Development of Novel Bis(indolyl)-hydrazide-Hydrazone Derivatives as Potent Microtubule-Targeting Cytotoxic Agents against A549 Lung Cancer Cells

Article abstract of DOI:10.1021/acs.biochem.5b01127

The biological significance of microtubules makes them a validated target of cancer therapy. In this study, we have utilized indole, an important pharmacological scaffold, to synthesize novel bis(indolyl)-hydrazide-hydrazone derivatives (NMK-BH compounds) and recognized NMK-BH3 as the most effective one in inhibiting A549 cell proliferation and assembly of tissue-purified tubulin. Cell viability experiments showed that NMK-BH3 inhibited proliferation of human lung adenocarcinoma (A549) cells, normal human lung fibroblasts (WI38) and peripheral blood mononuclear cells (PBMC) with IC₅₀ values of -2, 48.5, and 62 μM, respectively. Thus, the relatively high cytotoxicity of NMK-BH3 toward lung carcinoma (A549) cells over normal lung fibroblasts (WI38) and PBMC confers a therapeutic advantage of reduced host toxicity. Flow cytometry, Western blot, and immunofluorescence studies in the A549 cell line revealed that NMK-BH3 induced G2/M arrest, mitochondrial depolarization, and apoptosis by depolymerizing the cellular interphase and spindle microtubules. Consistent with these observations, study in cell free system revealed that NMK-BH3 inhibited the microtubule assembly with an IC₅₀ value of -7.5 μM. The tubulin-ligand interaction study using fluorescence spectroscopy indicated that NMK-BH3 exhibited strong and specific tubulin binding with a dissociation constant of -1.4 μM at a single site, very close to colchicine site, on β-tubulin. Collectively, these findings explore the cytotoxic potential of NMK-BH3 by targeting the microtubules and inspire its development as a potential candidate for lung cancer chemotherapy.

Full text of DOI:10.1021/acs.biochem.5b01127

Article
pubs.acs.org/biochemistry
Development of Novel Bis(indolyl)-hydrazide−Hydrazone
Derivatives as Potent Microtubule-Targeting Cytotoxic Agents
against A549 Lung Cancer Cells
Dipanwita Das Mukherjee,† N. Maruthi Kumar,‡ Mukund P. Tantak,‡ Amlan Das,† Arnab Ganguli,†
Satabdi Datta,† Dalip Kumar,*,‡ and Gopal Chakrabarti*,†
†Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35
Ballygunge Circular Road, Kolkata, WB 700 019, India
‡Department of Chemistry, Birla Institute of Technology and Science, Pilani, Rajasthan 333 031, India
S
* Supporting Information
ABSTRACT: The biological significance of microtubules
makes them a validated target of cancer therapy. In this
study, we have utilized indole, an important pharmacological
scaffold, to synthesize novel bis(indolyl)-hydrazide−hydrazone
derivatives (NMK-BH compounds) and recognized NMK-
BH3 as the most effective one in inhibiting A549 cell
proliferation and assembly of tissue-purified tubulin. Cell
viability experiments showed that NMK-BH3 inhibited
proliferation of human lung adenocarcinoma (A549) cells,
normal human lung fibroblasts (WI38) and peripheral blood mononuclear cells (PBMC) with IC50 values of ∼2, 48.5, and 62
μM, respectively. Thus, the relatively high cytotoxicity of NMK-BH3 toward lung carcinoma (A549) cells over normal lung
fibroblasts (WI38) and PBMC confers a therapeutic advantage of reduced host toxicity. Flow cytometry, Western blot, and
immunofluorescence studies in the A549 cell line revealed that NMK-BH3 induced G2/M arrest, mitochondrial depolarization,
and apoptosis by depolymerizing the cellular interphase and spindle microtubules. Consistent with these observations, study in
cell free system revealed that NMK-BH3 inhibited the microtubule assembly with an IC50 value of ∼7.5 μM. The tubulin−ligand
interaction study using fluorescence spectroscopy indicated that NMK-BH3 exhibited strong and specific tubulin binding with a
dissociation constant of ∼1.4 μM at a single site, very close to colchicine site, on β-tubulin. Collectively, these findings explore
the cytotoxic potential of NMK-BH3 by targeting the microtubules and inspire its development as a potential candidate for lung
cancer chemotherapy.
there is an urgent need for the development of more effective
icrotubules, the cylindrical polymers, composed of αβ-
Mtubulin subunits, are the major component of the and selective microtubule-targeting anti-mitosis agents against
lung cancer.
eukaryotic cytoskeleton and mitotic spindle that play a crucial
role in a variety of cellular processes, including mitosis.1−3
Many clinically used microtubule-targeting drugs such as Taxol
and vinblastine have been reported to interfere with the
microtubule dynamics, leading to mitotic block and apoptosis
in cancer cells.4−6 Recently, many natural products and their
derivatives such as genistein and WTC-1 with anti-tubulin
activity have also been recognized as potent cancer chemo-
therapeutic agents.7,8 Thus, the microtubule is regarded as one
of the most attractive molecular targets for anticancer drugs.
Globally, among all human cancers, lung cancer is the leading
cause of cancer mortality.9,10 On the basis of histology, lung
cancers are classified as small-cell lung cancer (SCLC) and non-
small cell lung cancer (NSCLC). However, 80−85% of all lung
cancer cases are attributed to NSCLC.11 Introduction of
microtubule-targeting chemotherapeutics caused a significant
improvement in remission of the disease, but the nonspecific
toxicity-related side effects and development of chemo-
resistance limit the clinical success of these drugs.11,12 Hence,
The indole ring system is one of the most crucial
heterocycles that has found versatile pharmacological implica-
tions in medicinal chemistry.13,14 Many of the natural and
synthetic indole-based heterocycles with various functionalities
are reported as potential anticancer molecules.15−17 Recently,
the diverse biological activities of hydrazide−hydrozones have
attracted the researchers to develop their heterocyclic analogues
as efficient chemotherapeutic agents.18−21
The aim of this study was to identify and elucidate the
cytotoxic mechanism of potential microtubule-targeting chemo-
therapeutic agents against a lung cancer (A549) cell line. To
achieve our goal, we chemically synthesized bis(indolyl)-
hydrazide−hydrazones, denoted as NMK-BH, from indole-
2(3)-carboxylic acid hydrazide and indole-3-carboxaldehyde,
Received: October 16, 2015
Revised: April 18, 2016
Published: April 25, 2016
© 2016 American Chemical Society
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a
Scheme 1. Synthesis of Bis(indolyl)-hydrazide−Hydrazone (NMK-BH1−6) Derivatives
a
Reagents and conditions: (a) diluted H2SO4, EtOH, reflux, 80−90%; (b) NH2NH2, H2O, EtOH, reflux, 85−90%; (c) DMF-POCl3, 90−95%; (d)
AcOH, EtOH, reflux, 70−90%.
wherein the indole rings were linked by an active
pharmacophore (−CO−NH−NCH−). Modifications were
focused on incorporation of different functional groups, an
electron-withdrawing (Br) and/or electron-releasing (−OCH3
or 4-OCH3C6H4) substituent, at different positions of the
indole (A or B) moiety, and altering the location of the indole
(A)−indole (B) linkage between positions C-2 and C-3 of the
indole ring in the bis(indolyl)-hydrazide−hydrazone skeleton.
