained acetic acid and sodium acetate [32]. The non-heterocyclic com-
pound 5 was prepared accidentally as mentioned before. However, it was also systematically
prepared by reaction of the oxazolone 3a with excess n-propylamine in ethanol at room tem-
perature. All new compounds (Table 1) were confirmed using NMR, IR and the molecular for-
mulae were established using HRMS. The purities were confirmed using LC/MS.
Biological Screening
Effect on purified recombinant P-glycoprotein (Pgp). First, the sub-molecular interac-
tion between test compounds and Pgp subunits was undertaken using human recombinant
Pgp membrane bound protein linked to ATPase enzyme subunits. It is known that Pgp inhibi-
tors such as verapamil (VRP) are supposed to increase ATPase activity leading to more ATP
consumption (33.1% less remaining ATP concentration compared to basal level). On the other
hand, direct ATPase inhibitors such as sodium vanadate would decrease ATP consumption
(33.9% more remaining ATP concentration compared to basal condition). Curox-1 (3a), and
Cur1-12V (5) showed pure ATPase stimulatory effects with 61.4% and 62.2% less remaining
ATP concentration, respectively. On the other hand, Cur-3, 7, 9 and 10 showed pure ATPase
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P-GP Inhibitors and Paclitaxel Synergism
Fig 2. Design of Pgp inhibitors by re-scaffolding of the linker region of curcumin and verapamil.
doi:10.1371/journal.pone.0168938.g002
inhibition up because they significantly left more remaining ATP concentration compared to
that left by basal ATPase activity (Fig 4a). Other curcumin analogues as well as curcumin itself
did not induce any significant change for ATP consumption rate. This might be attributed to
lack of interaction with either subunit of Pgp molecules or attributed to dual interaction with
both subunits.
Multidrug resistance in particular tumor types, such as solid tumors within gastrointestinal
tract, is highly attributed to impaired cellular pharmacokinetics and intracellular drug reten-
tion issues [33]. Therefore, our designed compounds to enhance the cellular entrapment of P-
glycoprotein substrates were tested within LS-174T colorectal cancer cells. Curox-1 (3a), Fer-
vox (3c), Cur-3 (4b), Cur-10 (4e) and Cur1-12V (5) significantly increased cellular retention
of DOX (Pgp probe) and increased its intracellular concentration from 1.9 0.2 nmole/cell to
2.9 0.13 nmole/cell, 2.9 0.2 nmole/cell, 3.0 0.45 nmole/cell, 4.1 0.6 nmole/cell and 2.9 0.16
nmole/cell, respectively (Fig 4b). On the other hand, Curox-3, Cinvox, Cur1-01, Cur-7, Cur-9
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P-GP Inhibitors and Paclitaxel Synergism
Fig 3. Chemical Synthesis Scheme. Reagents and Conditions: (a) Acetic anhydride, sodium acetate, heat.
(b) Aniline, acetic acid, sodium acetate, 110˚C, 24 hr. (c) Ammonium acetate, pyridine, 110˚C, 18 hr. (d) n-
PrNH2, EtOH, r.t., 2h.
doi:10.1371/journal.pone.0168938.g003
and curcumin (reference compound) did not induce any significant increase for the intracellu-
lar DOX concentration. Compounds 4f and 4g were excluded from this test due to poor solu-
bility at the assay conditions.
Cytotoxicity assessment of test compounds. Sulforhodamine B (SRB) assay was used to
assess the cytotoxicity of newly synthesized compounds against Pgp high-expressing colorectal
cancer cell line (LS-174T) over a concentration range 0.01–100 μM. LS-174T is known with its
high resistance (high R-fraction) and consequently high probability of tumor recurrence [34].
This parameter is complimentary to the primary goal of inhibition of Pgp, accumulation of
Pgp probe and modulation of resistance. Not all test compounds showed significant cytotoxic-
ity against LS-174T cells. Nonetheless, some compounds exhibited considerably low IC50
values (less than 10 μM) accompanied with very low R-values (less than 5%). For instance,
Curox-1 (3a), Curox-3 (3b), Fervox (3c), Cinvox (3d), Cur-10 (4e) and Cur1-12V (5) did not
show good cytotoxicity since their IC50 values were higher than 100 μM. On the other hand,
Cur-1, Cur-3 (4b), Cur-7 (4c), Cur-8 and Cur1-01 (4a) killed LS-174T cells with IC50 values of
7.6, 8.9, 35.9, 27.2 and 70.2 μM, respectively (Table 2). Interestingly, less than 5% of LS-174T
cells were resistant to Cur-1, Cur-3, Cur-7 and Cur-8.
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P-GP Inhibitors and Paclitaxel Synergism
Table 1. Structure and codes of the synthesized oxazolones and imidazolones.
Cpd No.
3a
Code
Curox-1
Curox-3
CinVox
FerVox
-
R1
H
R2
H
na
1
1
1
1
1
0
1
1
1
1
1
0
1
1
1
R3
H
R4
H
nb
1
1
0
0
0
1
1
1
1
0
0
1
1
0
1
3b
3c
H
H
OMe
OMe
OMe
H
OH
OH
OH
H
H
H
3d
3e
OMe
H
OH
H
3f
-
H
H
H
H
3g
4a
-
H
Cl
H
OMe
H
OH
H
Cur1-01
Cur-03
Cur-07
Cur-09
Cur-10
Cur-1
Cur-8
Cur1-12V
H
4b
4c
H
H
OMe
OMe
H
OH
OH
H
OMe
H
OH
H
4d
4e
H
H
H
H
4f
H
H
H
H
4g
5
H
Cl
H
OMe
H
OH
H
H
doi:10.1371/journal.pone.0168938.t001
Fig 4. (A) The effect of test compounds on the activity of P-glycoprotein efflux pump cell free isolated recombinant P-gp protein and
(B) within LS-174T cells. The influence of test compounds on the cellular pharmacokinetics via inhibiting the activity of Pgp pump. Verapamil
(VRP) is a standard direct Pgp blocker. Sod Vanadate is a standard ATPase inhibitor. Curcumin is the prototype compound in the design. The
asterisk (*) denotes significant difference from control.
doi:10.1371/journal.pone.0168938.g004
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P-GP Inhibitors and Paclitaxel Synergism
Table 2. Cytotoxic Activity of synthesized compounds against LS174T CRC cell lines.
