Synergistic interaction of N-3-Br-benzyl-noscapine and docetaxel abrogates oncogenic potential of breast cancer cells

Article abstract of DOI:10.1111/cbdd.13902

Noscapine, an opium alkaloid, was discovered to bind tubulin, arrest dividing cells at mitosis, and selectively induce apoptosis to cancer cells. N-3-Br-Benzyl-Noscapine (Br-Bn-Nos), one of the derivatives of noscapine, was demonstrated to have improved anticancer potential compared with noscapine. We approached to evaluate the single and combined effect of Br-Bn-Nos and docetaxel (DOX) based on molecular modeling and cellular study. The individual predicted free energy of binding (?G_bind,pred) for Br-Bn-Nos and DOX with tubulin was found to be ?28.89 and ?36.07?kcal/mol based on molecular mechanics generalized Born solvation area (MM-GBSA) as well as ?26.21 and ?34.65?kcal/mol based on molecular mechanics Poisson Boltzmann solvation area (MM-PBSA), respectively. However, the ?G_bind,pred of Br-Bn-Nos was significantly reduced (?33.02 and ?30.24?kcal/mol using MM-GBSA and MM-PBSA) in the presence of DOX on its binding pocket. Parenthetically, the ?G_bind,pred of DOX was significantly reduced (?37.17 and ?32.80?kcal/mol using MM-GBSA and MM-PBSA) in the presence of Br-Bn-Nos on its binding pocket. The reduced ?G_bind,pred in the presence of Br-Bn-Nos and DOX together indicated a combination effect of both the ligands. The combined interaction of both the agents onto tubulin dimmer was also determined experimentally using purified tubulin, in which a?combination regimen of Br-Bn-Nos and DOX reduced the fluorescence intensity of tubulin to a higher value (68%) compared with the single regimen. Further, isobologram analysis revealed the synergistic effect of Br-Bn-Nos and DOX in antiproliferative activity using MCF-7 cell line at 48?hr (sum FIC?=?0.49) and at 72?hr (sum FIC?=?0.62). The combination dose regimen of Br-Bn-Nos and DOX blocks the cell cycle progression at the G2/M phase and induces apoptosis to cancer cells more effectively compared with the single regimen. Taken together, our study provides compelling evidence that the anticancer potential of noscapine derivatives may be substantially improved when it is used in a combined application with DOX for breast cancer.

Full text of DOI:10.1111/cbdd.13902

Article type : Research Article
Synergistic interaction of N-3-Br-Benzyl-noscapine and docetaxel abrogates
oncogenic potential of breast cancer cells
Running title: Synergistic effect of N-3-Br-Benzyl-noscapine and docetaxel
Shruti Gamya Dash1, Srinivas Kantevari2, Swaroop Kumar Pandey3,4 and Pradeep Kumar Naik1*
1Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and
Bioinformatics, Sambalpur University, Jyoti Vihar, Burla, Sambalpur-768 019, Odisha, India
2Fluoro and agrochemicals Division, CSIR-Indian Institute of Chemical Technology, Hyderabad
500 007, India
3Bencos Research Solutions, New Link Road, Andheri West, Mumbai-400053, Maharashtra,
India.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13902
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4Present address: Department of Life Sciences and the National Institute for Biotechnology in the
Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
*For correspondence: Phone No.: +91-9479268802; E-mail: pknaik1973@gmail.com
ABSTRACT
Noscapine, an opium alkaloid was discovered to bind tubulin, arrest dividing cells at mitosis, and
selectively induced apoptosis to cancer cells. One of its derivatives, N-3-Br-Benzyl-Noscapine
(Br-Bn-Nos) was demonstrated to have improved anticancer potential compared to noscapine. We
approached to evaluate the single and combined effect of Br-Bn-Nos and docetaxel (DOX) based
on molecular modelling and cellular study. The individual predicted free energy of binding
(∆Gbind,pred) for Br-Bn-Nos and DOX with tubulin was found to be -28.89 kcal/mol and -36.07
kcal/mol based on MM-GBSA as well as -26.21 kcal/mol and -34.65 kcal/mol based on MM-
PBSA, respectively. However, the ∆Gbind,pred of Br-Bn-Nos was significantly reduced (-33.02
kcal/mol and -30.24 kcal/mol using MM-GBSA and MM-PBSA) in presence of DOX on its
binding pocket. Parenthetically, the ∆Gbind,pred of DOX was significantly reduced (-37.17 kcal/mol
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and -32.80 kcal/mol using MM-GBSA and MM-PBSA) in the presence of Br-Bn-Nos on its
binding pocket. The reduced ∆Gbind,pred in presence of Br-Bn-Nos and DOX together indicated a
combination effect of both the ligands. The combined interaction of both the agents onto tubulin
dimmer was also determined experimentally using purified tubulin, in which a combination
regimen of Br-Bn-Nos and DOX reduced the fluorescence intensity of tubulin to a higher value
(68%) compared to the single regimen. Further, isobologram analysis revealed the synergistic
effect of Br-Bn-Nos and DOX in anti-proliferative activity using MCF-7 cell line at 48 h (sum FIC
= 0.49) and at 72 h (sum FIC = 0.62). The combination dose regimen of Br-Bn-Nos and DOX
blocks the cell cycle progression at the G2/M phase and induced apoptosis to cancer cells more
effectively compared to the single regimen. Taken together, our study provides
compelling evidence that the anticancer potential of noscapine derivatives may be substantially
improved when it is used in a combined application with DOX for breast cancer.
Keywords: N-3-Br benzyl Noscapine, Molecular Docking, Combination therapy, Docetaxel,
Breast cancer, Isobologram
Introduction
Chemotherapeutics regimens currently available for breast cancer have been hindered by
poor selectivity towards cancerous cells and hence, associated with severe toxicity (Pace et al.
1996; Rowinsky, 1997; Crown and O'Leary, 2000; Theiss and Meller, 2000; Topp et al. 2000).