These newly synthesized compounds were well characterized
by infrared (IR), nuclear magnetic resonance (NMR) (1H and
13C), and mass spectral analysis.
The cytotoxic efficacies of the ligands (NMK-BH1−6) were
evaluated on the basis of their antiproliferative activity against
lung adenocarcinoma (A549) cells, and microtubule assembly
inhibitory activity indicated that NMK-BH3 is the most active
ligand of the series. NMK-BH3 also inhibited the proliferation
of different human cancer cell lines. High cytotoxicity of NMK-
BH3 toward cancer cell lines motivated the elucidation of the
possible cytotoxic mechanism of this ligand. Here, we are
reporting that the novel ligand, NMK-BH3, induced mitotic
arrest and mitochondria-mediated apoptosis in A549 cells
through tubulin binding. Confocal microscopy was employed
to visualize the microtubule depolymerization in cultured A549
cells, while the light scattering assay and transmission electron
microscopy (TEM) analysis established the inhibitory effect of
NMK-BH3 on microtubule assembly in cell free system. The
modes of interaction of the test compound with tubulin and its
binding site were studied using fluorescence spectroscopy, and
molecular docking was performed to confirm that NMK-BH3
binds to tubulin very close to the colchicine binding site on
tubulin.
instrument. High-performance liquid chromatography (HPLC)
analyses were performed on a Waters 515 HPLC system.
DMEM and an antibiotic and antimycotic solution were
purchased from Himedia. FBS and 0.25% trypsin-EDTA were
from GIBCO (Invitrogen, Auckland, New Zealand). DAPI,
colchicine, vinblastine, and podophyllotoxin were from Sigma-
Aldrich (St. Louis, MO). The annexin V-FITC-PI apoptosis
assay kit, primary antibodies to α-tubulin, Bax, p53, and Bcl 2
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Cleaved caspase 9 and cleaved caspase 3 were from Cell
Signaling Technology. The Bradford protein estimation kit and
TRITC-conjugated secondary antibodies were from Genei.
Flow cytometry was accomplished by FACScalibur (Becton-
Dickson), and data analysis was performed using CellQuest Pro
(Becton-Dickson). All other chemicals and reagents were
obtained from Sisco Research Laboratories.
General Procedure for Synthesis of Bis(indolyl)-
hydrazide−Hydazones (NMK-BH1−6). The intermediates
indole-2(3)-carbohydrazide (2) and indole-3-carboxaldehyde
(4) were prepared starting from commercially available indole,
as described previously.17,18,22,23 Indole-3-carboxylic acids 1
were prepared from the reaction of indole with trifluoroacetic
anhydride followed by hydrolysis with sodium hydroxide. To
obtain the corresponding ester, a solution of indole-3-carboxylic
acid 1 (1 mmol) in absolute ethanol (20 mL) was refluxed with
a catalytic amount of concentrated sulfuric acid for 20 h and the
residue was extracted with ethyl acetate (30 mL), washed with a
saturated sodium bicarbonate solution (20 mL), dried, and
evaporated. As shown in Scheme 1, a solution of the
appropriate ester (1 mmol) and hydrazine hydrate (2 mmol)
in ethanol (15 mL) was refluxed for 24 h and cooled, and the
solid that was obtained was filtered and recrystallized from
ethanol to give pure indole-2(3)-carbohydrazide (2) in 85−
90% yield.
EXPERIMENTAL PROCEDURES
■
Materials. All the reagents and solvents were purchased
from Merck and Aldrich. The reaction was monitored by thin
layer chromatography. Melting points were determined with
electrothermal capillary melting point apparatus (EZ melting)
and are uncorrected. IR and NMR (1H and 13C) spectra were
recorded on a Shimadzu FT-IR spectrophotometer and a
Bruker Advance II spectrometer, respectiely. Mass spectra were
recorded on a “Hewlett-Packard” HP GS/MS 5890/5972
For preparation of indole-3-carboxaldehydes 4, with
continuous stirring, phosphorous oxychloride (8.41 mL, 90
mmol) was added to 0 °C dimethylformamide (28 mL, 370
mmol) for 30 min followed by addition of a solution of indole 3
(85.47 mmol) in DMF (10 mL, 130 mmol) at room
temperature until it became a yellow paste. At the end of the
reaction, 30 g of crushed ice was added to the paste followed by
dropwise addition of a sodium hydroxide solution (1 N
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solution, 100 mL) while the mixture was being stirred. The
resulting suspension was heated rapidly to 90 °C, cooled at
room temperature, and refrigerated overnight. The product was
filtered, washed with water (2 × 100 mL), and air-dried to
afford the pure indole-3-carboxaldehydes 4 in 80−90% yields
(Scheme 1).
dye JC-1 (2 μg/mL) in culture medium for 30 min at 37 °C,
washed and resuspended in PBS, and analyzed for red and
green fluorescence by flow cytometry.29 The excitation
wavelength of 488 nm was used to quantify the mitochondrial
population with the green (JC-1 monomers) and red (JC-1
agrregates) fluorescence at emission wavelengths of 535 and
595 nm, respectively.
Finally, to the mixture of indole-2(3)-carboxylic acid
hydrazide 2 (1 mmol) and indole-3-carboxaldehyde 4 (1
mmol) in absolute ethanol (20 mL) was added a catalytic
amount of glacial acetic acid, and the mixture was refluxed at 80
°C for 5 h and cooled and the solid that separated out was
filtered and recrystallized from ethanol to afford bis(indolyl)-
hydrazide−hydrazones in 75−90% yields (Scheme 1).
Cell Culture. Human non-small lung adenocarcinoma
(A549), human breast carcinoma (MCF-7 and MDA-MB-
231), human ovarian carcinoma (PA-1), and human
hepatocellular carcinoma (HepG2) cells and normal human
lung fibroblast (WI38) cells were cultured in nutrient mixture
DMEM supplemented with 10% FBS, 3.7 g/L sodium
bicarbonate, and a 1% antibacterial and antimycotic solution
containing penicillin, streptomycin, and amphotericin B. Cells
were grown at 37 °C in a humidified atmosphere of 5% carbon
dioxide and 95% air in an incubator.
Isolation of Human Peripheral Blood Mononuclear
Cells (PBMC). Whole blood was collected from healthy human
volunteers, who were not taking any medication. PBMC were
isolated by the difference in gradient density of Ficoll-
Histopaque (Histopaque 1077, Sigma-Aldrich). Whole blood
was layered on the top of an equal volume of Ficoll-Histopaque
and centrifuged at 400g for 30 min at room temperature. The
PBMC found at the plasma−Ficoll-Histopaque interphase was
carefully collected with a Pasteur pipet, washed twice with PBS
(300g for 5 min), and resuspended in DMEM supplemented
with 10% FBS, 3.7 g/L sodium bicarbonate, and a 1%
antibacterial and antimycotic solution containing penicillin,
streptomycin, and amphotericin B.