Cpd No
3a
Cpd Code
Curox-1
Curox-3
Fervox
Cur1-01
Cur-3
IC50 (μM)
>100
>100
>100
70.2
8.9
(±)SE
N/A
N/A
N/A
2.7
R-fraction (%)
N/A
N/A
N/A
N/A
1.5
3b
3c
4a
4b
4c
0.4
Cur-7
35.9
N/T
2.6
0.0
4d
4e
Cur-9
N/A
N/A
1.3
N/A
N/A
0.8
Cur-10
Cur-1
>100
7.6
4f
4g
5
Cur-8
27.2
>100
6.1
0.0
Cur1-12V
N/A
N/A
doi:10.1371/journal.pone.0168938.t002
Chemomodulatory effect of Cur-3 and Cur1-12V to paclitaxel (PTX) within colorectal
cancer cells. Attributed to their clear Pgp interaction properties as well as their considerable
effect on Pgp substrate cellular accumulation, Cur-3 (ATPase inhibitor, cytotoxic) and Cur1-
12V (Direct Pgp inhibitor, no cytotoxic activity) would be good candidates to improve the
activity of Pgp substrate drugs (such as paclitaxel) within Pgp expressing tumor cell types
(such as LS-174T colorectal cancer cells). Regardless the significant cytotoxic activity of Cur-3,
it is a thousand fold weaker cytotoxic than PTX (7.9 0.5 nM). In combination with PTX, Cur-
3 (4b) did not increase the potency of PTX against LS-174T (Fig 5a). This can be observed
from a slight increase of IC50 of PTX to 23.8 8.1 nM. It can be even declared that Cur-3 antag-
onized the potency of PTX against LS-174T with combination index value equals 3.27. How-
ever, Cur-3 abolished the resistance of LS-174T to PTX from R-value of 45.7 3.6% to 2.5
1.1%. Conversely, the Pgp direct competitive inhibitor Cur1-12V (10 μM) significantly
increased PTX potency against LS-174T cells; IC50 values of PTX were 7.9 0.5 nM and 4.3 1.1
nM alone and in combination with Cur1-12V, respectively (Fig 5b). In addition, Cur1-12V
significantly decreased the resistance of LS-174T to PTX from R-value of 45.7 3.6% to 16.2
6.6% for PTX alone and in combination with Cur1-12V, respectively (Fig 5). A similar
result was observed in a combination of Cur1-12V with PTX at the same ratio used for Cur-3
(Table 3).
Fig 5. The effect of serial equitoxic concentrations (ratio 1:100) of Cur3 (A), and 10 μM (B), and serial
concentration of Cur1-12V (ratio 1:100) (C) on the cytotoxicity of PTX in LS-174T cells. Cells were exposed to serial
dilution of PTX (●), Chemosensetizer (▼) or their combination (○) for 72 h. Cell viability was determined using SRB
assay.
doi:10.1371/journal.pone.0168938.g005
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P-GP Inhibitors and Paclitaxel Synergism
Table 3. Chemomedulatory effects of test compounds on the cytotoxicity parameters of PTX against LS-174T cell line.
Compound
Cytotoxic parameter
LS-174T cells
R-fraction (%)
HCT-116 cells
R-fraction (%)
IC50
IC50
PTX
7.9±0.5 nM
8.9±0.4 μM
>100 μM
45.7±3.6%
0.92±0.5%
N/A
102.7±12.7 nM
12.23±1.6 μM
>100 μM
43.9±1.7%
4.1±1.1%
N/A
Cur-3
Cur1-12V
PTX+Cur-3 (1:100)
PTX+Cur1-12V (10 μM)
PTX+Cur1-12V (1:100)
23.8±8.1 nM
4.3±1.1 nM
4.9±0.9 nM
2.5±1.1%
16.2±6.6%
28.1±2.1%
63.6±4.2 nM
12.6±2.9 nM
17.5±3.1 nM
2.7±1.1%
40.4±1.6%
38.6±1.9%
doi:10.1371/journal.pone.0168938.t003
Similar to LS-174T cell line, Cur-3 exerted humble cytotoxic effect against HCT-116 cells
(12.23 1.6 μM) compared to PTX (102.7 12.7 nM). However, Cur-3 killed HCT-116 with
very low resistance remaining fraction (4.1 1.1%). On the other hand, Cur1-12V did not man-
age to kill HCT-116 up to 100 μM for 72h. Ultimately, PTX faced resistance fraction up to 43.9
1.7% within HCT-116 cells (Fig 6). Interestingly, equitoxic combination of Cur-3 enhanced
the potency of PTX against HCT-116 (63.6 4.2 nM) as well as decreased the resistance fraction
to 2.7 1.1% (Fig 6a). Additionally, Cur1-12V (10 μM) increased the potency of PTX against
HCT-116 cells (12.6 2.9 nM); while it did not significantly influence their resistance fraction
(40.4 1.6%) (Fig 6b). Similar results was observed when combining Cur1-12V with PTX using
1:100 ratio (Fig 6c).