Over the past few decades, these inadequate scenarios have pushed extensive research on
finding more specific and less toxic drugs for cancer. Noscapine, a safe anti-tussive agent, is an
excellent choice for cancer treatment because of its mechanism of action, which is not detrimental
to healthy cells (Ye et al. 1996; Zhou et al. 2006). It doesn't significantly alter tubulin's stable
monomer/polymer ratio over a broad range of concentrations (Ye et al. 1996; Zhou et al. 2003).
This is a distinct benefit over currently available antimicrotubule medications, which either
prevent microtubule disassembly (taxanes, epothilone) or tubulin assembly (vincas, eribulin,
estramustine) and thus do not cause hemo and neuronal toxicity due to its inherent mechanism of
action. To optimize its anticancer activity, we strategically developed more effective derivatives
by changing the scaffold structure for therapeutics applications. A battery of noscapinoids has
already developed, some of which have shown impressive anticancer activity in comparison to
noscapine (Naik et al. 2011; Naik et al. 2012; Manchukonda et al. 2013; Manchukonda et al. 2014;
Santoshi et al. 2015; Mahaddalkar et al. 2017). However, complete remission of cancer cells was
not achieved even at a higher concentration based on in vivo animal study (Zhou et al. 2003;
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2006). Therefore, new treatment modalities such as combination therapy could be beneficial for
breast cancer.
The combination therapy of anti-microtubule agents is an undiscovered source of
chemotherapeutic resources. Presence of multiple drug binding sites on the tubulin, suggests that
a reasonable combination of two or more drugs of this class may increase the efficacy of
anticancer drugs and diminish toxic side effects, thereby improving the therapeutic index. In this
study, we approach to evaluate the combined effect of N-3-Br-Benzyl-noscapine and docetaxel
towards better anticancer activity. The drug combination significantly reduces the cancer cell
growth (sum FIC <1), which shows the synergism between N-3-Br-Benzyl-noscapine and
docetaxel in their anticancer activity.
Materials and methods
A. Molecular modeling evaluation of N-3-Br-Benzyl Noscapine as tubulin binding agent and
having combination effect with docetaxel
(a) Ligand preparation
The molecular structure of Br-Bn-Nos and docetaxel (DOX) were built using maestro
molecular builder (Schrodinger). The constructed structures were energy minimized using
Macromodel (version 17.4, Schrodinger) and OPLS 2005 force field. A PRCG algorithm with
1000 steps and an energy gradient of 0.001 was used for energy minimization. Using Ligprep, a
suitable bond order was allocated to each ligand. In addition, the ligands were DFT optimized
using Jaguar (version 17.4, Schrödinger, LLC) applying Becke's three-parameter exchange
potential and the Lee-Yang-Parr correlation functional (B3LYP) (Lee et al. 1988; Becke, 1993)
with a basis set 3-21G*(Binkley et al. 1980; Gordon et al. 1982). The various conformations of the
molecules were generated using Ligprep (Schrodinger).
(b) Protein preparation
The co-crystal structure of amino-noscapine and tubulin (PDB ID: 6Y6D, resolution 2.20
Å) (Oliva et al. 2020) was used for the molecular docking and predicting the binding affinity of
Br-Bn-Nos and DOX. The hydrogen atoms were added and the protein structure was prepared
based on the multistep procedure of the protein preparation wizard (Schrodinger). Briefly, it
involves minimization of added hydrogen atoms and optimization of hydrogen bonding networks.
Further, refinement of the structure was achieved by molecular dynamic simulation of 100 ns
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using GROMACS 5.1.5 with similar parameters setting as reported earlier (Santoshi and Naik,
2014).
(c) Molecular docking
The various conformations of Br-Bn-Nos generated above were docked onto the
noscapinoids binding site (Oliva et al. 2020) at the interface of αβ-tubulin heterodimer (PDB ID:
6Y6D) prepared above. The binding site was specified by creating two grid boxes around it. An
inner grid box of size 14Å x 14Å x 14Å was defined at the centroid of the noscapinoids binding
site by selecting the co-complex ligand, amino-noscapine using the Glide grid-receptor generation
program. This box defines the search space in which the diameter midpoint of each docked ligand
is required to be present. Further, an outer grid box was also defined with a size of ≤ 24 Å of the
co-complexed ligand, amino noscapine. It defines the volume within which all ligand atoms of a
valid pose must be located. Similarly, the various conformations of the DOX generated above
were docked onto the paclitaxel binding site of tubulin reported earlier (Nogales et al. 1998;
Synder et al.2000; Canales et al. 2011; Winefield et al.2008). We have used the published co-
crystal structure of paclitaxel-tubulin (PDB ID: 1TUB) (Nogales et al. 1998) to extract the binding
site amino acids and their coordinates and mapped onto the above prepared protein structure using
SiteMap (Schrodinger software package) to define the taxotere binding site. Further, this binding
site was specified in the docking of DOX by creating two grid boxes using the grid receptor
generation program (Schrodinger software package). An inner grid box of size 14Å x 14Å x 14Å
was created at the centroid of the taxotere binding site to define the search space in which the
diameter midpoint of each docked ligand is required to be present. Further, an outer grid box with
a size of 20Å x 20Å x 20Å was defined within which all ligand atoms of a valid pose must be
located. For the molecule docking of Br-Bn-Nos and DOX onto their respective sites we have used
Glide-XP algorithm (Halgren et al. 2004) using Schrodinger package and evaluated their binding
poses using Glide XPScore function (Friesner et al. 2004; Halgren et al. 2004). Three cycles of
molecular docking were performed to elucidate their binding affinity in single as well as in their
combination. In the first cycle, both the ligands were docked individually into their respective
binding site. In the second cycle, to the co-complex of Br-Bn-Nos and tubulin, DOX was docked
to determine the combined effect of DOX with Br-Bn-Nos. Similarly, in the third cycle, to the co-
complex of DOX and tubulin, Br-Bn-Nos was docked to determine its combination effect with
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docetaxel. For the ligand docking method, the scale factor of 0.4 for van der Waals radii was
implemented to protein atoms with exact partial charges less than or equal to 0.25. Out of the
10000 poses sampled, 1000 were extracted by minimization (conjugate gradients) and favorable
Glide docking performance was further evaluated by 30 structures with the lowest energy
conformation. The single best conformation for each ligand was used for further analysis.
