Design and biological evaluation of novel tubulin inhibitors as antimitotic agents using a pharmacophore binding model with tubulin

Article abstract of DOI:10.1021/jm050761i

Although the structure has been elucidated for the binding of colchicine and podophyllotoxin as potent destabilizer for microtubule formation, very little is known about MDL-27048, a competitive inhibitor for colchicine and podophyllotoxin. The structural

Full text of DOI:10.1021/jm050761i

5664
J. Med. Chem. 2006, 49, 5664-5670
Design and Biological Evaluation of Novel Tubulin Inhibitors as Antimitotic Agents Using a
Pharmacophore Binding Model with Tubulin
Do Yoon Kim,† Kyun-Hwan Kim,*,‡ Nam Doo Kim,§ Ki Young Lee,| Cheol Kyu Han,§ Jeong Hyeok Yoon,§
Seung Kee Moon, Sung Sook Lee, and Baik L. Seong*,†,#,X
Department of Biotechnology, College of Engineering, Yonsei UniVersity, Seoul 120-749, Korea, Department of Pharmacology, School of
Medicine, Konkuk UniVersity, Chungju 380-701, Korea, Institute of Life Science and Biotechnology, Yonsei UniVersity, 134 Shinchon-Dong,
Seodaemun-Gu, Seoul 120-749, Korea, Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale
UniVersity School of Medicine, New HaVen, Connecticut 06510, R&D Center, Equispharm Company, Limited, 3F Sung-ok B/D, 4-1
Sunae-dong, Bundang-gu, Sungnam-shi, Kyunggi-do 463-825, Korea, CKD Research Institute, Chonan Post Office Box 74, Chonan 330-600,
Korea, and Protheon, B120E Yonsei Engineering Complex, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, Korea
ReceiVed August 3, 2005
Although the structure has been elucidated for the binding of colchicine and podophyllotoxin as potent
destabilizer for microtubule formation, very little is known about MDL-27048, a competitive inhibitor for
colchicine and podophyllotoxin. The structural basis for the interaction of antimitotic agents with tubulin
was investigated by molecular modeling, and we propose binding models for MDL-27048 against tubulin.
The proposed model was not only consistent with previous competition experiment data between colchicine
and MDL-27048, but further suggested an additional binding cavity on tubulin. Based on this finding from
the proposed MDL-tubulin complex, we performed molecular design studies to identify new antimitotic
agents. These new chalcone derivatives exerted growth inhibitory effects on all four human hepatoma and
one renal epithelial cell lines tested and induced strong cell cycle arrest at G2/M phase. Furthermore, these
compounds exhibited a strong inhibitory effect on tubulin polymerization in vitro. Therefore, we suggest
that the validated MDL-27048 model would serve as a potent platform for designing new molecular entities
for anticancer agents targeted to microtubules.
Introduction
tinguished by the site of binding on the tubulin molecule. Rep-
resentative examples are taxol, vinblastine, and colchicines
binding sites, respectively (Figure 1). Among these binding sites,
the taxol binding site has been well-characterized with the crystal
structure of tubulin7 and partially overlaps with that of other
compounds such as epothilones8 or eleutherobin.9 The vinblas-
tine binding site is not precisely known, except for a single
cross-linking experiment that identified the residues 175-213
of â-tubulin as the binding region. Because these residues are
located in the plus end of the microtubule, this result suggests
that the vinblastine binding site is exposed to the plus end of a
microtubule surface.10 Colchicine is the first drug that is known
to bind tubulin, and the colchicine binding site has been
characterized recently from a complex with colchicine and a
stathmin-like domain.20 The development of ketones as tubulin-
binding agents such as chalcones has been reviewed recently.22
Microtubules are ubiquitous, essential cytoskeletal polymers
in all eukaryotic cells controlling various cellular functionss
transport of material within the cell, movement of cytoplasmic
organelles or vesicles, and proper progression through cell
division.1 The function of microtubules is strongly associated
with their stability. The proteins that stabilize microtubules
include the classic microtubule-associated proteins (MAPs)a that
are thought to bind along the length of the microtubule polymer
and enhance its stability. In the case of proteins that destabilize
microtubules, Op18 protein, also referred to as stathmin, and
XKCM1 have been recently identified.2 Furthermore, exogenous
ligands can also have dramatic effects on microtubules, often
interacting directly with tubulin to either stabilize (such as pac-
litaxel)3 or destabilize microtubules (such as colchicines, MDL-
27048).4,5 These ligands disrupt the dynamics of polymerization
and depolymerization of microtubules involved in cell division,1
and the interference with the dynamics of tubulin and cell divi-
sion has been proven clinically useful for designing anticancer
agents.6 These agents can be grouped into three classes, as dis-
Although the structure has been elucidated for binding with
colchicine and podophyllotoxin as a potent destabilizer for
microtubule formation, very little is known about the structural
basis for other destabilizing agents such as MDL-27048, a
competitive inhibitor with colchicine and podophyllotoxin.11 In
this study, we propose the binding model of MDL-27048 against
tubulin by molecular modeling and validate the model by
designing and testing new chalcone derivatives. A pharmacoph-
ore model was built by analyzing colchicine and podophyllo-
toxin interaction with tubulin, projected into the MDL-binding
model and combined with the receptor shape information of
the X-ray tubulin structure. The model was then used to screen
new tubulin binding agents, potentially as antimitotic agents.