Cytotoxicity Assay. The growth inhibitory effects of the
ligands on human cancer cell lines and normal cells were
assessed by the MTT assay.24,25 Briefly, cultured A549, MCF-7,
MDA-MB-231, PA-1, HepG2 and WI38 cells and PBMC, in
logarithmic phase, were seeded on a 96-well microtiter plate (1
× 104 cells/mL) and treated with varying concentrations of the
ligands for 48 h. Then, 20 μL of the MTT solution (5 mg
mL−1) was added to each well followed by dissolution of the
formazan crystals formed in 100 μL of DMSO. Thereby, the
absorbance was measured at 570 nm on an enzyme-linked
immunosorbent assay reader (Multiskan EX, Labsystems). Data
were calculated as the percentage of inhibition by the following
formula:
Western Blot Analysis. The whole cell extracts of NMK-
BH3-treated A549 cells were prepared as described previ-
ously.30 For isolation of soluble and polymer fractions of
tubulin, the cells were lysed with hypotonic lysis buffer [20 mM
Tris-HCl (pH 6.8), 1 mM MgCl2, 2 mM EGTA, and 0.5% NP-
40] and centrifuged at 15000g for 15 min at room
temperature.31 The supernatant containing soluble tubulin
was separated from the pellet containing polymerized tubulin,
and the pellet was dissolved in the hypotonic lysis buffer. The
protein concentrations were estimated by the Bradford
method.32 An equal amount of protein (50 μg) was resolved
via sodium dodecyl sulfate−polyacrylamide gel electrophoresis
(10 to 12% gel) and electrotransferred to nitrocellulose
membranes. Subsequently, the membranes were blocked
overnight at 4 °C in 5% BSA and probed with the respective
primary antibodies followed by the appropriate HRP-
conjugated secondary antibody according to the dilution
specified by the manufacturer. The chemiluminescent signal
was detected using Super Signal West Pico Chemiluminescent
Substrate (Pierce, Rockford, IL).
Cell Cycle Analysis. A549 cells were grown in the presence
of different doses of NMK-BH3 for 18 h, harvested, fixed in ice-
cold methanol for 30 min at 4 °C, and incubated with a 1 mg/
mL RNase A solution at 37 °C for 4 h. Flow cytometric analysis
of the DNA content, stained with propidium iodide, was
performed with a flow cytometer.27,33
Immunofluorescence Microscopy. A549 cells, grown as a
monolayer on a glass coverslip, were treated with NMK-BH3
for 18 h. Subsequently, the cells were washed (with PBS), fixed
(2% formaldehyde for 30 min), and permeabilized (0.1%
sodium citrate and 0.1% Trition X for 15 min), and nonspecific
binding was blocked with 5% BSA. The cellular microtubule
was probed with the anti-α-tubulin primary antibody (1:200
dilution) followed by the TRITC-conjugated secondary
antibody (1:100 dilution), and the nuclear DNA was
counterstained with DAPI. The images were obtained with an
Olympus LSM IX81 confocal microscope and analyzed with
Olympus Fluoview.
Tubulin Preparation. Goat brains were subjected to two
cycles of temperature-dependent assembly and disassembly in
PEM buffer [100 mM PIPES (pH 6.9), 1 mM MgSO4, and 1
mM EGTA] in the presence of 1 mM GTP.25,34 Tubulin was
prepared by a third cycle of the temperature-dependent
assembly process in glutamate buffer [monosodium glutamate,
3 mM MgCl2, and 1 mM EGTA (pH 6.8)] upon addition of 1
mM GTP.35
% inhibition = 100 − (A /A ) × 100 %
[
]
(1)
t
s
where At and As are the absorbance of the test substances and
solvent control, respectively.7,26,27
Microtubule Polymerization Assay. Microtubule poly-
merization, in the absence and presence of NMK-BH
compounds, in microtubule assembly glutamate buffer upon
addition of 1 mM GTP was monitored turbidimetrically at 340
nm for 20 min at 37 °C using a JASCO V-630 UV−visible
spectrophotometer.36 A similar experiment was performed with
different concentrations of NMK-BH3, and the concentration
of NMK-BH3 required to inhibit tubulin polymerization by
50% compared to the control (i.e., IC50 value) was
determined.8
Apoptosis Assay. Apoptosis analysis was performed as
described previously.28 Briefly, A549 cells treated with different
concentrations of NMK-BH3 (0- 10 μM) were centrifuged,
resuspended in 500 μL of Ca+-enriched binding buffer at a
density of 1 × 106 cells/mL, labeled with annexin-V and PI,
according to the manufacturer’s protocol, and analyzed for
apoptosis by flow cytometry.
Determination of the Mitochondrial Transmembrane
Potential (Δψm). A549 cells treated with various doses of
NMK-BH3 for 24 h were stained with a fluorescent cationic
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Electron Microscopy. Tubulin was polymerized with GTP
(1 mM) in the presence of different concentrations of NMK-
BH3 at 37 °C, fixed with 0.5% glutaraldehyde, and negatively
stained with 1% uranyl acetate. The samples were viewed using
a Philips Fei Technai Spirit electron microscope. Images were
recorded at 43000× magnifications.
Fluorescence Measurements. All the fluorescence
measurements were taken in a Hitachi F-3010 fluorescence
spectrophotometer using a 1 cm path length cell. The recorded
fluorescence values were corrected for the inner filter effect
using the Lakowicz equation:
vinblastine, and subsequently, the fluorescence spectra were
recorded.
Modified Dixon Plot. The fluorescence of the mixture of 2
μM tubulin and varying concentrations of podophyllotoxin (0−
40 μM) in the presence of 3, 5, and 10 μM NMK-BH3 was
recorded at the emission maxima (440 nm) upon excitation at
350 nm. The experiment was repeated thrice, and the inhibitory
concentration (Ki) was calculated from a modified Dixon
plot.40
Nature of the NMK-BH3−Tubulin Interaction. The
fluorescence intensity of the NMK-BH3−tubulin complex (10
μM NMK-BH3 and 10 μM tubulin) was monitored at 440 nm,
with respect to time, in the absence or presence of 40 μM
vinblastine and 40 μM podophyllotoxin upon excitation at 350
nm. To calculate the off rate constant (koff), data were fitted to
the equation ln(F0 − Ft) = Kofft + constant, where F0 and Ft are
the fluorescence of NMK-BH3−tubulin complex (10 μM
NMK-BH3 and 10 μM tubulin) at infinite time and time t,
respectively.38
Molecular Modeling. In our study, we utilized the crystal
structure of αβ-tubulin [Protein Data Bank (PDB) entry 1JFF]
as a template for docking studies.41,42 The molecular structures
of NMK-BH3 and NMK-BH5 were drawn by using Chem
Draw and converted to .mol files. The docking was performed
using algorithm-based ligand docking program Discovery
Studio 2.5. The energy-minimized structures of tubulin (PDB
entry 1JFF) and the ligands were generated by using the
prepare protein module (using the CHARMm force field) and
the prepare ligand module of Discovery Studio 2.5, respectively.
In both cases, all parameters were set to default values (Table
S6B of the Supporting Information). The force field generated
42 different binding pockets on tubulin, and we used the most
stable (lowest-energy conformation) conformation of the
ligand to dock on those 42 different pockets by using the
cdocker protein ligand interaction module of Discovery Studio.