(d) Molecular dynamics simulation
The docked complex of (a) Br-Bn-Nos with tubulin, (b) DOX with tubulin, (c) both Br-Bn-
Nos and DOX in combination with tubulin, and (d) the tubulin only was considered for the MD
simulation using Amber 16 (Case et al. 2016). The parameters for both the ligands including DOX
and Br-Bn-Nos along with GTP and GDP were calculated using Amber 16 software Antechamber
system (Wang et al. 2006). All charges for atomic points were determined using the charging
model AM1-BCC (Jakalian et al. 2002). In Amber16, topologies and internal coordinates were
created for all complexes using tleap programme. Missing hydrogen was added, and Protein and
Ligand parameters were assigned using FF14SB and GAFF force fields, respectively (Maier et al.
2015). TIP3P water model in a truncated octahedron at 12 Å distance between the protein atoms
and the wall of the box (Jorgensen et al. 1983) was added with dissolved counter ions to neutralize
the system. Once the topologies and internal coordinates for all complexes have been obtained,
three rounds of minimization have been carried out for each complex in order to relax the system.
Position constraints of 10 kcal/Å2 and 2 kcal/Å2 have been imposed on the protein system for the
first and second rounds, respectively, to relax the water molecules around the protein. In the third
round, no restrictions were imposed. After removal of bad contacts through minimization, all four
molecular systems were balanced at 300 K and 1 atm at 500 ps. The balanced systems were then
run at 100 ns each with a time step of 2 fs. The cut-off for non-bonded interaction was 10 Å during
simulations, electrostatics was measured using Particle Mesh Ewald (PME) and bonds were
restricted using shake algorithm (Ryckaert et al. 1977; Darden et al. 1993; Essmann et al. 1995).
The simulation temperature was regulated with the Langevin thermostat. Coordinates for each
molecular system were written every 20 ps.
(e) Predicted free energy of binding using MM-GBSA and MM-PBSA
The predicted free energy of binding (ΔGbind,pred) of Br-Bn-Nos and DOX in their single
binding and in co-binding with tubulin was determined as the ensemble average of the binding
free energy of a total of 250 snapshots from the last 5 ns of the MD simulation trajectory of their
respective molecular systems. We have used molecular mechanics generalized Born solvation area
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(MM-GBSA) and molecular mechanics Poisson Boltzmann solvation area (MM-PBSA) methods
(Kollman et al. 2000; Massova and Kollman, 2000) to obtain ΔGbind,pred as explained below:
ΔGbind,pred = ΔGcomplex – [ΔGRec + ΔGlig]
G = Egas +Gsol -TS.
Egas = Eint +Eele +Evdw
Gsol = GPB(GB) + Gsol-np
Gsol-np= γSAS
Where, G is Gibbs free energy, Egas is the gas phase energy calculated as the sum of internal
energy (Eint), energy generated as a result of the electrostatic interaction (Eele) and the van der
Waals interaction (Evdw). Gsol is the solvation free energy calculated as the sum of polar (GPB(GB)
)
and nonpolar contributions (Gsol-np). Polar interaction contribution (GPB(GB)) was calculated as the
summation of electrostatic contribution (Eele) and polar solvation contribution (GPB(GB)). The
nonpolar solvation contribution (Gsol-np) is approximated as linearly dependent on the solvent
accessible surface area (SAS) and γ is the surface tension constant that was set to 0.0072 kcal
mol−1 Å −2 (Massova and Kollman, 2000).
B. Biology
(a) Cell lines and Chemicals
Noscapine and docetaxel were purchased from Sigma. The novel derivative of noscapine,
Br-Bn-Nos was chemically synthesized by reaction scheme mentioned below (Manchukonda et
al., 2013) and HPLC purified (purity > 96%). The human breast cancer cell line MCF-7 was
acquired from the cell registry of the National Centre for Cell Science in Pune, Maharashtra, India.
All the chemical reagents and media used for cell culture were obtained from Mediatech, Cellgro.
The cells were grown in a 5% CO2 and 95% humidity in Dulbecco's modified Eagle medium
(DMEM, Pan Biotech) at a favorable temperature of 37 °C, supplemented with 10 % fetal bovine
serum (FBS) and antibiotics. For bioassays using trypsin-EDTA (0.25%), having a confluence
with 70-80 % cells were sub cultured. Cell at 70-80% confluence was sub-cultured using trypsin-
EDTA (0.25%) for assays.
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Reaction Scheme: Synthesis of N-3-Br benzyl noscapine: (i) a: m-CPBA, DCM; b: 2N HCl; C:
FeSO4.7H2O; (ii) 3-Bromo benzyl bromide, KI, K2CO3, Acetone, RT, 97%.
(b) Cell viability assay
The cell proliferation analysis was conducted in 96-well plates using a human breast
cancer cell line, MCF-7 at a density of 3 X 103 cells/well. The assay was performed with Br-Bn-
Nos at variable concentrations of 5, 10, 25, and 50 µM. Similarly, the cells were incubated with
DOX at a variable concentration of 0.001, 0.01, 0.1 and 1 µM. After 24 h and 48 h of treatment,
the viability of the cell was checked by 3-(4, 5-dimethylthiazol-2-yl)-2, 5, ditetrazolium bromide
(MTT) assay. The plate was read at a wavelength of 570 nm in a (Varioskan, Thermo Scientific)
flash multimode reader. Fifty percent inhibitory concentration (IC50) of tested drugs was obtained
by transferring the data into a graphic program (e.g., Excel) and expressed as a percentage of the
untreated controls and then evaluated by Logit regression analysis using a pre-programmed excel
spreadsheet obtained from MMV group at Swiss Tropical Institute, Basel, Switzerland (Pandey et
al. 2016).