The biological activity of these new chalcone derivatives was
confirmed by tubulin polymerization inhibition assay and the
cytotoxic activity against human hepatoma and kidney cell lines.
The present MDL-27048 binding model and pharmacophore are
* To whom correspondence should be addressed. Department of Bio-
technology, College of Engineering, Yonsei University, Seoul 120-749,
Korea (B.L.S.). Tel.: +82-2-2123-2885 (B.L.S.); +82-43-840-3729 (K.-
H.K.). Fax: +82-2-362-7265 (B.L.S.); +82-43-851-9329 (K.-H.K.). E-
mail: blseong@yonsei.ac.kr (B.L.S.); khkim10@kku.ac.kr (K.-H.K.).
† Department of Biotechnology, Yonsei University.
‡ Konkuk University.
§ Equispharm Company, Limited.
| Yale University School of Medicine.
CKD Research Institute.
# Protheon.
X Institute of Life Science and Biotechnology, Yonsei University.
a Abbreviations: MAPs, microtubule-associated proteins; RMSD,
root-mean-square deviation; ATCC, American Type Culture Collection;
DMEM, Dulbecco’s modified Eagles medium; SBDD, structure-based drug
design.
10.1021/jm050761i CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/19/2006

Interaction of Antimitotic Agents with Tubulin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5665
through van der Waals interactions, similar to colchicine and
podophyllotoxin interaction with tubulin.20
Except for colchicine, no information has been available on
the interaction of the destabilizing agent, including MDL-27048,
with tubulin. Therefore, we screened and tested new chalcone
derivatives to validate our MDL-27048 binding model. First,
we built a pharmacophore model by analyzing colchicine and
podophyllotoxin interaction with tubulin. As depicted in Figure
4A, the derived pharmacophore consists of two hydrophobic
centers (yellow sphere) and one polar interaction site (yellow
cylinder). The hydrophobic center that is located in the middle
of trimethoxyphenyl moiety of colchicine and podophyllotoxin
is surrounded by Leu â242, Ala â250, Leu â255, Ala â316,
Val â318, and Ile â378 residues. The other hydrophobic center
makes contact with Met â259, Ala â316, and a carbon chain of
Lys â352 residues. The yellow cylinder represents a possible
polar interaction with the Cys â241 residue, and the distance
between the cylinder and the sulfur atom in Cys â241 is 2.9 Å.
These three characteristics represent the minimum interactions
in the colchicine binding site. As shown in Figure 4B, the MDL-
27048 binding model is matched properly with the proposed
pharmacophore. The hydrophobic center located in the middle
of trimethoxyphenyl moiety in podophyllotoxin corresponds to
the 2,5-dimethoxyphenyl moiety in MDL-27048 and the other
hydrophobic center matched with the 4-dimethylamino phenyl
moiety in MDL-27048. The polar interaction between C2-
oxygen in the A-ring of colchicine and Cys â241 residue also
corresponds to the interaction between C5-oxygen in MDL-
27048 and Cys â241.20
Figure 1. Chemical structures of tubulin inhibitors. MDL-27048 (trans-
1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-pro-
pen-1-one) and podophyllotoxin ([5R-(5R,5aâ,8aR,9R)]-5,8,8a,9-
tetrahydro[2,3-d]-1,3-dioxol-6(5aH)-one), competitive inhibitors of
colchicine ((S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo-
[a]-heptalen-7-yl)acetamide), interact with the colchicine binding site.
We observed an additional cavity within the colchicines
binding site, as described with a red circle in Figure 4B, mainly
composed of Val â238, Leu â242, and Leu â255 (Figure 3).