When the ligand is bound to any one pocket of tubulin, it gives
a maximum of 10 poses. We generated the cdocker energy of
each pose. We also measured the binding energy of each pose
of the tubulin−ligand interaction by using the calculate binding
energy module of Discovery Studio. This module used the
following formula:
F
= F
antilog[(Aex + Aem)/2]
(2)
corrected
observed
where Aex and Aem are the absorbance at the excitation and
emission wavelength, respectively.37 The absorbance measure-
ments were performed using a JASCO V-630 UV−visible
spectrophotometer in a quartz cuvette (1 cm path length).
Because NMK-BH3 has an absorbance maximum at 350 nm,
the fluorescence emission spectra were recorded from 400 to
500 nm using an excitation wavelength of 350 nm.
To analyze the time dependence of binding of NMK-BH3
with tubulin, 5 μM NMK-BH3 was added to 1 μM tubulin and
the fluorescence of the resulting NMK-BH3−tubulin complex
was recorded at 440 nm upon excitation at 350 nm for 30 min
at 25 °C.
Association Kinetics. For the study of association kinetics,
5 μM NMK-BH3 and 1 μM tubulin were used. The value of
ln(Emax − Et) was plotted against time, where Emax is the
maximal amount of enhanced fluorescence and Et is the
enhanced fluorescence at time t. The biphasic plot was analyzed
according to the method of Lambire and Engelborghs.
Emax − Et = A × e−α/t + B × e−β/t + C
(3)
where A and B are the amplitudes and α and β the observed
rate constants for the fast and slow phase, respectively. The
amplitude of the fast phase was low relative to that of the fast
phase. The apparent association rate constant (Kon) was
calculated as Kon = α/c, where α is the slope of the plot of
ln(Emax − Et) versus time and c is the concentration of the
ligand, NMK-BH3.38
To determine the specificity of the interaction of NMK-BH3
with tubulin, the fluorescence of NMK-BH3 (5 μM) in the
presence of tubulin and certain other proteins, each at 5 μM, at
the emission maxima (440 nm) was recorded.
The binding parameters for binding of NMK-BH3 to tubulin
were obtained by Scatchard plot analysis.25,38,39 The concen-
trations of the free and bound ligand were calculated from two
titrations of tubulin and NMK-BH3 at 25 °C for 10 min. In one
titration, NMK-BH3 (2 μM) was incubated with varying
concentrations of tubulin (0−5 μM), while in the other
titration, tubulin (2 μM) was incubated in the presence of
different concentrations of NMK-BH3 (0−10 μM). The
Scatchard plot was constructed from these fluorescence data,
using excitation and emission wavelengths of 350 and 440 nm,
respectively.
Determination of the NMK-BH3 Binding Site. For the
competition experiment, the tubulin−NMK-BH3 complex was
formed by incubating 10 μM tubulin and 10 μM NMK-BH3 at
25 °C for 10 min. Thereby, the preformed tubulin−NMK-BH3
complex was incubated in the absence and presence of varying
concentrations of podophyllotoxin (0−40 μM) or 40 μM
binding energy = protein−ligand complex energy
− (protein energy + ligand energy)
The cdocker energy and the binding energy for the tubulin−
ligand interaction were represented as means the standard
error of the mean (SEM) obtained from the valid binding
poses.
Data Analysis. The data are represented as means SEM.
The statistical analysis was conducted with one-way analysis of
variance, and P values of <0.05 were considered to be
statistically significant. The experiments were repeated at least
thrice.
RESULTS
■
Design and Synthesis of Bis(indolyl)-hydrazide−
Hydrazones (NMK-BH Compounds). In search of a novel
and more efficient microtubule-targeting chemotherapeutic
agent against lung cancer, we have synthesized six novel
bis(indolyl)-hydrazide−hydrazone (NMK-BH) compounds
based on the indole scaffold. Modifications were focused on
incorporation and variation of the nature and location of
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Table 1. In Vitro Cytotoxicity and Microtubule Polymerization Inhibitory Activity of Bis(indolyl)-hydrazide−Hydrazones
(NMK-BH1−6) and Colchicine
a
b
IC50 is the concentration of the compound required to inhibit the proliferation of A549 lung adenocarcinoma cells by 50%. Percent inhibition of
microtubule polymerization in the presence of the respective ligand (at 10 μM) and 5 μM colchicine, compared to control (without ligand). Data are
represented as averages of five independent experiments.
functional group substituents and altering the position of the
indole linkage.
All the compounds were synthesized following the procedure
outlined in Scheme 1. The structural elucidations were
ascertained on the basis of their IR, NMR (1H and 13C), and
MS spectral data (sections S1A and S1B of the Supporting
Information), and purity (≥98%) was determined using HPLC
(section S1B of the Supporting Information).
Screening of Bis(indolyl)-hydrazide−Hydrazones
Based on Cytotoxicity and Tubulin Polymerization
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Figure 1. Antiproliferative effect of NMK-BH3 on lung cancer and normal lung fibroblast cells and PBMC. Cytotoxicity of varying doses (0−100
μM) of NMK-BH3 on (A) A549 cells, (B) WI38 cells, and (C) PBMC upon 48 h treatment. Cell viability was assessed by the MTT assay, and data
are represented as means SEM [*P < 0.05 vs control (untreated cells)]. (D) Relative cell viability of A549 and WI38 cells and PBMC in the
presence of different concentrations (0−100 μM) of NMK-BH3 upon 48 h treatment. Representative data from three independent experiments (n =
3).
Inhibitory Potential. The antiproliferative activities of the
synthesized bis(indolyl)-hydrazide−hydrazone derivatives were
evaluated against human non-small lung adenocarcinoma
(A549) cells by using the MTT assay. As shown in Table 1,
NMK-BH-2, NMK-BH3, and NMK-BH-4 showed remarkable
cytotoxicity against A549 cells with IC50 (50% inhibitory
concentration) values of ∼7.5 0.6, ∼2 0.4, and 6 0.5 μM,
respectively, while NMK-BH-1, -5, and -6 were inactive with
IC50 values of >15 μM.
We further examined the potency of the bis(indolyl)-
hydrazide−hydrazone compounds on the basis of their effect
on the assembly of goat brain purified tubulin in cell free system.
The data listed in Table 1 indicate that with the exception of
NMK-BH-5, the other ligands of the series inhibited the
microtubule assembly with values ranging from 10 to 62%.
NMK-BH3 (10 μM) was the most effective compound in
inhibiting the tubulin polymerization by 62 2%, while NMK-
NMK-BH3 against MCF-7, MDA-MB-231 (human breast
adenocarcinoma), HepG2 (human hepatocellular carcinoma),
and PA-1 (human ovarian carcinoma) cells were determined to
be ∼4.5 0.5, ∼6 0.5, ∼10 1, and 22 2 μM, respectively
(section S2a of the Supporting Information). These results
indicated that in addition to A549 cells, NMK-BH3 also
exhibited significant growth inhibitory activity against MCF-7,
MDA-MB-231, and HepG2 cells while it was inactive against
PA-1 cells.