(c) In vitro interaction of N-3-Br-Benzyl-noscapine and docetaxel
The cells were treated with a combination regimen of both Br-Bn-Nos and DOX by taking
variable concentrations (50 µM Br-Bn-Nos+0.001µM DOX, 25 µM Br-Bn-Nos+0.01 µM DOX,
10 µM Br-Bn-Nos+0.1 µM DOX and 5 µM Br-Bn-Nos+1 µM DOX) directly onto the medium
after 12h of cell attachment. The first and the last of these preparations had either Br-Bn-Nos or
DOX alone. The cancer cells, MCF-7 at a density of 3 X 103 cells/well were exposed for 48 h and
72 h to these dilutions followed by MTT assay. The IC50 was determined by logit regression
analysis. The fractional inhibitory concentration (FIC) was interpreted by the following formula:
퐶표푛푐.표푓 푑푟푢푔 푖푛 푐표푚푏푖푛푎푡푖표푛 푡표 푝푟표푑푢푐푒 퐼퐶50
퐹퐼퐶 =
퐶표푛푐. 표푓 푑푟푢푔 푎푙표푛푒 푟푒푞푢푖푟푒 푡표 푝푟표푑푢푐푒 퐼퐶50
The sum FIC value for each of the preparations determined by the following formula was used to
classify the drug–drug interaction.
퐼퐶50 표푓 푑푟푢푔 퐴 푖푛 푐표푚푏푖푛푎푡푖표푛
퐼퐶50 표푓 푑푟푢푔 퐴 푎푙표푛푒
퐼퐶50 푑푟푢푔 퐵 푖푛 푐표푚푏푖푛푎푡푖표푛
퐼퐶50 표푓 푑푟푢푔 퐵 푎푙표푛푒
푆푢푚 퐹퐼퐶 =
+
Sum FIC < 0.5 represents substantial synergism, sum FIC < 1 represents synergism, sum FIC = 1
represents additive interaction, sum FIC ≥ 1 represents antagonism. An Isobologram was plotted
to show the drug interaction as per the method proposed earlier (Pandey et al. 2016).
(d) Cell cycle analysis
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In Dulbecco's Modification of Eagle's Medium (DMEM), MCF7 cells were cultivated with
4.5 g/l glucose and L-glutamine, supplemented by 10% bovine fetal serum and 1%
penicillin/streptomycin. MCF-7 cells (1X105) were seeded in a 6-well culture plate overnight and
then treated with the indicated concentration of Br-Bn-Nos (20 μm) and DOX alone (0.1 μm) and
combination regimen (25 μm Br-Bn-Nos+0.01 μm DOX). Staining solution RNase (5µg/ml),
propidium iodide (5 µg/ml) and Triton X (0.1%) were used to determine the cell cycle phase and
analyzed by flow cytometer (FACS Calibur) to estimate the percentage of cells in the different
stages of the cell cycle. The experiment was performed in triplicates.
(e) Annexin V apoptosis assay
Choline phospholipids, like the phosphatidylcholine and sphingomyelin (PS), are subjected
on the outer leaflet throughout apoptosis, whereas amino phospholipids (phosphatidylserine,
phosphatidylethanolamine) are only found on the cytoplasmic edge of the lipid bilayer. Detection
of PS by fluorochrome-tagged 36 KDa anticoagulant protein Annexin V allows for accurate
estimation of apoptotic incidence. MCF-7 cells (5 X 104) were treated with Br-Bn-Nos (20 μm),
DOX (0.1 μm) or in their combination regimen (25 μm Br-Bn-Nos+0.01 μm DOX) for apoptosis
study using the Annexin V (BD Pharmingen, San Diego, CA, USA) binding assay according to the
manufacturer’s instructions. The cells were then analyzed using the BD FACS Calibur (San Jose,
CA, USA).
(f) Tubulin purification
Two cycles of temperature- and GTP-dependent polymerization and depolymerization in
the presence of 1 M glutamate were used to isolate microtubules from the goat brain (Hamel and
Lin, 1981). Purified tubulin was extracted from the microtubule proteins by phosphocellulose
chromatography based on the methods and procedures discussed earlier (Panda et al. 2000; Joshi
and Zohu, 2001) and the amount of purified tubulin was estimated using the Bradford method as
well as by SDS PAGE (Bradford, 1976). Aliquots were frozen in liquid nitrogen and preserved at -
80 degrees Celsius until required.
(g) Tryptophan fluorescence quenching assay with purified tubulin
Tubulin (2 µM) was treated with desired concentrations of Br-Bn-Nos (20 μM and 50 μM),
DOX (0.1 μM) and in combination regimen (DOX 0.01 μM+Br-Bn-Nos 25 μM) in a water bath
(35 °C; 45 min). They were then excited at 295 nm and the emission reading at 310–400 nm was
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obtained. A FlouroMax® 4 spectrofuorometer (Horiba Scientifc, Edison, NJ) supported by
FluorEssence 3.5 software was used for the spectrofluorimetric titrations.
(h) ANS binding assay with purified tubulin
Fluorescence probe 8-anilino-1-napththalene sulfonate (ANS) is mostly used for the
detection and analysis of conformational changes in proteins. Tubulin (2 µM) was incubated with
25 μM concentrations of Br-Bn-Nos, 0.1 μM concentration of DOX and in their combination
regimen (DOX 0.01 μM+Br-Bn-Nos 25 μM) for 30 min at 35 °C in PEM buffer. ANS (50 µM)
was then added to the samples and the samples were incubated in the dark (15 min; 25 °C). After
the incubation, they were excited at 350 nm and the emission spectra were taken at 410–470 nm
using a Flourolog 3 spectrofluorometer (Horiba Scientific, Edition, NJ) assisted by fluorescence
3.5 software. The assays were repeated two times.