Discussion
Our objective of this research was to introduce molecular frameworks supported by a simple,
yet efficient, protocol for in vitro screening of MDR modulators. Our design was inspired
by curcumin because it is established chemosensitizer as supported by several researches.
The re-scaffolding process of the central part furnished compounds with good activity in che-
mosensitazation of resistant CRC cancer cells against PTX. The screening protocol was
designed as follows: First to identify Pgp inhibitors by measuring the ATP consumption rate.
This method is useful not only in quantifying the Pgp inhibition, but also in identifying the
mechanism of action. This assay is sub-cellular, therefore, the protein expression inhibitors are
excluded from its scope. The test could differentiate between compounds that bind directly to
Fig 6. The effect of serial equitoxic concentrations (ratio 1:100) of Cur3 (A), and 10 μM (B), and serial concentration of
Cur1-12V (ratio 1:100) (C) on the cytotoxicity of PTX in HCT-116 cells. Cells were exposed to serial dilution of PTX (●),
Chemosensetizer (▼) or their combination (○) for 72 h. Cell viability was determined using SRB assay.
doi:10.1371/journal.pone.0168938.g006
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P-GP Inhibitors and Paclitaxel Synergism
the efflux substrate site and those acting on ATPase subunit leading to deprivation of the efflux
pump from necessary energy source. Complimentary and parallel to ATP consumption test,
we conducted cellular entrapment test that measures the accumulation of a Pgp probe (sub-
strate) inside cells that are overexpressing Pgp. The short incubation time (2 h) also eliminated
the possibility of protein expression mechanism. The ATPase activity test gives an indication
of binding to Pgp and its inhibition; but the entrapment test answers the question whether this
binding is translated into accumulation of a drug inside the resistant cells. Combining the
results of the two tests, from our perspective, is a short way to decide which compounds should
be the focus of next investigations (Combination with PTX). We determined that a successful
candidate should lead to accumulation of Pgp substrate (probe) inside Pgp expressing cells sig-
nificantly. In addition such a compound should show a confirmed Pgp inhibition through a
defined mechanism as interpreted from the ATP consumption test. Our observation was that
five (out of 10) test compounds increased cellular permeability of DOX. However, only Curox-
1, Cur-3, Cur-10 and Cur1-12V met the criteria because they either significantly increased
ATP consumption (Curox-1 and Cur1-12V) or decreased ATP consumption (Cur-3 and Cur-
10). Curox-1 and Fervox increased cellular retention of DOX. However, they were excluded
because their effect on the purified Pgp was not clear, as there was no significant change in
ATP consumption rate compared to control. Our interpretations is that these two compounds
either act as dual inhibitors (inhibit both substrate site and the ATPase simultaneously), or
they inhibit MDR transporter(s) other than Pgp.
The structure-activity relationship (SAR) for activities on the purified Pgp protein is clear.
The subclass oxazolones (Curox-1, Curox-3, Cinvox and Fervox) generally preferred the Pgp
substrate site over the ATPase subunit. All of these compounds increased ATP consumption
rate either significantly (Curox-1) or slightly (others). All the compounds that have unsubsti-
tuted nitrogen (NH) at position 1 of the imidazolone ring (Cur-3, 7, 9 and 10) showed obvious
affinity towards the ATPAse subunit indicating suitability of the presence of hydrogen bond
donor at this position for binding. Meanwhile, the N-phenyl analogue (Cur1-01) was not sig-
nificant inhibitor of the Pgp and did not enhance the cellular entrapment of DOX either, indi-
cating bulk intolerance at this position of the heterocyclic ring. The open non-heterocyclic
analogue Cur-12V was unique structure in the tested set of compounds. It showed also direct
inhibition of the Pgp (similar to oxazolone analogues e.g. Curox-1) as well as significant
increase in the retention of DOX. This interesting compound is planned for further studies
because it has obvious drug-like structure. Other than clear SAR among different core struc-
tures, the length of the linker (6-atom vs. 8-atom series) had smaller impact on activities within
the same chemical species.