Differential Cytotoxic Potential of NMK-BH-3 be-
tween Human Cancer Cell Lines and Normal Cells. As
the nonspecific cytotoxicity of the anticancer drugs is one of the
major obstacles in cancer chemotherapy, we examined the
specificity of NMK-BH3 toward A549 (human lung adeno-
carcinoma) cells compared to WI38 (normal human lung
fibroblast) cells. NMK-BH3 inhibited the proliferation of A549
cells and WI38 cells (Figure 1A,B and section S2b of the
Supporting Information) in a dose-dependent manner.
Comparison of the IC50 values demonstrated that NMK-BH3
was ∼25 times less cytotoxic toward WI38 cells (IC50 = 48.5
μM) than toward A549 cells (IC50 = 2 μM) (Figure 1D).
Further, we have also investigated the cytotoxicity of NMK-
BH3 on PBMC, and the IC50 was observed at a concentration
of 62 μM, which is ∼30-fold higher than that of A549 cells
(Figure 1C,D and section S2b of the Supporting Information).
Moreover, the comparative study of the IC50 values revealed
that the toxicities of NMK-BH3 toward MCF-7, MDA-MB-231,
HepG2, and PA-1 cells were ∼14-, ∼10-, ∼6-, and ∼3-fold
lower, respectively, when compared to that for PBMC. Thus,
we concluded that NMK-BH3 exhibits significant cytotoxicity
toward different human cancer cell lines while inducing
minimal toxicity in normal cells. Because lung cancer is the
BH2 and NMK-BH4 inhibited tubulin self-assembly by 44
2
and 53 3%, respectively, at 10 μM. However, NMK-BH1 and
NMK-BH6 (10 μM) did not exhibit discernible tubulin
polymerization inhibitory activity.
These results suggest that the growth inhibitory activities of
the compounds follow a good correlation with their abilities to
inhibit tubulin polymerization. Thus, on the basis of the order
of the inhibitory effect on A549 cell proliferation and tubulin
polymerization (NMK-BH3 > NMK-BH4 > NMK-BH2 >
NMK-BH1 = NMK-BH6 > NMK-BH5), compound NMK-
BH3 was selected as the lead ligand for further study.
Moreover, NMK-BH3 also inhibited the proliferation of
several other types of human cancer cell lines in a dose-
dependent manner (section S2a of the Supporting Informa-
tion). The half-maximal inhibitory concentrations (IC50) of
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Figure 2. NMK-BH3 induced dose-dependent apoptosis in A549 cells. Control and 24 h NMK-BH3-treated cells were labeled with Annexin-V-
FITC and PI, and the percentage of cells found in different regions of the dot plot was obtained by flow cytometric analysis. The experiment was
repeated thrice.
most common cause of cancer-related motility worldwide, we
used A549 cells to understand the mechanism of cytotoxicity of
NMK-BH3 for further study.
apoptosis being executed via the mitochondrial intrinsic
pathway.
Effect of NMK-BH-3 on the Activation of the Caspase-
Dependent Intrinsic Apoptotic Pathway. To elucidate the
detailed signaling pathway of NMK-BH-3-induced cell death,
the expression of apoptosis-related proteins was analyzed by
Western blot. We observed that NMK-BH3 treatment led to a
remarkably increased level of expression of Bax, with a
concomitant reduction in the level of Bcl-2 expression at
both tested concentrations (5 and 10 μM). Moreover, the levels
of expression of p53, caspase 3, and caspase 9 were found to
increase in a dose-dependent manner upon exposure to NMK-
BH3 (Figure 3C).
Effect of NMK-BH3 on Cell Cycle Progression of A549
Cells. The cell cycle progression of A549 cells in response to
NMK-BH3 was assessed by flow cytometric analysis of the
cellular DNA content. Treatment with NMK-BH3 was found
to induce significant dose-dependent G2/M arrest, which was
accompanied by remarkable reduction in G0/G1 and S phase
populations of A549 cells as shown in Figure 4A and section
S3C of the Supporting Informatio. The NMK-BH3-induced
cell cycle arrest pattern was comparable to that observed in the
presence of colchicine, a classical microtubule-disrupting agent
(sections S3B and S3C of the Supporting Information).
Effect of NMK-BH3 on the Cellular Microtubule
Organization of A549 Lung Cancer Cells. As NMK-BH3
successfully inhibited the assembly of purified tubulin and
induced strong G2/M arrest, we monitored the effect of NMK-
BH3 on the integrity of the mitotic spindle and cellular
microtubule network of A549 cells by confocal microscopy. In
Effect of NMK-BH-3 on Apoptosis. The lung adenocarci-
noma (A549) cells treated with varying concentrations of
NMK-BH3 for 24 h were subjected to biparametric flow
cytofluorimetric analysis of apoptosis using the annexin V-PI
assay.28 We found that NMK-BH3 caused significant
accumulation of early (annexin-V+-PI−) and late (annexin-V+-
PI+) apoptotic populations of A549 cells, compared to control,
in a dose-dependent fashion (Figure 2). Approximstely 25% of
the A549 cells were apoptotic (annexin V positive) when
treated with 2 μM NMK-BH3, while 5 and 10 μM ligand
induced apoptosis in ∼41 and ∼54% of the A549 cells,
respectively, compared to 2% in control cells. Under similar
conditions, remarkable populations of colchicine-treated cells
(positive control) were undergoing early and late apoptosis,
respectively (section S3A of the Supporting Information).
Effect of NMK-BH-3 on Mitochondrial Integrity. The
mitochondrion plays a pivotal role in induction of apoptosis.43
Hence, we quantified Δψ of A549 cells treated with NMK-BH3
using JC-1 by flow cytometry.44,45 JC-1 is a potential sensitive
fluorescent probe, which forms red fluorescent J-aggregates at
high ψ but exists as green fluorescent monomeric form at low
ψ. NMK-BH3 treatment caused concentration-dependent
depolarization of mitochondria as indicated by the decrease
in the red:green fluorescence ratio of JC-1 (shown in Figure
3A,B). This result was in accordance with the induction of
apoptosis, as mentioned above, and, thus, hinted at the
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Figure 3. NMK-BH3-induced mitochondria mediated apoptosis in A549 cells. (A) Dose-dependent mitochondrial depolarization. The cells treated
with the indicated doses of NMK-BH3 for 24 h were stained with JC-1 and flow cytometrically analyzed and are represented as (A) a dot plot
showing the percentage of cells with intact (red) mitochondria and depolarized (green) mitochondria and (B) a histogram representing the change
in the of red/green mean fluorescence intensity (R/G MFI) ratio. (C) Dose-dependent expression of apoptosis-related proteins. The whole cell
lysate of 24 h NMK-BH3-treated cells was subjected to Western blot analysis for detection of Bax, Bcl-2, p53, cleaved caspase 9, and cleaved caspase
3 with β-actin as the loading control. Data are plotted as means SEM [*P < 0.05 vs control (untreated cells)].
vehicle (0.1% DMSO)-treated control cells, spindle micro-
tubules showed normal bipolar organization with proper
chromosomal alignment at the metaphase plate (Figure 4B)
and the interphase microtubules were radial, intact, and well-
organized (as shown in Figure 5A). However, in the presence
of NMK-BH3 (2 and 5 μM), the dose-dependent increase in
the extent of formation of aberrant multipolar spindles was
observed, while at a high concentration (10 μM), the spindle
microtubules were either multipolar in organization or
completely absent (Figure 4B). As a consequence, misalign-
ment of the chromosome of NMK-BH3-treated cells from the
metaphase plate was apparent. It was also accompanied by
concentration-dependent reduction in interphase microtubule
density at the periphery and disorganization of the central
interphase microtubule network (Figure 5A).