Results
In a quest of increasing the anticancer activity of the noscapine, the –NCH3 group of
isoquinoline ring has been substituted with various functional groups based on in silico
combinatorial approach and screened out potent derivatives for chemical synthesis and
experimental evaluation (Manchukonda et al. 2013). Many of these derivatives have shown
anticancer activity at a lower concentration compared to noscapine. One of such derivative, N-3-
Br-Benzyl noscapine has revealed higher tubulin binding affinity compared to noscapine
(Manchukonda et al. 2013) and showed anti-proliferative activity using MDA-MB-231 breast
cancer cell line (Cheriyamundath et al. 2019). This derivative of noscapine is different from the
first generation noscapine derivatives (including 9-Br-noscapine) in which modification have been
done at C-9 position of the isoquinoline ring (Figure 1A,B). Here in this study, we have used N-3-
Br-Benzyl derivative of noscapine (Figure 1C) to evaluate its combined effect with docetaxel
towards better therapeutic outcome by minimizing the concentration of docetaxel to preclude its
side effect.
B. (a) Molecular modelling
In order to determine the predictive binding affinity of Br-Bn-Nos and DOX individually
and in their combination, we have docked them at their respective binding pockets on tubulin in
three cycles of molecular docking. In the first cycle, DOX and Br-Bn-Nos were docked into their
respective binding pockets independently. Both Br-Bn-Nos and DOX docked well into their
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binding site with a docking score of -4.99 and -5.88 kcal/mol respectively (Table 1). In the second
cycle, the co-complex of tubulin-DOX was taken and Br-Bn-Nos was docked onto its binding site
to determine the change in docking score in presence of DOX. The docking score of Br-Bn-Nos
was reduced to -6.03 kcal/mol, indicating improvement in its binding affinity in presence of DOX.
Similarly in the third cycle, DOX was docked into its binding site in the co-complex of tubulin-Br-
Bn-Nos to determine its change in docking score in presence of Br-Bn-Nos. The presence of Br-
Bn-Nos further reduced the docking score of DOX to -6.54 kcal/mol. The reduced docking score
in presence of Br-Bn-Nos and DOX together indicated the combined effect of both the ligands.
Table 1: Molecular docking results (Glide XPscore) and the relevent energy parameters of Br-Bn-
Nos and DOX in single as well as in combination with tubulin.
Glide XPscore
(kcal/mol)
-4.99
Glide Evdw
(kcal/mol)
-31.67
Glide Ecoul
(kcal/mol)
-8.29
Glide Energy
(kcal/mol)
-35.97
Ligands
Br-Bn-Nos
DOX
-5.88
-42.26
-11.96
-50.22
Br-Bn-Nos docked with
Tubulin_DOX complex
DOX docked with Tubulin_
Br-Bn-Nos complex
-6.03
-6.54
-39.07
-41.24
-5.46
-9.23
-52.53
-59.24
(b) MD simulation of the complex
The binding of both Br-Bn-Nos and DOX with tubulin were evaluated independently (Tub-
Br-Bn-Nos and Tub-DOX complexes) as well as in combination with tubulin (Tub-DOX+Br-Bn-
Nos) by a molecular dynamic simulation of 100 ns. To analyse the system's stability, the root
means square deviations (RMSD) of Cα-atoms were computed for all frames over the entire
duration of simulation (Figure 2). The fluctuation in the RMSD of Cα carbon atoms was very
small after equilibration and all of the systems were found to be stable after 20 ns of simulation.
The root mean square fluctuations (RMSF) of Cα-atoms were also measured to see if there were
any changes in residue flexibilities during the entire duration of MD simulation (Figure 3). The
residues with higher RMSF tend to show more flexibility. Both Br-Bn-Nos and DOX were found
to bind with tubulin during the entire duration of the simulation. However, the topmost five
ligand-tubulin complexes based on the lowest total energy from the MD simulation trajectory were
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obtained to generate the average structure in order to analyze the binding mode of ligands. Both
Br-Bn-Nos and DOX were found to accommodate well inside the binding cavity. The Br-Bn-Nos
docked well at the interface of α- and β- tubulin, whereas the binding of DOX is biased more
towards β-tubulin (Figure 4a, b). Their binding mode with the tubulin was represented by ligplots
in single as well as in combination (Figure 5a-d). The binding site amino acids involved in the
binding of Br-Bn-Nos independently and in the presence of DOX were found albite different,
which may be due to the change in conformation of tubulin in presence of DOX. None of the
binding site amino acids were involved in hydrogen bonding with the Br-Bn-Nos when it was
docked individually onto tubulin (Figure 5a). In contrast, the binding site amino acids Asp 357(D),
Cys 356(D), Cys 241(D), and Gln 247(D) were involved in making 6 hydrogen bonding with Br-
Bn-Nos in presence of DOX (Figure 5b). Parenthetically, the binding site amino acids Arg 278(D)
and Arg 284(D) are involved in 2 hydrogen bonding with DOX when it was docked individually
onto tubulin (Figure 5c). In contrast, the binding site amino acids Gly 370(D), Arg 278(D), Arg
284(D), and Gln 281(D) are involved in making 9 hydrogen bonds with DOX in presence of Br-
Bn-Nos (Figure 5d).
(c) Calculated free energy of binding of Br-Bn-Nos and DOX with tubulin
The free energy of binding and its respective components of both Br-Bn-Nos and DOX
with tubulin were calculated independently as well as in combination and presented in Table 2.