After combining with receptor shape information of X-ray
tubulin structure, the proposed pharmacophore was projected
to the MDL-27048 binding structure and used to screen new
tubulin binding agents.
To validate our proposed model, we virtually screened from
our natural compound database of pharmacophores (total 9720
compounds) for the proposed colchicine binding site described
in Figure 4B. Twenty-four compounds were selected. We then
initially tested these compounds with cell proliferation assay
on HL-60 cell line within the concentration range of 0.003 to
10 µg/mL for each test compound (data not shown). We finally
selected five compounds, 1 (CHA101), 2 (CHA102), 3 (CHA103),
4 (CHA105), and 5 (CHA106), which showed the highest
activity against HL-60 growth inhibition. These new chalcone
derivatives had different molecular sizes (Figure 5) and were
found to be novel compounds hitherto without any previous
reports as tubulin inhibitors.
For virtual screening of new derivatives, we considered pri-
marily the two major interaction points (two hydrophobic inter-
actions among three interaction points as discussed before) and
an additional binding pocket (shown with red circle; Figure 4B).
Therefore, all five compounds, 1-5, fitted into the proposed
MDL-27048 binding pocket comprising the two hydrophobic
characteristics and an additional binding pocket. A representative
binding model is shown for both 1 and 2, respectively (Figure
6). It is interesting to note that 4 and 5 were similar in the sense
that they carry almost the same size of moiety into the additional
binding pocket, whereas 4 carried extra biphenyl moiety in the
classic chalcone binding pocket (Figures 5 and 6).
Figure 2. Comparison of podophyllotoxin in X-ray structure (PDB
code 1SA1: cyan) and in AutoDock result (colored gray). X-ray
structure of podophyllotoxin is represented by ball-and-stick. Only top
cluster of AutoDock results (94 conformers) are shown with wire form.
expected to provide a useful basis for further designing of new
derivatives intervening the dynamics of microtubule formation.
Results and Discussion
To test whether the AutoDock program is feasible for ligand
binding to tubulin, the podophyllotoxin-colchicine complex
structure (PDB code: 1SA1) was initially chosen and the
docking structure of podophyllotoxin was compared with its
crystallographic structure (Figure 2). The top configuration
cluster among 100 independent podophyllotoxin conformers was
composed of 94 conformers. This top cluster showed the highest
frequency of occurrence, so we accepted top cluster configu-
ration as podophyllotoxin binding model. The binding mode of
the lowest energy structure in the top docking cluster and that
of crystallographic structure to tubulin were very similar
(RMSD: 1.07 Å). This result demonstrates that AutoDock
analysis is suitable for the identification of the colchicine binding
site in tubulin. Based on this result, the MDL-27048 binding
model was generated using the same condition used for
podophyllotoxin binding. The top configuration cluster among
100 independent MDL-27048 conformers was composed of 42
conformers, and the structure of lowest energy in the top docking
cluster was shown in Figure 3A. As depicted in Figure 3B,
MDL-27048 shared the binding pocket with colchicine, and the
overlapping of the binding sites explained the competition
among colchicine, podophyllotoxin, and MDL-27048 in tubulin
binding.11 Interaction of MDL-27048 involves amino acid
residues Cys â241, Lys â352, Val â318, Leu â255, Leu â242,
Leu â248, Ala â250, Ala â316, Ile â378, and Met â259, mostly
Interestingly, the newly discovered compounds carry striking
resemblance to the enone analogues of curcumin as a chalcone
core that has been previously reported as an inhibitor of

5666 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Kim et al.
Figure 3. (A) Model of MDL-27048 and tubulin complex. (B) Comparison of colchicine, podophyllotoxin, and MDL-27048 binding model with
tubulin. Cyan, yellow, and purple represent colchicine, podophyllotoxin, and MDL-27048, respectively.
endothelial cell proliferation.23 This chalcone core, referred to
as compound 6 in that paper, lacks the group that occupies the
additional binding site (Figures 5 and 6). Therefore, our
compound virtually screened in this study is expected to exhibit
the improved activity over compound 6 lacking the group
occupying the new binding site. For this reason, we used
compound 6 as a control for the cell proliferation assay (Figure
7) and the in vitro tubulin polymerization assay (Figure 9).