The cytotoxicity of the synthesized compounds is of secondary importance after their Pgp
inhibitory and chemomodulatory influence. However, the cytotoxic activities of test com-
pounds alone should be in consideration upon running the combination tests with PTX. It
was interesting to find that SAR pattern of the cytotoxic activities is similar to that of Pgp activ-
ities. The ATPase-inhibitory NH imidazolone series Cur-1, 3, 7 and 9 exhibited good to mod-
erate antiproliferative effects against LS-174T CRC cells. Furthermore, some of them (Cur-1
and Cur-3) showed low micromolar IC50 at 7.6 and 8.9 μM respectively. Not surprisingly, the
R-value that measures the level of resistance was very low in all cases. This is expected because
LS-174T cells are over expressing Pgp and possess inherent resistance to cytotoxic agents. Our
finding is that the cytotoxic compounds (Cur-3 and 7) might be considered as auto-chemosen-
sitizers, causing accumulation of their own molecular populations inside cancer cells. On the
other hand, the oxazolones as well as the open non-heterocyclic analogue Cur1-12V did not
show any significant anticancer activities under 100 μM. Thus, it was interesting to compare
the effect of both compounds (Cur-3 and Cur1-12V), as representatives of two distinct classes,
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P-GP Inhibitors and Paclitaxel Synergism
on the resistance of LS-174T CRC cell lines. Cur-3 (4b) embodies the ATPase inhibitor sub-
class, causes accumulation of Pgp probe and has considerable cytotoxicity at low micromolar
level. Cur12-V embodies direct Pgp inhibitor subclass, also causes considerable retention of
Pgp probe but it is not cytotoxic. For this assay, we ignored all compounds that showed lower
potency in cellular entrapment assay (Curox-3, Cinvox, Cur1-01, Cur-7 and Cur-9), caused
color interference with assays (Curox-1) or suffered from poor solubility (Cur-1, Cur-8 and
Cur-10).[35, 36]
Paclitaxel (PTX) is a commonly used chemotherapeutic agent as first line treatment of
many solid tumors.[37] However, its efficacy in gastrointestinal tumors is hampered by MDR
type of resistance.[38–40] For instance, our assay showed that PTX is quite potent against S-
174T CRC cancer cell lines with IC50 at nanomolar level (7.9 0.5 nM) but its resistance frac-
tion is very high (R-value is 45.7 3.6%). Unlike other reports, our research was focused on the
effect of the chemosensitizers Cur-3 and Cur1-12V on the R-value when given in combination
with PTX.[41–46]. This is because the decrease in the IC50 does not necessarily mean that the
resistance is subsequently suppressed. However, the change in IC50 should not be ignored to
guard against antagonistic activities and negative drug-drug interactions. Combinations of
PTX with test compounds resulted in diminishing the resistance fraction from 45.7% to 2.5%
and 16.2% for Cur-3 and Cur1-12V, respectively. Since PTX is proven to be a substrate to Pgp
efflux pump, the resistance change is likely due to the impact of our compounds on the Pgp
(both proven to inhibit the Pgp). To confirm results, we repeated the same combination test
protocol against another resistant CRC cell line, the HCT-116. Similar results were obtained
except that CUR1-12V decreased the IC50 but did not significantly affect the resistant fraction.
Inhibition of other MDR proteins such as MRP1 and BCRB should be included in future
investigations as they were not in the scope of this particular research. Nonetheless, our com-
pounds clearly caused modulation of the MDR leading to sensitization of the tumor cells
towards PTX.
Conclusion
Our design, based on curcumin as lead and safe natural product, furnished serious new molec-
ular frameworks to modulate multidrug resistance as potential adjuvant treatment of tumors
overexpressing Pgp. The initial investigation, though simple, introduced a model of how to
quickly and effectively quantify the chemosensitizing properties of new compounds based on
changes in resistance (primarily) and potency (conjunctively).
Indeed it is too early to state that we finished investigation of our two lead compounds as
further studies should be conducted on utility on non-Pgp expressing cells as well as different
resistant cell lines. Our scope was to build a framework (compounds and efficient assay) as a
start. The two lead compounds, either the cytotoxic Cur-3 or the non-cytotoxic Cur1-12V,
should be subjected to further studies for optimization and development as MDR modulators.
Just to mention some, future investigations should include effect on other types of cancer that
are over expressing as well as those not expressing (Pgp-knockout) cells. Effect of our com-
pounds on Pgp protein expression (curcumin-like effect), and inhibition assay against other
MDR protein such as MRP-1 and BCRP are important to clarify the scope and the exact mech-
anism of the newly discovered lead compounds.
Experimental
Melting points (m.p.) were determined in open capillary tubes using Electrothermal apparatus
(Stuart, UK) and are uncorrected. IR spectra were recorded using the potassium bromide method
on Perkin-Elmer 1650 spectrophotometer and expressed in wave number (υmax) cm−1.). In this
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P-GP Inhibitors and Paclitaxel Synergism
report, we only listed the important IR stretching bands, including NH, OH, CH, C = O, C = N
and/or C = C. 1H-NMR and 13C-NMR spectra were measured in deuterated chloroform
(CDCl3) or deuterated dimethyl sulphoxide (DMSO-d6), on Avance III 850 MHz, Avance III
600 MHz or AV-400 MHz spectrometers (Bruker, Billerica, Massachusetts, USA), at the Fac-
ulty of Science Spectroscopy Center, King Abdulaziz University, Jeddah, Saudi Arabia. Chemi-
cal shifts were expressed as ppm against TMS as internal reference. LC/MS analyses were
performed on an Agilent 6320 Ion Trap HPLC–ESI-MS/DAD (Santa Clara, CA, USA) with
the following settings: The analytes were separated using an Macherey-Nagel Nucleodur-C18
column (150 mm length × 4.6 mm i.d., 5 μm) (Macherey-Nagel GMBH & Co. KG, Duren, Ger-
many). Mobile phase; isocratic elution using a mixture of isopropanol and 0.01M ammonium
acetate in water (65: 35, v/v). The flow rate was 0.4 mL/min.; total run time = 20 min. Purities
are reported according to percentage of Peak Areas at wavelength 244 nm. Reaction progress
and compound purity were monitored by Thin Layer Chromatography (TLC) using silica gel
matrix, L × W 5 cm × 20 cm, fluorescent indicator (Machery-Nagel Co., Germany). Column
chromatography was performed on silica gel 60 (particle size 0.06 mm—0.20 mm). High-reso-
lution mass spectrometry (HRMS) was performed in the Faculty of Science, King Abdulaziz
University on Impact II™ Q-TOF spectrometer (Bruker, Germany). All fine chemicals and sol-
vents were purchased from the authorized local distributors of Sigma Aldrich (USA), Acros
(Belgium), Alfa-Aeser (England) and Panreac (Spain). Some reagents were purchased and
imported from Molport (England). Sulforhodamine-B was purchased from Sigma-Aldrich
(St. Louis, MO, USA). Tricloroacetic acid (TCA) and other materials used in biological screen-
ing were of the highest available commercial grade. Vacuum evaporation were performed on
Bu¨chi Rotavapor R-215. Solvent and chemicals waste were collected and disposed according to
KAU Laboratory Safety and Environmental Protection Guidelines.