The last 250 frames from the last 5 ns of trajectory were considered to calculate the ensemble
average of the free energy of binding using both MM-GBSA and MM-PBSA methods. The free
energy of binding for Br-Bn-Nos and DOX with tubulin was found to be -28.89 and -36.07
kcal/mol based on MM-GBSA as well as -26.21 and -34.65 kcal/mol based on MM-PBSA,
respectively. Further, the free energy of binding of Br-Bn-Nos was reduced to -33.02 and -30.24
kcal/mol using MM-GBSA and MM-PBSA calculation when DOX was present on its binding
pocket, indicating combination effect of both the ligands. Parenthetically, the free energy of
binding of DOX was reduced to -37.17 and -32.80 kcal/mol using MM-GBSA and MM-PBSA,
respectively when Br-Bn-Nos was present in binding tubulin. For all complexes, the binding
energy was decomposed into its various energy components (the electrostatic, van der Waals, and
solvation). Both van der Waals (ΔEVDW) and the electrostatic component (ΔEELE) were observed to
make very significant contributions to the free energy of binding. However, the net polar
contribution (ΔG(ele,PB/GB) = ΔEele + ΔG(PB/GB)) was rendered unfavorable due to very large penalty
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imposed by the desolvation component (ΔGPB/GB) while the net nonpolar component (ΔEvdw) and
(ΔGsol-np) were observed to make a highly favourable contribution to the free energy of binding.
Table 2: Predicted free energy of binding (∆Gbind,pred) and its various componentes (kcal/mol) of
Br-Bn-Nos and DOX in single as well as in combination binding with tubulin. The values in bold
represent the (∆Gbind,pred of molecules with tubulin based on MM-GBSA and MM-PBSA methods.
Energy
Component
ΔEVDW
Tub_DOX+
Br-Bn-Nos
-41.24
Tub_Br-Bn-Nos+
DOX
Br-Bn-Nos
Dox
-31.61
-286.66
-333.46
308.85
-3.62
-42.26
-21.21
-57.26
39.70
-4.61
-39.07
-30.00
-65.40
32.98
ΔEELE
-303.55
-348.88
307.49
-3.69
ΔEGAS
ΔGGB-Polar
ΔGSOL-NP
ΔGSOL-GB
ΔGbind-GBSA
ΔGPB
-4.65
279.35
-28.89
323.16
-3.54
29.19
-36.07
30.62
-4.51
323.90
-33.02
31.03
-37.17
36.69
335.94
-3.36
ΔGSOL-NP
ΔGSOL-PB
ΔGbind-PBSA
-4.29
279.22
-26.21
29.61
-34.65
320.48
-30.24
33.69
-32.80
B. Biology
(a) Inhibition of cellular proliferation
The Br-Bn-Nos inhibited proliferation of MCF-7 cells in a dose-dependent manner with
IC50 values of 11.5 μM and 7.71 μM respectively at 48 h and 72 h (Figure 6a). Similarly, the DOX
showed IC50 values of 0.39 μM and 0.016 μM against MCF-7 cells respectively at 48 h and 72 h
(Figure 6b). In order to minimize the toxicity of DOX and to enhance the antiproliferative efficacy
we have examined the antiproliferative activity with a combination of a lower concentration of
DOX and a higher concentration of Br-Bn-Nos. Approximately, 50% inhibition of cellular
proliferation was achieved in a combination regimen of Br-Bn-Nos (5 µM) and DOX (1µM) after
48 h and 72 h post-treatment (Figure 6c). Further, the interaction of both the agents has been
analyzed on the basis of their sum FICs and isobologram plot (Figure 7). The sum FICs value was
found to be 0.49 and 0.62 respectively at 48 h and 72 h. The isobologram of N-3-Br-Benzyl-
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noscapine with docetaxel suggests that the combination has synergistic antiproliferative activity
both at 48 h and 72 h exposure (sum FIC <1).
(b) Cell cycle effect
Both noscapinoids and docetaxel have been reported to bind tubulin and inhibit the cell
cycle progression at G2/M phase, followed by induction of apoptosis to cancer cells (Ye et al.
1998; Dash et al. 2020). We examined the inhibition in cell cycle progression of MCF-7 cells with
the treatment of Br-Bn-Nos and DOX in single as well as in combination regimens. The cells were
treated with 20 μM of Br-Bn-Nos and 0.1 μM of DOX in the single regimen, whereas 25 μM Br-
Bn-Nos+0.01 μM DOX for the combination regimen. The cell cycle distribution was evaluated via
flow cytometric analysis. The presence of 2N DNA indicates that the cells are in the G1 phase,
while the accumulation of duplicated 4N DNA indicates that the cells are in G2 and M phases.
Accumulation of DNA in between 2N and 4N peaks represent, the cells are in the S phase. In
contrast, less than 2N DNA indicates the apoptotic cells in which the DNA is degraded to different
extents (sub-G1 phase). As shown in Figure 8, there is a high accumulation of cells in the G2/M
phase. In contrast to the G2/M phase block, cells with sub-G1 phase were found to increase
compared to the control after 48 h of treatment in single and in a combination regimen. The sub-
G1 population with the treatment of Br-Bn-Nos was increased to 15.4%, whereas with DOX it was
increased to 16.1% and in combination, it further increased to 23.2% in comparison to control.
The combined effect of both Br-Bn-Nos and DOX on cell cycle progression could be useful for
the induction of apoptosis.
(c) Induction of apoptosis
Induction of apoptosis to MCF-7 cells was investigated using PE Annexin V and 7-AAD
apoptosis kit. The representative dot-plots illustrating apoptotic status was shown in Figure 9. The
percentage of apoptotic cells (early apoptotic and late apoptotic cells) treated with Br-Bn-Nos (25
μm) + DOX (0.01 μm) was 24.1% and 20.3%, which was significantly high compared to single
regimen treatment with 20 μm of Br-Bn-Nos (11.0 % and 3.73 %) or 0.1 μm of DOX (10.9 % and
3.80 %) respectively after 48h in comparison to controlled untreated cells. This study
demonstrated that the proposed combination effect of Br-Bn-Nos and DOX would not only
potentially induced apoptosis to the cancer cell, but also provide a promising prospect of order to
reduce the toxicity of DOX.