A direct cell proliferation/cytotoxicity assay was performed
using four independent human hepatoma and one renal epithelial
cell lines within the range of 0.01 to 100 µM of test compounds,
using compound 6 as positive control. As shown in Figure 7,
strong inhibitory effects were observed with compounds 1, 2,
and 5 for both Huh7 and 293T cell lines. Most importantly,
these three compounds showed manifest improved activity over
compound 6. We obtained similar results from the cell prolifera-
tion assay with other human hepatoma cell lines, HepG2, SNU-
368, and SNU-398 (data not shown). The IC50 value of test
compounds and control are summarized in Table 1. The
antiproliferative activities of compounds 1, 2, and 5 were better
than that of the control compound, compound 6, consistent with
the interaction with the newly identified binding site in this
study. However, the antiproliferative effects of 3 and 4 were
much lower in all test cell lines. These results validate the newly
discovered binding pocket in tubulin. Parallel analysis of MDL-
27048, previously described as a potent antimitotic agent,22
exhibited a relatively higher IC50 value on our cell proliferation
assay (data not shown), consistent with previously observed
resistance of human hepatoma cells to tubulin interfering
agent.24
To determine whether the cytotoxic effects induced by the
treatment of the three chalcone derivatives (1, 2, and 5) were
caused by cell cycle arrest at a certain phase or not, we
performed cell cycle analysis using flow cytometry. Indeed, the

Interaction of Antimitotic Agents with Tubulin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5667
Table 1. The results also confirm that the cytotoxic effect is
mediated by direct interaction of the compounds with a new
binding pocket in tubulin. The IC50 value for tubulin binding
was higher than cell proliferation (Figure 9), reminiscent of
previous studies.22
Conclusions
A molecular docking study suggests that MDL-27048 binding
to tubulin involves Cys â241, Lys â352, Val â318, Leu â255,
Leu â242, Leu â248, Ala â250, Ala â316, Ile â378, and Met
â259, mostly through van der Waals interactions, in much the
same way observed in colchicine and podophyllotoxin binding.
This binding structure is in agreement with previous experi-
mental data relevant to colchicine and MDL-27048 binding to
tubulin.11 Consistent with direct competition between the two
compounds,11 here we proposed a new binding model for MDL-
27048 that overlaps with colchicines, and the binding involves
three major interactions between MDL-27048 and tubulin
(Figure 4B). The major interaction in this model is first of all
in good agreement with the known interactions with classic
ligands (colchicine and podophyllotoxin in this study). We
further validated the binding model by designing new chalcone
derivatives and testing the cytotoxic activities, cell cycle
analysis, and the inhibition of tubulin polymerization in vitro.
Among these, compounds 1, 2, and 5 consistently exhibited
potent activities and merits for the further evaluation as novel
antimitotic agents. Furthermore, the present MDL-27048 binding
model and the proposed pharmacophore would provide a useful
guideline for the future design of new chemical entities of
microtubule-targeted anticancer agents.
Experimental Section
Materials. Compounds 1 and 2 were purchased from Sigma-
Aldrich (www.sigmaaldrich.com), and compound 3 was purchased
from Timtec (www.timtec.net). Compounds 4 and 5 were purchased
from Asinex (www.asinex.com). All test compounds used in this
study were dissolved in DMSO as 10 mM stock. The solubilities
of 1, 2, and compound 6 were higher than 100 µM, whereas those
of 3, 4, and 5 were near 20 µM in aqueous solution.
Figure 4. (A) Pharmacophore model of colchicine (pink) and podo-
phyllotoxin (cyan). (B) Red circle proposes additional binding pocket.
Pharmacophore binding model is shown under the backdrop of
colchicine binding site shape information. MDL-27048 model and
podophyllotoxin are represented with blue and pink, respectively.
Synthesis of 1-Phenyl-3-pyridin-2-yl-propenone (Compound
6). Compound 6 was synthesized by the method of Robinson and
co-workers.23 An amount equal to 20 mL of 10% aqueous sodium
hydroxide solution and 2 mL of methanol were added to a flask,
and the mixture was cooled to 10 °C with stirring. An aliquot of
6.2 mL (0.065 mol) of pyridine-2-carboxaldehyde was added in
one portion. Acetophenone in the amount of 3.8 mL (0.032 mol)
was then added in small portions over a period of an hour,
maintaining the temperature at around 10 °C. The contents of the
flask were stirred for 5 h, and the reaction mixture was poured
onto 30 mL of water. The resulting solid was filtrated and washed
with 20 mL of water. The solid was then dried and recrystallized
three times from hexane and diethyl ether to give 2.7 g (75%) of
1-phenyl-3-pyridin-2-yl-propenone. 1H NMR (CDCl3): 7.30 (t, 1H,
CHPhH-m), 7.50 (m, 3H, COPhH-m and CHPhH-m), 7.60 (t, 1H,
COPhH-p), 7.75 (m, 2H, CHPhH-o,p), 8.11 (m, 3H, COPhH-o and
CH), 8.69 (dd, J ) 4.2 Hz, 1H). MS(FAB) m/z 210 (M + H+).