Chemical Synthesis
Acylglycines 1a-c (all are reported compounds) are prepared according to known procedure
of amide formation by reaction of (E)-acid chlorides and ethyl glycinate hydrochloride fol-
lowed by saponification of the product with NaOH at 40˚C. Compounds 3e,[47] 3f,[48] and
4d[49] were prepared according to general procedures mentioned below in the synthesis of
other new compounds of the this research and their spectral data were found identical to that
of literatures. The intermediate 3g was prepared and used directly without further characteri-
zation for the next step.
General procedure for preparation of oxazolones 3a-3g. A mixture of the (E)-arylglycine
(0.01 mol), and the (E)-arylcarbaldehyde (2a) (0.011 mol), anhydrous sodium acetate (0.05 g)
and acetic anhydride (5ml) was warmed on a boiling water bath with occasional stirring for 30
h, cooled and left at 4˚C for 18 h. The produced yellow solid mass was filtered under vacuum
until complete dryness. The solid was washed with cold ethanol-water mixture, the solvent was
removed and the solid was dried by vacuum filtration for sufficient time. The solid was washed
twice with petroleum ether and the crude product was crystalized from ethanol.
(Z)-4-[(E)-3-phenylallylidene]-2-[(E)-styryl)oxazol-5(4H)-one (3a). This compound
was prepared in 83% yield as a yellow solid, m.p. 150–151˚C.[50] 1H NMR (850 MHz, DMSO-
d6) δ 7.78–7.86 (m, 1H), 7.71 (d, J = 16.09 Hz, 1H), 7.67 (d, J = 7.27 Hz, 2H), 7.60–7.65 (m, 1H),
7.55 (dd, J = 11.42, 15.57 Hz, 1H), 7.44–7.50 (m, 5H), 7.35–7.44 (m, 2H), 7.21 (d, J = 11.42 Hz,
1H), 7.08 (d, J = 16.61 Hz, 1H); IR (FT-IR, cm−1): 3053.13, 1775.82, 1639.51, 1591.55, 1563.78,
1452.71, 1316.4; LC-MS (ESI), RT = 13.4 min, m/z 301.9 [M + H]+.
(Z)-2-[(E)-4-hydroxy-3-methoxystyryl]-4-[(E)-3-phenylallylidene]oxazol-5(4H)-one
(3b). This compound was prepared in 83% yield as a yellow-brown solid in 70% yield; m.
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P-GP Inhibitors and Paclitaxel Synergism
p. 149–151˚C. 1H NMR (850 MHz, DMSO-d6) δ 7.69 (d, J = 16.09 Hz, 1H), 7.60–7.67 (m, 3H),
7.44–7.50 (m, 3H), 7.35–7.44 (m, 3H), 7.14–7.21 (m, 2H), 3.80–3.94 (m, 4H); IR (FT-IR,
cm−1): 3060.7, 3005.17, 1758.15, 1644.56, 1614.27, 1503.2, 1417.37, 1273.48, 1200.28; LC-MS
(ESI), RT = 7.7 min, m/z 347.9 [M + H]+.
4-[(Z)-4-hydroxy-3-methoxybenzylidene)-2-[(E)-styryl]oxazol-5(4H)-one (3c). This
compound was prepared in 83% yield as a yellow solid powder was obtained in 61% yield,
m.p. >250˚C (dec). 1H NMR (850 MHz, CDCl3) δ 8.00–8.05 (m, 1H), 7.73 (d, J = 16.09 Hz,
1H), 7.62–7.66 (m, 3H), 7.42–7.50 (m, 3H), 7.18 (s, 1H), 7.15 (d, J = 7.78 Hz, 1H), 6.84 (d,
J = 16.09 Hz, 1H), 3.95–4.03 (m, 3H); IR (FT-IR, cm-1): 1763.2, 1642.03, 1599.12, 1513.29,
1422.42, 1311.35, 1265.91, 1200.28; LC-MS (ESI), RT = 8.5 min, m/z 321.9 [M + H]+; 13
C
NMR (214 MHz, CDCl3) δ 168.7, 167.3, 163.4, 151.4, 144.1, 142.2, 134.6, 133.5, 132.5, 130.9,
130.4, 129.1, 128.2, 126.0, 123.2, 115.4, 113.4, 56.0, 20.7.
4-[(Z)-4-hydroxy-3-methoxybenzylidene]-2-[(E)-4-hydroxy-3-methoxystyryl]oxazol-5
(4H)-one (3d). This compound was prepared in 83% yield as a yellow solid in 77% yield, m.
p. 174–176˚C. 1H NMR (850 MHz, CDCl3) δ 7.98 (s, 1H), 7.64–7.71 (m, 2H), 7.20–7.26 (m,
1H), 7.06–7.20 (m, 3H), 6.77 (d, J = 16.09 Hz, 1H), 3.95–4.09 (m, 3H), 3.87–3.95 (m, 3H), 3.85
(s, 1H); IR (FT-IR, cm-1): 2942.06, 2848.66, 1785.92, 1758.15, 1649.61, 1594.07, 1513.29,
1417.37, 1369.41, 1270.96; LC-MS (ESI), RT = 5.0 min, m/z 368.0 [M + H]+; 13C NMR (214
MHz, CDCl3) δ 168.8, 168.7, 167.2, 163.3, 151.6, 151.4, 143.3, 142.2, 141.9, 133.5, 133.5, 132.5,
130.6, 125.9, 124.8, 123.5, 123.4, 123.4, 123.2, 121.6, 121.5, 115.4, 113.6, 111.4, 111.3, 56.1, 56.1,
56.0, 56.0, 20.7, 20.7.