(c) Both Br-Bn-Nos and DOX bind tubulin in single and in combination regimen
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In order to experimentally validate our findings based on molecular modelling study with
respect to the tubulin-binding affinity of Br-Bn-Nos and DOX in single as well as in their
combination, we determined the quenching of fluorescence intensity of tubulin with the treatment
of both the agents in single as well as in combination regimen. Due to the presence of the aromatic
amino acid tryptophan, the tubulin is autofluorescence in nature. Any chemical compounds that
bind with tubulin and alter its conformation leads to a decrease in its intrinsic fluorescence. The
relative percentage of decrease in fluorescence intensity was 31.01% with the treatment of 25 µM
of Br-Bn-Nos and 58% with the treatment of 0.1 µM of DOX, suggesting that both the agents bind
to tubulin with different affinity. Further, the fluorescent intensity of tubulin was reduced to a
higher value of 68% with the combination treatment of Br-Bn-Nos (25µM) and DOX (0.1µM)
(Figure 10). Significant reduction in tubulin fluorescence intensity in the combination regimen of
Br-Bn-Nos and DOX indicate co-binding of both the ligands with tubulin.
(d) Effect of Br-Bn-Nos and DOX on conformational changes of tubulin
In order to further investigate the structural changes on tubulin due to binding of Br-Bn-
Nos and DOX in single as well as in combination regimen, we probed the tertiary structure of the
protein using an ANS-binding assay. ANS is a fluorescent probe, whose fluorescence improves
when attached to protein. ANS, when bound to hydrophobic patches on proteins, shows enhanced
fluorescence. An increase in ANS fluorescence of tubulin suggests a loss of protein structural
integrity. Purified tubulin with the treatment of Br-Bn-Nos (25μM) and DOX (0.1 μM) showed an
increase in tubulin-ANS fluorescence intensity (Figure 11). It displayed a 29.8% increase in
fluorescence intensity at 25 µM Br-Bn-Nos, and 40.39% in presence of DOX (0.1 µM) compared
to unbound tubulin. Similarly, in combination treatment with Br-Bn-Nos (25 μM) and DOX (0.1
μM) the tubulin-ANS fluorescence intensity was further increased to 57.50% respectively,
indicating a gradual incremental perturbation of the structural integrity of the tubulin by the
binding of both the agents together.
Discussion
Poor clinical prospects of the current chemotherapy have triggered the development of new
treatment modalities such as combination therapy for breast cancer. It is becoming well-
appreciated that a toxic drug at its maximum tolerated dose given intermittently is not necessarily
better and there exists an opportunity to reduce its dose levels by using combination regimens of
drugs that display synergistic interactions (Jordan and Wilson, 2004). The presence of diverse
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drug binding sites on tubulin suggests that rational combination of two or more drugs of anti-
microbial therapy might be able to enhance the anti-cancer efficacy and reduce toxic side effects,
thereby improving the therapeutic index. Tubulin binding drugs such as taxanes and vinca
alkaloids are the most efficient anticancer agents for breast cancer treatment (Chougule et
al.2011). However, due to the emergence of drug resistance and the subsequent serious side
effects, their therapeutic utility is limited (Doddapaneni et al. 2016). In contrast, noscapine, a less
toxic orally administered antimicrotubular agent, clearly indicates antitumor activity in vitro and
in vivo against a number of cancers (Laden et al. 2002; Zohu et al.2002; Zhou et al. 2006).
Therefore, to decrease the severe side effects of docetaxel and increase its anti-cancer
effectiveness we adopt a combination treatment with one of the derivatives of noscapine i.e. Br-
Bn-Nos. Similar studies have been conducted previously to demonstrate the combination effect of
DOX and other anticancer agents for better therapeutic efficacy (Dash et al. 2020; Galsky and
Vogelzang, 2010; Fisusi and Akala, 2019).
In this study, we have demonstrated the combined effect of Br-Bn-Nos and DOX based on
molecular modeling and cellular study. Molecular docking of Br-Bn-Nos and DOX, individually,
revealed a docking score of -4.99 kcal/mol and -5.88 kcal/mol respectively. However, the docking
score of Br-Bn-Nos was significantly reduced (-6.03 kcal/mol) when it was docked onto a co-
complex of tubulin-DOX and similarly docking score of DOX was reduced to a greater extent (-
6.54 kcal/mol) when it was docked onto a co-complex of tubulin-Br-Bn-Nos, indicating co-
binding of both the agents simultaneously to tubulin at two different binding pockets. Further, the
significant reduction in free energy of binding based on MD simulation in combination with MM-
GBSA and MM-PBSA calculation in presence of Br-Bn-Nos and DOX together compared to their
individual binding with tubulin indicated a combination effect of both the ligands. The result
obtained from theoretical prediction was validated through experiments by tubulin-binding assay
and tubulin-ANS binding assay. Combination treatment of Br-Bn-Nos and DOX reduced the
fluorescence intensity of tubulin to a higher value compared to their single regimen treatment,
indicating the combined effect of both the agents. Similarly, a significant increase in fluorescent
intensity of tubulin-ANS with the treatment of Br-Bn-Nos and DOX in combination regimen
compared to single regimen treatment suggest co-binding of both the agents to tubulin. The
combined effect of both Br-Bn-Nos and Dox was also revealed from the cellular study using the
MCF-7 cancer cell line. Combination treatment of Br-Bn-Nos and DOX was found to be more
effective in inhibiting cellular proliferation, blocking of cell cycle progression and induction of
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apoptosis compared to single-drug treatment. Further, the isobologram analysis (sum FIC <1) of
docetaxel with Br-Bn-Nos indicate significant synergism at 48 h and 72 h exposures.
In conclusion all these results taken together established a proof-of-concept that a rational
combination of Br-Bn-Nos and DOX could generate synergistic effects on cancer treatment, which
is a highly promising and novel approach for the treatment of breast cancer. Further studies need
to be conducted to better understand the growth inhibition molecular mechanism in the combined
treatment of breast cancer cells.
Acknowledgments
We would like to acknowledge the financial assistant provided by the Department of
Science and Technology, Government of India (Award no- DST/INSPIRE/Code No.: IF170022)
for providing student research fellowship to Shruti Gamya Dash and also the financial support by
OHEPEE, Govt. of Odisha through World Bank. We are grateful to Dr. Manu Lopus and UM-
DAE Centre for Excellence in Basic Sciences for providing extended facilities.