Molecular Modeling. Molecular modeling studies were con-
ducted on several R12000 SGI Octane workstations with the
software package InsightII (Accelrys, Inc.), and the structure-based
virtual screenings were performed using Equispharm’s in-house
software packages on a Linux workstations (http://www.equisp-
harm.com; the in-house programs, PharmoMap and PharmoScan,
will be published elsewhere). The molecular visualization tool was
performed by InsightII package.
flow cytometric analysis showed that all three compounds
strongly induced G2/M arrest in SNU-398 cells, and the effect
was observed in a dose-dependent manner in the test range of
50-5000 nM (Figure 8). These results confirmed and further
extended the growth inhibitory effects of the three chalcone
derivatives on hepatoma cells.
The results suggest that the additional binding pocket of
tubulin identified in the present study can accommodate
compounds 1, 2, and 5. Compound 3 may not fit properly into
this pocket because of a relatively long ethyl ether group
extended from the phenyl moiety (Figure 5). We, therefore,
suggest that, for further design of tubulin-targeted antimitotic
agents, careful considerations should be given to the size of
the moiety fitting the newly identified binding pocket.
Finally, we tested whether the compounds directly interact
with tubulin and inhibit tubulin polymerization in vitro. As
shown in Figure 9, strong inhibitory effect was observed with
three derivatives. Compounds 1 and 2 and compound 6 showed
50% tubulin polymerization inhibition at 19.8, 15.7, and around
30 µM (IC50), respectively. It was difficult to evaluate the IC50
value of compound 5 because of poor solubility in our
experimental setting. It should be noted that the IC50 of
compound 6 was higher than compounds 1 and 2, further
extending data in the cell proliferation assay (Figure 7) and
Molecular Docking Study. To find out the docking structures
of MDL-27048 into the previous defined colchicine binding region
of tubulin,20 automated docking simulation was implemented with

5668 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Kim et al.
Figure 5. New chalcone derivatives as screened from chemical DB based on the MDL-27048 binding model. The enone analogue of curcumin as
a chalcone core (compound 6 in the text) was presented. Compound 6 was reported as an inhibitor of endothelial cell proliferation.23
Figure 6. Model of new chalcone derivatives and tubulin complex. 1 (A) and 2 (B), shown in green, interact with both the classic colchicines
binding site and the additional pocket newly identified in this work (see Figure 4). Binding model of compound 6 (purple) was presented for
comparison.
the program AutoDock version 3.0.12-14,19 The starting conforma-
tions of MDL-27048 were optimized with quantum mechanical
calculations using Gaussian 98 (6-31g* base set).21 All ligand atomic
charges were assigned using the Gasteiger-Marsili formation,
which is the type of atomic charges used in calibrating the
AutoDock empirical free energy function.15 Atomic solvation
parameters and fragmental volumes were assigned to the protein
atoms using the AutoDock utility, AddSol. All ligand atoms but

Interaction of Antimitotic Agents with Tubulin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5669
Figure 7. Cytotoxic effect on human hepatoma and renal epithelial
cell lines by chalcone derivatives. (A) Huh7 cells grown in 24-well
plates were treated with 10 µM of indicated compounds for 48 h. The
phase-contrast microscopic examination of treated cells was shown
(200× magnification). Results are representative of three independent
experiments. (B) The effects of chalcone derivatives on cell growth
were evaluated by cell proliferation assay, as described in Experimental
Section. Huh7 and 293T cells were treated with 20 µM of indicated
compounds for 48 h and a proliferation assay was performed. Results
are an average of triplicate experiments, and the standard deviation is
shown in a bar.
no protein atoms were allowed to move during the docking
simulation, and the rotatable bonds in the ligand were defined using
an AutoDock utility, AutoTors. The interaction energy between
ligand and protein was evaluated using atom affinity potentials that
are precalculated on a grid.16 The grid maps were calculated using
AutoGrid. We used this grid map with 60 points in each x, y, and
z direction, equally spaced at 0.375 Å. Docking was performed
using the Lamarckian genetic algorithm in AutoDock 3.0. Each
docking experiment was performed 100 times, yielding 100 docked
conformations. Parameters used for the docking were as follows:
population size of 50; random starting position and conformation;
maximal mutation of 2 Å in translation and 50 degrees in rotations;
elitism of 1; mutation rate of 0.02 and crossover rate of 0.8; and
local search rate of 0.06. Simulations were performed with a
maximum of 1.5 million energy evaluations and a maximum of
27 000 generations. The pseudo-Solis and Wets local search method
was included with the default parameters. Final docked conforma-
tions were clustered using a tolerance of 1 Å RMSD.