Preparation of (Z)-3-phenyl-5-[(E)-3-phenylallylidene]-2-(E)-styryl-3,5-dihydro-4H-
imidazol-4-one (4a). The oxazolone 3a (1.5 g, 0.005 mol) was dissolved in 15 mL acetic acid
containing 0.5 g sodium acetate and heated. Aniline (0.512 g, 0.5 mL, 0.0055 mol) added to the
heated mixture and temperature was adjusted to 100˚C for 8 hrs. Most of solvent was removed
by vacuum evaporation and the residue was poured into ice water containing 10% of HCl. The
precipitated solid was collected by vacuum filtration, washed with water, sodium bicarbonate
then water. The dried crude solid was purified by column chromatography (gradient: hexane
to hexane-dichloromethane to dichloromethane) to afford 0.43 g (23%) of pure product 4a as
page yellow solid, m.p. 180–185˚C. 1H NMR (850 MHz, DMSO-d6) δ 7.93 (d, J = 15.57 Hz,
1H), 7.69 (d, J = 7.27 Hz, 2H), 7.57–7.63 (m, 3H), 7.51–7.57 (m, 2H), 7.36–7.50 (m, 10H), 7.11
(d, J = 11.42 Hz, 1H), 6.59–6.65 (m, 1H); IR (FT-IR, cm−1): 3058.18, 3027.89, 2995.07, 1705.14,
1621.84, 1576. 40, 1495.62, 1379.50, 1331.54, 1210.37; LC-MS (ESI), RT = 13.9 min, m/z 377.0
[M + H]+.
Parallel synthesis of compounds 4b-4g. Anhydrous ammonium acetate (0.46 g, 0.005
mol) was placed 25 mL-tube of Thermal Integrity Reaction Station, added the appropriate
azlactone (0.003 mol) and pyridine (10 ml) under inert air. The device was adjusted for 18 h
run at 100˚C for 18 and stirring. The solvent was removed by evaporation under vacuum.
The residue was subjected to flash column chromatography (silica gel, mixtures of petroleum
ether/dichloromethane, 1:4, v/v) to afford the desired pure compounds (4b-g).
(Z)-5-[(E)-3-(4-hydroxy-3-methoxyphenyl)allylidene]-2-(E)-styryl-3,5-dihydro-4H-imi-
dazol-4-one (4b). The product was dark red solid and it was obtained in 55% yield, m.p. 97–
100˚C. 1H NMR (600 MHz, DMSO-d6) δ 7.52–7.72 (m, 3H), 7.32–7.51 (m, 4H), 7.14–7.26 (m,
2H), 6.94–7.14 (m, 2H), 6.76–6.94 (m, 2H), 3.76–3.96 (m, 3H); IR (FT-IR, cm−1): 3060.71,
2934.49, 2838.57, 1768.25, 1712.71, 1642.03, 1578.92, 1465.33, 1445.14, 1432.51, 1116.97; High
Resolution MS (EI+, m/z) (M+H)+ Calcd. for C21H19N2O3, 447.1351, found 447.1382. 13
C
NMR (214 MHz, DMSO-d6) δ 170.7, 158.3, 149.1, 148.5, 144.5, 143.8, 143.0, 140.7, 139.9,
139.8, 135.5, 135.2, 130.8, 130.5, 130.0, 129.6, 129.6, 129.5, 129.4, 128.7, 128.5, 128.3, 128.2,
128.1, 128.0, 122.6, 121.2, 119.6, 117.5, 116.3, 110.7, 56.0, 49.0.
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P-GP Inhibitors and Paclitaxel Synergism
5-[(Z)-4-hydroxy-3-methoxybenzylidene)-2-[(E)-4-hydroxy-3-methoxystyryl]-3,5-dihy-
dro-4H-imidazol-4-one (4c). The product was brick red and it was obtained in 53% yield,
m.p 113–116˚C. 1H NMR (850 MHz, DMSO-d6) δ 7.87–8.00 (m, 1H), 7.72–7.83 (m, 1H), 7.58
(d, J = 16.09 Hz, 1H), 7.45–7.52 (m, 1H), 7.20–7.27 (m, 1H), 7.14 (s, 1H), 6.87–6.98 (m, 2H),
6.78–6.87 (m, 1H), 3.80–3.92 (m, 6H); IR (FT-IR, cm−1): 2936.76, 2844.68, 1594.22, 1509.4,
1429.43, 1269.49, 1208.91; LC-MS (ESI), RT = 3.0 min, m/z 367.9 [M + H]+. High Resolution
MS (EI+, m/z) (M+H)+ Calcd. for C20H19N2O5, 367.1288, found 367.1290.
5-(Z)-benzylidene-2-(E)-styryl-3,5-dihydro-4H-imidazol-4-one (4d). The product was
yellowish-brown solid obtained in 42% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.62 (d,
J = 7.53 Hz, 2H), 7.52–7.58 (m, 1H), 7.51 (s, 1H), 7.35–7.48 (m, 5H), 7.26–7.35 (m, 1H), 7.16–
7.24 (m, 1H), 7.02–7.12 (m, 1H), 6.87 (dd, J = 1.88, 15.81 Hz, 1H); High Resolution MS (EI+,
m/z) molecular ion requires for C18H14N2O, 275.1140, found 275.1176.