These observations were supported by the immunoblot
analysis of the tubulin wherein incubation of A549 cells with
increasing concentrations of NMK-BH3 led to a significant
decrease in the polymeric tubulin fraction and a remarkable
increase in the size of the soluble pool of tubulin subunits
(Figure 5B).
Effect of NMK-BH3 on the Assembly of Tissue-Purified
Tubulin. We also tested the effect of NMK-BH3 on the rate
and extent of polymerization of brain tissue purified tubulin in a
cell free system wherein the turbidity of the microtubules was
monitored at 340 nm. This wavelength was selected instead of
the usual wavelength of 350 nm as NMK-BH3 exhibited
significant absorbance at 350 nm. Figure 6A revealed that
NMK-BH3 inhibited tubulin polymerization with a half-
maximal inhibitory concentration (IC50) of 7.5
0.3 μM,
and 85% inhibition of tubulin assembly was achieved at 15 μM.
TEM analysis of microtubules further confirmed that at 7.5
μM, NMK-BH3 effectively inhibited the tubulin assembly and
formed fewer microtubules compared to a dense network of
intact microtubules in the control. Moreover, at a high
concentration (15 μM), NMK-BH3 induced significant
aggregation of tubulin heterodimers (Figure 6B).
NMK-BH3 Binds to Tubulin. The compound NMK-BH3
absorbs light with absorbance maxima at 350 nm (section S4A
of the Supporting Information). NMK-BH3, although negli-
gibly fluorescent in aqueous solution, exhibited significant
concentration-dependent fluorescence enhancement in the
presence of purified tubulin (0−10 μM). The NMK-BH3−
tubulin complex exhibited fluorescence emission maxima at 440
nm upon excitation at 350 nm (section S4B of the Supporting
Information). Hence, we used this phenomenon of fluores-
cence enhancement to characterize the interaction of NMK-
BH3 with tubulin.
To determine the nature of binding of NMK-BH3 to tubulin,
we monitored the fluorescence of the ligand (5 μM) in the
presence of 1 μM tubulin for different time intervals (0−30
min). We observed that within 3 min of incubation, the
fluorescence of NMK-BH3 was increased by ∼13-fold and this
enhancement becomes saturated upon further incubation for up
to 30 min (Figure 7A) This result indicates that the binding of
NMK-BH3 with tubulin is fast in nature.
The kinetic profile for the binding of NMK-BH3 with
tubulin, as shown in Figure 7B, was analyzed by the
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Figure 4. Effect of NMK-BH3 on spindle microtubules. (A) NMK-BH3-induced concentration-dependent G2/M cell cycle block. Cytofluorimetric
analysis of the PI-stained DNA content of untreated and 18 h NMK-BH3-treated A549 cells. The average percentages of cells in each phase of the
cell cycle are shown. (B) Disorganization of spindle microtubules of A549 cells by NMK-BH3 upon 18 h treatment. Immunofluorescence images of
the microtubules (red) and nucleus (blue) were obtained by an Olympus Confocal microscope. The scale bar equals 10 μm. Representative data
from three independent experiments.
biexponential equation, as explained in Experimental Proce-
dures. The apparent association constants (Kon) as calculated
from the biexponential equation were 2511 150 M−1 s−1 for
the fast phase and 31 5 M−1 s−1 for the slow phase.
To check the specificity of the interaction of NMK-BH3 with
tubulin, we determined the increase in fluorescence intensity of
NMK-BH3 in the presence of tubulin, BSA, lysozyme, and
lactate dehydrogenase (LDH) each at 5 μM. As shown in
Figure S4C, there was a prominent increase (∼30 fold) in the
fluorescence of NMK-BH3 in the presence of tubulin compared
to insignificant fluorescence promotion with BSA, lysozyme,
and LDH, indicating that NMK-BH3 binds specifically to
tubulin.
tubulin (2 μM) in the presence of increasing concentrations of
the ligand (0−10 μM) is depicted in Figure 7C. Data from the
Scatchard plot, as shown in Figure 7D, suggest that the
dissociation constant (Kd) for interaction of NMK-BH3 with
tubulin was 1.4 0.2 μM. The x-axis intercept of 0.8 0.1
reflected the fact that NMK-BH3 has a single binding site on
tubulin.
NMK-BH3 Binding Site on Tubulin. Colchicine and
vinblastine are two classical microtubule-depolymerizing agents
that bind to the characteristic binding site on tubulin.1,46 As
NMK-BH3 also caused microtubule destabilization upon
tubulin binding, we were interested in identifying the NMK-
BH3 binding site on tubulin.
Scatchard Plot Analysis of NMK-BH3 and Tubulin
Interaction. The binding affinity and stoichiometry of binding
of NMK-BH3 to tubulin were calculated by double titration of
NMK-BH3 and tubulin at 25 °C. The concentration-dependent
increase in the fluorescence intensity of NMK-BH3-bound
However, the fluorescence emission spectra of the tubulin−
NMK-BH3 complex were found to overlap with that of the
tubulin-bound colchicine complex. So, we have used
podophyllotoxin (instead of colchicine) and vinblastine for
the binding studies. Podophyllotoxin is a microtubule-
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Figure 5. Effect of NMK-BH3 on interphase microtubules. (A) Dose-dependent disruption of the cellular microtubule network by NMK-BH3 upon
18 h treatement. Immunofluorescence images of the microtubules (red) and nucleus (blue) were obtained by an Olympus confocal microscope. The
scale bar equals 10 μm. (B) NMK-BH3-mediated depolymerization of cellular microtubules. Western blot analysis showed the expression of soluble
tubulin, polymerized microtubules, and total tubulin.
destabilizing drug that binds to the colchicine site on tubulin
without inducing fluorescence.47 Figure 8A shows the
concentration-dependent inhibition of the increment of
NMK-BH3−tubulin complex fluorescence by podophyllotoxin
where 40 μM podophyllotoxin decreased the fluorescence of
the NMK-BH3 (10 μM)−tubulin (10 μM) complex by 85%.
Furthermore, modified Dixon plot analysis also indicated that
podophyllotoxin competitively inhibited binding of NMK-BH3
to tubulin with an inhibitory concentration (Ki) of 3 0.3 μM
(Figure 8B). In contrast, vinblastine (40 μM) did not
significantly influence the fluorescence emission profile of the
NMK-BH3 (10 μM)−tubulin (10 μM) complex (section S5A
of the Supporting Information). These results clearly rule out
the possibility of binding of NMK-BH3 to the vinblastine site
and emphasize the fact that it binds to tubulin at a site either
identical to or overlapping with the colchicine binding site on
tubulin.