Conflict of Interest
The authors declare no conflict of interest.
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Legends of Figures
Figure 1: Structures of the (A) first generation noscapinoids that identifies the 9th position
modification in the noscapine scaffold, (B) molecular structure of the 9-Br-Noscapine and (C) N-
3-Br Benzyl Noscapine.
Figure 2: Root mean square deviations (RMSD) of Cα carbon atoms of tubulin only and in
complex with Br-Bn-Nos (Tubulin+ Br-Bn-Nos), with docetaxel (Tubulin+DOX) and with both
docetaxel and Br-Bn-Nos (Tubulin+DOX+Br-Bn-Nos) during 100 ns of MD simulation. The
relative fluctuation in the RMSD of the Cα atoms is very small after ~ 20 ns of the simulation. The
time step of 20 ps was used during the simulation. The topmost 5 frames from the MD simulation
trajectory with lowest total energy were considered to generate the average structure.
Figure 3: Root mean square fluctuation (RMSF) of the residues of tubulin of the docked ligands
in the bound form and in the unbound form of tubulin heterodimer. Different levels of flexibility
of these residues were noticed in the bound form of tubulin with Br-Bn-Nos and DOX in single as
well as in combination. Most of the residues showed flexibilities >5 Å in case of tubulin bound
with Br-Bn-Nos and DOX as compared to the free tubulin heterodimer, indicating that these
residues seem to be more flexible as a result of binding.
Figure 4. (a) Both Br-Bn-Nos and DOX are well accomodated inside their respective binding site
of tubulin. (b) Snapshot of both the ligands obtained. The binding site is represented as
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macromodel surface according to α- and β- tubulin (α-tubulin is represented in blue colour and β-
tubulin is represented in brown colour).
Figure 5. The ligplot analysis showing the binding mode of (a) Br-Bn-Nos with tubulin, (b) Br-
Bn-Nos with the co-complex of Tubulin-DOX, (c) DOX with tubulin, and (d) DOX with the co-
omplex of tubulin-Br-Bn-Nos. The binding mode of Br-Bn-Nos was different when docked to
tubulin and to the co-complex of tubulin-DOX, in which different number of hydrogen bondings
were involved with the binding site amino acids. Similarly, the binding mode of DOX was also
different when docked to tubulin and to the co-complex of tubulin-Br-Bn-Nos, in which different
number of hydrogen bonding involved with the binding site amino acids. The hydrogen bonds
formed (if any) are represented as dotted lines and the value represents the bond distance.
Figure 6. Br-Bn-Nos and DOX in single as well as in combination regimen at different
concentrations inhibit cellular proliferation of human breast cancer cell, MCF-7 after 48 h and 72
h treatment. The IC50 value amounted to 11.5 µM and 7.71 µM, respectively for 48h and 72h with
Br-Bn-Nos. Similarly, the IC50 value amounted to 0.39 µM and 0.016 µM, respectively for 48h
and 72h with DOX. In contrast, approximately 50% inhibition of cellular inhibition was achieved
in a combination regimen of Br-Bn-Nos (5µM) and DOX (0.001µM) after 48 h and 72 h post-
treatment.
Figure 7. Isobolograms showing in vitro interactions between Br-Bn-Nos and DOX. Sum FIC <
0.5 represents substantial synergism, sum FIC < 1 represents synergism, sum FIC = 1 represents
additive interaction, and sum FIC >1 represents antagonism between two drugs.
Figure 8. Figure A-D depict represented figures of analyses of cell cycle distribution in a two-
dimensional disposition as determined by flow cytometry in MCF-7 cells treated with 20 µM of
Br-Bn-Nos, 0.1 µM of DOX as single regimen and 25 µM of Br-Bn-Nos + 0.01 µM of DOX in
combination regimen. Both Br-Bn-Nos and DOX inhibit cell cycle progression at mitosis followed
by the appearance of a characteristic hypodiploid (sub-G1) DNA peak, indicative of apoptosis.
Figure 9. Analysis of apoptosis cell death induced by Br-Bn-Nos alone and in combination with
DOX based on flow cytometric analysis. PE conjugate of Annexin V was used in combination
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with 7-Amino-Actinomycin (7-AAD) to distinguish among 3 subpopulations: PE-- and 7-AAD --
population indicates viable cells (bottom left quadrant); PE -- and 7-AAD + population indicates
early apoptotic cells (lower right quadrant); PE + and 7-AAD + population indicate late apoptotic
cells (top right quadrant).
Figure 10. Decrease of fluorescence intensity of tubulin by Br-Bn-Nos and DOX in single as well
as in combination regimen. Tubulin (2.0 μM) was incubated with Br-Bn-Nos (25 µM) and DOX
(0.1 µM) alone as well as in combination regimen (25 µM of Br-Bn-Nos and 0.01 µM of DOX)
and the emission spectra were collected (310 nm – 400 nm). Both Br-Bn-Nos and DOX in single
as well as in combination regimen showed a concentration-dependent quenching of the intrinsic
tubulin fluorescence emission intensity indicating the binding of both Br-Bn-Nos and DOX to
tubulin. The more reduction in tubulin fluorescence intensity in combination regimen of both Br-
Bn-Nos and DOX, compared to their single binding, revealed combination effect with the tubulin.
The graph is a representative of three independent experiments.
Figure 11. Enhancement of tubulin-ANS fluorescence by Br-Bn-Nos and DOX in single as well
as in combination regimen. Tubulin (2.0 µM) was incubated without (control) or with Br-Bn-Nos
(25 µM), DOX (0.1 µM) and in their combination regimen (Br-Bn-Nos 25 µM+DOX 0.01 µM),
followed by incubation with ANS (50 µM). The samples were excited at 380 nm and the emission
spectra were collected (390 to 500 nm). The increase is more in tubulin-ANS fluorescence in
combination regimen of both Br-Bn-Nos and DOX, compared to their single binding, and revealed
combination effect with the tubulin. The graph is a representative of three independent
experiments.
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