(Z)-2-phenyl-5-[(E)-3-phenylallylidene]-3,5-dihydro-4H-imidazol-4-one (4e). The
product was yellowish crystals and it was obtained in 55% yield. 1H NMR (850 MHz, DMSO-
d6) δ 8.30–8.43 (m, 3H), 8.19 (d, J = 7.79 Hz, 2H), 7.67 (t, J = 7.01 Hz, 1H), 7.62 (t, J = 7.27 Hz,
2H), 7.57 (d, J = 8.30 Hz, 2H), 7.00–7.10 (m, 3H); IR (FT-IR, cm−1): 3154.11, 3116.24, 3060.71,
1700.09, 1642.03, 1591.55, 1493.10, 1450.19, 1359.31, 1263.39; LC-MS (ESI), RT = 4.2 min, m/z
301.
(Z)-5-[(E)-3-phenylallylidene]-2-(E)-styryl-3,5-dihydro-4H-imidazol-4-one (4f). The
product was yellowish solid and it was obtained in 59% yield; m.p. 243–244˚C. 1H NMR (850
MHz, DMSO-d6) δ 11.80 (s, 1H), 11.74 (s, 1H), 7.68 (d, J = 7.27 Hz, 1H), 7.64–7.67 (m, 1H),
7.61–7.64 (m, 1H), 7.54–7.61 (m, 2H), 7.40–7.50 (m, 4H), 7.35–7.39 (m, 1H), 7.27 (d, J = 15.57
Hz, 1H), 7.00 (d, J = 16.61 Hz, 1H), 6.87–6.94 (m, 1H); IR (FT-IR, cm−1): 3138.96, 3085.95,
3025.37, 1695.04, 1639.51, 1601.64, 1571.35, 1525.91, 1442.61, 1316.39, 1276.01, 1202.80;
LC-MS (ESI), RT = 4.2 min, m/z 30.
2-(E)-4-chlorostyryl-5-[(Z)-4-hydroxy-3-methoxybenzylidene]-3,5-dihydro-4H-imida-
zol-4-one (4g). The product was yellowish brown solid and it was obtained in 78% yield. 1H
NMR (600 MHz, DMSO-d6) δ ppm 3.85 (s, 3 H) 6.87 (d, J = 7.91 Hz, 1 H) 6.92 (s, 2 H) 7.04 (d,
J = 16.56 Hz, 1 H) 7.52 (d, J = 8.28 Hz, 1 H) 7.60 (d, J = 16.56 Hz, 1 H) 7.64–7.79 (m, 2 H) 8.02
(s, 1 H); High Resolution MS (EI+, m/z) molecular ion requires for, C19H15 ClN2O3, 355.0805,
found 355.0852.
(2E,4E)-2-cinnamamido-5-phenyl-N-propylpenta-2,4-dienamide (5). The azlactone 3a
(0.01 mol) was placed in 20 mL ethanol and propyl amine (0.02 mol) and the mixture was left
to stir at r.t. for 2 h. After completion of the reaction as monitored by TLC (2% MeOH in
dichloromethane), the precipitated solid compound 5 was collected by filtration, washed with
ethanol and crystallized from a mixture of dicloromethane and methanol. The yellowish crys-
talline solid was obtained in 61% yield; m.p. 205–207˚C. 1H NMR (850 MHz, DMSO-d6) δ
9.68 (s, 1H), 8.02 (dd, J = 5.45 Hz, 1H), 7.64 (d, J = 7.27 Hz, 2H), 7.50–7.56 (m, 3H), 7.45–7.49
(m, 2H), 7.40–7.45 (m, 1H), 7.34–7.40 (m, 2H), 7.25–7.32 (m, 1H), 7.02 (dd, J = 11.42, 15.57
Hz, 1H), 6.83–6.94 (m, 2H), 6.72 (d, J = 11.42 Hz, 1H), 3.10 (q, J = 6.75 Hz, 2H), 1.47 (sxt,
J = 7.27 Hz, 2H), 0.87 (t, J = 7.53 Hz, 3H); IR (FT-IR, cm-1): 3367.08, 2956.22, 2931.57,
2876.79, 2057.8, 1657.9, 1644.2, 1614.07, 1531.9, 1482.6, 1329.21, 1279.91, 1211.43; LC-MS
(ESI), RT = 3.3 min, m/z 361.0 [M + H]+.
Biological Screening
Cell culture. Human colorectal cancer cell line (LS-174T), was generously gifted from
Professor Vladimir P. Torchilin (Northeastern University, Boston, MA, USA); Human colo-
rectal cancer cell line (HCT-116 was obtained from VACCERA, Giza, Egypt. Both cell lines
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P-GP Inhibitors and Paclitaxel Synergism
were maintained in RPMI-1640 media; supplemented with 100 μg/mL streptomycin, 100
units/mL penicillin and 10% heat-inactivated fetal bovine serum. Cells were propagated in a
humidified incubator at 37˚C with 5% (v/v) CO2 atmosphere.
Assessment of Pgp activity in-situ. To assess the effect of test compounds on the efflux
pumping activity of Pgp in tumor cells, the Pgp substrate, doxorubicin (DOX), was used as a
probe and verapamil (VRP) was used as standard Pgp blocker. Briefly, exponentially growing
cells were plated in 6-well plates in plating density of 105cells/well. Cells were exposed to DOX
(10 μM), DOX (10 μM) and test compound (10 μM), or DOX (10 μM) and VRP (10 μM) for 2
h at 37˚C and subsequently extracellular DOX containing media was washed trice with ice
cold PBS. Intracellular DOX were extracted after cell lysis by incubation with SDS (2% w/v),
saturated aqueous solution of ZnSO4 (100 μl), Acetonitrile (500 μl) and acetone (500 μl) for 30
min at 37˚C. After centrifugation, supernatant was collected and DOX concentration was mea-
sured spectroflourometrically at λex/em 482/550 nm [43, 51].