Nature of the Binding of NMK-BH3 to Tubulin. In an
attempt to define the nature of binding of NMK-BH3 to
tubulin and confirm its binding site, the change in fluorescence
emission of the preformed NMK-BH3 (10 μM)−tubulin (10
μM) complex was monitored in the absence and presence of 40
μM podophyllotoxin and 40 μM vinblastine, with respect to
time. Figure 8C demonstrates that podophyllotoxin, but not
vinblastine, strongly reduced the fluorescence intensity of the
NMK-BH3−tubulin complex in a time-dependent manner,
indicating that NMK-BH3 reversibly binds to tubulin at the
colchicine site. Further, the off rate constant (Koff) for NMK-
BH3 from its site on tubulin was calculated as described in
Experimental Procedures and was estimated to be 0.005 s−1
(Figure S5B).
In Silico Prediction of the NMK-BH3 Binding Site on
Tubulin. To predict the binding site of NMK-BH3 on tubulin,
we performed the molecular docking study using Discovery
Studio 2.5. For our study, we utilized the crystal structure of
αβ-tubulin (PDB entry 1JFF) to investigate the possible
binding site of NMK-BH3 on the tubulin heterodimer. By using
specific modules (described in Experimental Procedures), the
cdocker energy obtained for NMK-BH3 was found to be 14.5
0.3 kcal/mol, whereas the binding energy calculated from the
stated formulas (Experimental Procedures) was found to be
−48.6 1 kcal/mol. To validate our docking conditions, we
performed a similar simulation with another ligand NMK-BH5,
which did not affect tubulin polymerization, to bind at the same
site. Very interestingly, we observed that for NMK-BH5 the
cdocker energy was −15.4
0.2 kcal/mol and the binding
0.5 kcal/mol, which indicates unstable
energy was 8.5
interaction.
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Figure 6. NMK-BH3 inhibits tubulin polymerization in cell free system. (A) Polymerization of tubulin dimers was performed in the presence of
varying concentrations (0−15 μM) of NMK-BH3, and the level of polymerized tubulin was monitored turbidimetrically at 340 nm. (B)
Transmission electron microscopic analysis. The inhibitory effect of 0, 7.5, and 15 μM NMK-BH3 on the assembly of brain-purified tubulin was
visualized by TEM. The scale bar equals 50 nm.
Figure 7. Characterization of NMK-BH3−tubulin interaction. (A) Time-dependent change in the fluorescence intensity of 5 μM NMK-BH3 in the
presence of 1 μM tubulin. (B) Association kinetics of the NMK-BH3−tubulin complex (5 μM NMK-BH3 and 1 μM tubulin). (C) Concentration-
dependent binding of NMK-BH3 with tubulin. Fluorescence spectra of tubulin (2 μM) upon incubation with (a) 0, (b) 1, (c) 2.5, (d) 5, (e) 7.5, and
(f) 10 μM NMK-BH3 are shown. (D) Scatchard plot of the binding of NMK-BH3 to tubulin. Excitation and emisson maxima are 350 and 440 nm,
respectively.
Further analysis of the binding site revealed that NMK-BH3
binds to a pocket, comprising β sheet (B8), α helix (H9 and
H10), and H9- and H10-associated loops, present on the
intermediate domain of the β subunit of tubulin (Figure 9A).
The proximity of the amino acids of the binding pocket to the
NMK-BH3 structure facilitates the establishment of the
noncovalent interaction, thereby stabilizing the protein−ligand
complex. The amino acids constituting the NMK-BH3 binding
pocket and their respective location are shown in section S6A
of the Supporting Information. Moreover, the binding of NMK-
BH3 to tubulin was strengthened by the formation of a
hydrogen bond with the Asn339 residue, located on the H10
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Figure 8. NMK-BH3 reversibly binds to tubulin at the colchicine site. (A) Concentration-dependent quenching of the fluorescence of the tubulin−
NMK-BH3 complex (10 μM tubulin and 10 μM NMK-BH3) by podophyllotoxin. Emission spectra of the tubulin−NMK-BH3 complex in the
presence of (a) 0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 30, and (g) 40 μM podophyllotoxin were plotted after excitation of the sample at 350 nm. (B)
Modified Dixon plot showing the concentration-dependent decrease in the fluorescence intensity of the tubulin (2 μM)−NMK-BH3 complex by
varying concentrations of podophyllotoxin (0−40 μM) in the presence of three sets of concentrations of NMK-BH3 (3, 5, and 10 μM). (C)
Reversibility of tubulin−NMK-BH3 interaction. The time-dependent change in the tubulin (10 μM)−NMK-BH3 (10 μM) complex fluorescence
intensity was recorded in the absence and presence of 40 μM vinblasine and 40 μM podophyllotoxin upon excitation at 350 nm.
associated loop of β-tubulin. Figure 9B shows that NMK-BH3
binds to tubulin at the site that is very close to the colchicine
binding site on β-tubulin.
activity. It was documented that the presence of an additional
electron-releasing 4-methoxyphenyl (NMK-BH5) or methoxy
(NMK-BH6) substituent on the same or adjoining indole rings
of the NMK-BH2 structure was detrimental to their
cytotoxicity. However, substitution with an electron-releasing
methoxy group at the C-5 or C-6 position of indole B, in
compound NMK-BH3 or NMK-BH4, was found to improve
their cytotoxic activity. This study also highlighted that
changing the position of the indole linkage from C-3 (in
NMK-BH1, -2, -5, and -6) to C-2 (in NMK-BH3 and NMK-
BH4) in indole A could contribute to the increased activity of
the rotamers. Overall, these findings (Table 1) suggested that
NMK-BH3 with an electron-releasing 5-methoxy substituent
and indole rings linked through the C-2 position of indole A is
the most potent cytotoxic ligand of the series. NMK-BH3 also
exhibited significant growth inhibitory activity against other
human cancer cell lines: MCF-7, MDA-MB-231, and HepG2.
Moreover, the relatively high antiproliferative potential of
NMK-BH3 toward human lung cancer (A549) cells and other
cancer cell lines compared to normal cells called for
investigation of its detailed mode of action.
In A549 lung cancer cells, NMK-BH3 induced dissipitation
of mitochondrial transmembrane potential and apoptotic cell
death. These results were supported by the previously
documented crucial role of mitochondria in MTA-induced
cancer cell death and close interaction of mitochondria with the
cytoskeleton.44,48,49 We also found that NMK-BH3-treated
A549 cells exhibited significantly increased levels of expression
of Bax, p53, caspase 3, and caspase 9 along with a concomitant
decrease in the level of Bcl-2 expression. These observations
DISCUSSION
■
The microtubules, being a critical component of the mitotic
spindle and cytoskeleton, are regarded as one of the sought-
after targets for therapeutic intervention. The clinical success of
the microtubule-targeting anticancer drugs warrants the
innovation of novel anti-mitosis agents that could overcome
the limitations of the currently available chemotherapeutics.