Assessment of Pgp ATPase activity. Assessment of Pgp-attached ATPase activity was
performed using Pgp-Glo™ Assay Systems (Promega Corporation, Madison, Wisconsin, USA).
Briefly, in 96-well plates, Pgp-Glo™ Assay Buffer was mixed with sodium vanadate, VRP, or
test compound (10 μM). Recombinant human Pgp membrane fraction was incubated with the
reaction mixture at 37˚C for about 5 minutes, and then the reaction was initiated by adding
10μl of 25mM MgATP and incubating for 40 minutes at 37˚C. An identical reaction mixture
without drug treatment (NTC) was assayed in parallel. The reaction was stopped and the
remaining un-consumed ATP was detected using luciferase firefly luminescent signal and
ATP standard curve was plotted. The remaining concentrations of ATP were expressed as (p.
mole/μg Pgp molecules). Sodium vanadate (Na3VO4) was used as selective inhibitor of Pgp
related ATPase enzyme, and samples treated with Na3VO4 have greater luminescent signal
than un-treated samples (NTC). In addition, VRP inhibits pgp molecules via covalent binding
and competing with other substrates, hence, stimulating ATPase activity resulting in more
consumption of ATP molecules compared to NTC.
Cytotoxicity assays. The cytotoxicity of the test compounds were tested against exponen-
tially growing LS-174T cells by sulforhodamine B (SRB) assay as previously described [52].
Briefly, sub-confluent cells were collected using 0.25% Trypsin-EDTA and plated in 96-well
plates at 1000–2000 cells/well. Cells were exposed to test compound for 72 h and subsequently
fixed with TCA (10%) for 1 h at 4˚C. After washing trice, cells were exposed to 0.4% SRB solu-
tion for 10 min in dark and subsequently washed with 1% glacial acetic acid. After drying over-
night, Tris-HCl was used to dissolve the SRB-stained cells and color intensity was measured at
540 nm.
The dose response curve of each compound was analyzed using Emax model:
ꢀ
ꢁ
m
½DŠ
%Cell viability ¼ ð100 À RÞ Â 1 À
þ R
m
m
Kd þ ½DŠ
Where [R] is the residual unaffected fraction (the resistance fraction), [D] is the drug concen-
tration used, [Kd] or IC50 is the drug concentration that produces a 50% reduction of the maxi-
mum inhibition rate and [m] is a Hill-type coefficient. Absolute IC50 is defined as the drug
concentration required to reduce absorbance by 50% of that of the control (i.e., Kd = absolute
IC50 when R = 0 and Emax = 100-R) [43].
Chemomodulatory effect of CUR3 and CUR1-12V to paclitaxel within colorectal cancer
cells. The chemomodulatory effect of the non cytotoxic compound, CUR1-12V, to paclitaxel
(PTX) within colorectal cancer cells was determined using the Pgp inhibitory concentration
of CUR1-12V (10 μM) along with serial concentrations of PTX. Chemomodulatory effect of
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P-GP Inhibitors and Paclitaxel Synergism
CUR3 to PTX within was determined using equitoxic combination analysis between PTX and
CUR3 (exposure ratio 1:100) as previously described [53]. In addition, chemomodulatory
effect of CUR1-12V to PTX at exposure ratio of 1:100 was assessed as well for comparison with
CUR3. Briefly, exponentially growing cells were seeded in 96-well plates (2000 cells/well) and
exposed to PTX and CUR3 or CUR1-12V for 72 h. Cells were subsequently subjected to SRB
assay as described in the previous section. Combination index (CI-value) was calculated and
used to define the nature of drug interaction (synergism if CI-value < 0.8 as; antagonism if CI-
value > 1.2; and additive if CI-value ranges from 0.8–1.2).
CI-value was calculated from the formula:
IC50ofdrugðxÞcombination IC50ofdrugðyÞcombination
CI À value ¼
þ
IC50ofdrugðxÞalone
IC50ofdrugðyÞalone
Statistical analysis. Data are presented as mean SEM using GraphPad prism™ software
(GraphPad software Inc., La Jolla, CA, USA) for windows version 5.00. Analysis of variance
(ANOVA) with Tukey’s post hoc test was used for testing the significance using SPSS1 for
windows, version 17.0.0. p<0.05 was taken as a cut off value for significance.
Supporting Information
S1 File. Cytotoxicity dose-response curves.
(DOCX)
S2 File. Spectra of synthesized compounds.
(DOCX)
Acknowledgments
The authors are grateful for Professor Alaa Khedr, Dept of Pharmaceutical Chemistry, Faculty
of Pharmacy, King Abdulaziz University for performing LC/MS analyses.
Author Contributions
Conceptualization: MEE AMA.
Data curation: MEE AMO AMA.
Formal analysis: AMA MTK HAA.
Funding acquisition: MEE.
Investigation: MEE AMO MTK HAA AMA.
Methodology: MEE AMO AMA.
Project administration: MEE.
Resources: MEE.
Supervision: MEE AMO AMA.
Validation: MEE MTK AMA.
Visualization: MEE AMA.
Writing – original draft: MEE MTK AMA.
PLOS ONE | DOI:10.1371/journal.pone.0168938 January 9, 2017
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P-GP Inhibitors and Paclitaxel Synergism
Writing – review & editing: MEE AMA.
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