Recently, indole heterocycles are attracting attention in
cancer chemotherapy because of their ability to serve as an
excellent scaffold for development of potential anticancer
agents.13,14,17,18 As lung cancer is the most prominent cause of
worldwide cancer deaths, in this study, we attempted to
develop a novel and potent anti-tubulin chemotherapeutic
agent against A549 lung cancer cells. Hence, we synthesized six
bis(indolyl)-hydrazide−hydrazones (NMK-BH compounds) by
linking indole rings by an active pharmacophore (−CO−NH−
NCH−) linker, and modifications were focused on
incorporation and variation of the nature and location of
functional group substituents and altering the position of the
indole linkage. The ligands were screened for cytotoxic
potential based on antiproliferative activity against A549 cells
and microtubule polymerization inhibition. SAR studies
indicated that NMK-BH1, without any functional group
substituent, was inactive while incorporation of the electron-
accepting 5-bromo substituent in NMK-BH2 resulted in
modest cytotoxicity and tubulin polymerization inhibitory
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Figure 9. In silico prediction of the NMK-BH3 binding site on the tubulin surface. (A) Ribbon diagram depicting in silico binding of NMK-BH3 on
the tubulin heterodimer, where red stands for α-tubulin monomer and blue stands for β-tubulin monomer. (i) Three-dimensional diagram
representing the binding site of NMK-BH3 on tubulin heterodimer. Red represents the polar domains, blue the nonpolar domains, and white the
neutral domains on the protein surface. (ii) Magnified view of the interaction of NMK-BH3 (ball and stick) with the β tubulin subunit of tubulin
heterodimer. (iii) Ball-and-stick figure showing the position of the amino acids that stabilize NMK-BH3 on the protein surface. The green dotted line
denotes the hydrogen bond. (B) Crystal structure representing the proximity of the NMK-BH3 (red) and colchicine (yellow) binding site on tubulin
heterodimer. Predictions were performed using the molecular docking software Discovery Studio 2.5.
were consistent with the earlier reports of the apoptosis signal
transduction pathway in response to microtubule-targeting
drugs.5,50−53 Moreover, NMK-BH3 also led to mitotic block at
the G2/M phase along with disorganization of the mitotic
spindle and depolymerization of the interphase microtubule
network, which was further confirmed by an increase in the
number of soluble tubulin dimers and a decrease in the number
of polymeric microtubules of A549 cells and the inhibition of
brain microtubule assembly, like other microtubule active
drugs.1,46,54 However, induction of mitochondrial depolariza-
tion and apoptosis occurred upon 24 h treatment, while cellular
microtubule depolymerization and mitotic arrest were apparent
18 h postincubation with NMK-BH3, indicating that prolonged
accumulation of the cells at mitosis, by disruption of the cellular
microtubule, could be a preceding event that triggers the
activation of the apoptosis pathway. Thus, the mode of action
of NMK-BH3 was similar to those of many other conventional
anti-tubulin agents.48,50,55
Compound NMK-BH3, although negligibly fluorescent in
aqueous solution, exhibits remarkable fluorescence enhance-
ment upon tubulin binding. A similar phenomenon is also
known in connection with colchicine and could be attributed to
the immobilization of the chromophoric group and conforma-
tional change in the drug and protein.56−58 The specificity of
interaction of NMK-BH3 with tubulin was evidenced by its
significant fluorescence promotion in the presence of tubulin
compared to that in the presence of other proteins like BSA,
lysozyme, and LDH. Thereby, NMK-BH3 inhibited micro-
tubule assembly with high-affinity binding (Kd = 1.4 μM) to
goat brain purified tubulin heterodimers at a single site.
According to fluorescence spectroscopic studies, podophyl-
lotoxin competitively displaced NMK-BH3 from the tubulin−
ligand complex (Ki = 3 μM), indicating that NMK-BH3 may
reversibly bind to tubulin at a site that is similar or overlapping
with the colchicine site, like DAT1 or CXI-benzo-84.25,59 The
molecular docking studies also revealed that the NMK-BH3
binding pocket, located on β-tubulin, comprised of β sheet
(B8), α helix (H9 and H10), and H9- and H10-associated
loops; H9 loop associated amino acids Gln293, Phe296,
Asp297, Ala298, Lys299, Pro307, and Arg308 were also
reported to be vital for colchicine binding.42 Moreover, the
interaction was stabilized by hydrogen bond formation with
Asn339 of β-tubulin. Thus, the analogy between the
biochemical results and docking simulations strengthens the
possibility of location of the NMK-BH3 binding site in the
proximity of the colchicine site on tubulin. On the basis of the
experimental and in silico results, we might predict that the
structural conformation of the NMK-BH ligands plays
important roles in their cytotoxic efficacy as well as tubulin
binding ability. The ligand NMK-BH5 constitutes a bulky 4-
methoxyphenyl substituent, which was absent in other
compounds such as NMK-BH2, NMK-BH3, and NMK-BH4
(Table 1). Interestingly, these compounds exhibited superior
cytotoxic potential and tubulin binding ability compared to
those of NMK-BH5. Hence, it might be inferred that the
presence of such a bulky functional group resulted in steric
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hindrance for the compound, and the binding efficiency to
tubulin is thus reduced.
phosphate-buffered saline; PI, propidium iodide; SAR,
structure−activity relationship; SCLC, small cell lung cancer;
SEM, standard error of mean; TEM, transmission electron
microscopy; Δψ, change in the mitochondrial membrane
potential; ψ, mitochondrial membrane potential.
In summary, this study explores the synthesis and screening
of novel bis(indolyl)-hydrazide−hydrazone derivatives and
identifies NMK-BH3 as the most selective and efficient
cytotoxic agent, endowed with a remarkable ability to induce
mitotic block and apoptosis in lung adenocarcinoma (A549)
cells by targeting the cellular microtubule system through
tubulin binding. Thus, NMK-BH3 could be a promising
candidate for further investigation and development of
improved chemotherapeutics for more efficient lung cancer
therapy in the future.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.5b01127.
■
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AUTHOR INFORMATION
Corresponding Authors
■
*Department of Biotechnology and Dr. B. C. Guha Centre for
Genetic Engineering and Biotechnology, University of Calcutta,
35 Ballygunge Circular Rd., Kolkata, WB 700 019, India.
Telephone: 91-33-2461-5445. Fax: 91-33-2461-4849. E-mail:
gcbcg@caluniv.ac.in.
*Department of Chemistry, Birla Institute of Technology and
Science, Pilani, Rajasthan 333031, India. Telephone: 91-159-
624-5073, ext. 279. Fax: 91-1596-244183. E-mail: dalipk@
pilani.bits-pilani.ac.in.
Funding
This work was funded by grants from the Department of
Biotechnology, Government of India (BT/PR12889/AGR/36/
624/2009) to G.C. We thank the DBT-CU-IPLS confocal
facility developed via the grant from the Department of
Biotechnology, Government of India (BT/PR14552/INF/22/
123/2010). D.D.M. was supported by a Senior Research
Fellowship from CSIR, Government of India, and S.D. was
supported by a Senior Research Fellowship, CSIR, Government
of India.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
BSA, bovine serum albumin; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate;
JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-car-
bocyanine; LDH, lactate dehydrogenase; MTA, microtubule-
targeting agent; NSCLC, non-small cell lung cancer; PBS,
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