Virtual Screening. The MDL-27048 docking model and the
podophyllotoxin-colchicine complex structure (PDB code: 1SA1)
with tubulin were utilized for rapid virtual screening of the
compound database. The pharmacophore was analyzed using
PharmoMap, an in-house software package developed by Equisp-
harm for structure-based virtual screening. The constructed Phar-
moMap is the feature-based pharmacophore in which the pharma-
cophoric points are represented by chemical features, such as
hydrogen bond acceptors/donors or hydrophobic features. The
pharmacophore models were employed as a search query to identify
inhibitors targeted to a colchicine binding site from a 3D small
molecule database, which comprises commercially available com-
pounds of chalcone moieties (total 9720 compounds). The virtual
screening for colchicines binding pocket of tubulin was carried out
Figure 8. Inhibition of cell cycle progression by chalcone derivatives.
(A) Cell cycle analysis after treatment of chalcone derivatives using
flow cytometry. SNU-398 hepatoma cells were treated with chalcone
derivatives for 48 h and subjected to cell cycle analysis. Each cell cycle
phase and percentages of G2/M phase cells are indicated. (B) Cell cycle
arrest at G2/M phase by chalcone derivatives treatment. Percentages
of G2/M phase cells obtained from panel A were plotted.
using the PharmoScan system, a structure-based virtual screening
tool developed by Equispharm. Compounds of unfavorable interac-
tions with the binding site or adopting unrealistic conformations
were screened against the process of pharmacophore mapping into
the colchicines binding site of tubulin. The twenty-four compounds
were finally selected for cell proliferation assay in vitro.
Cell Lines. Human hepatoma cell lines (HepG2 and Huh7) and
renal epithelial (293T) cell lines were purchased from the American
Type Culture Collection (ATCC, Rockville, MD). SNU-398 and
SNU-368 hepatoma cell lines were obtained from the Korean Cell
Line Bank, Seoul, Korea.17,18 These cell lines were maintained in
Dulbecco’s modified Eagles medium (DMEM; Flow Laboratories),
supplemented with 10% fetal bovine serum/glutamine/antibiotics
in a humidified atmosphere of 5% CO2 at 37 °C.
Cell Proliferation Assay. Cells (2 × 104 cells) were seeded into
96-well plates. After 24 h, the cells were grown in the presence or
absence of drugs for 48 h. After removing 100 µL supernatant from
each well, 20 µL of CellTiter 96Aqueous solution (Promega, USA)
was added into each well of the 96 well plate containing 100 µL
of culture medium, and the cells were further incubated for 2 h at
37 °C in a humidified atmosphere of 5% CO2. The reduction of
absorbance at 490 nm was measured.
Cell Cycle Analysis. The effects of compounds on cell cycle
were analyzed using flow cytometry. Briefly, hepatoma cells (5 ×

5670 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Kim et al.
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Figure 9. Inhibition of tubulin polymerization by chalcone derivatives.
The effects of chalcone derivatives on tubulin polymerization were eval-
uated as described in Experimental Section. Compounds 1, 2, and 5 and
compound 6 (control)23 were tested at different concentrations (0, 3, 10,
and 30 µM). IC50 value was calculated based on the tubulin polymeri-
zation assay. The solubility of compound 5 was near 20 µM in aqueous
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reported.23
Table 1. Antiproliferative Activities of Newly Identified Chalcone
Derivatives
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E.; Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian
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(15) Gasteiger, J.; Marsili, M. Iterative partial equalization of orbital
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cell proliferation IC50 (µM)
compounds
Huh7
293T
1
2
3
4
8.4
8.2
9.6
13.7
>100
>100
9.8
>100
>100
9.9
5
compound 6
14.3
13.8
105) were seeded into 12-well plates, grown for 24 h and further
grown in the presence of drug or carrier alone for 48 h. Cells were
harvested, washed twice with PBS, stained with the cell cycle
analysis kit (Becton Dickinson, Mountain View, CA), and analyzed
with flow cytometry (Becton Dickinson). The percentages of cells
in different phases of the cell cycle were estimated using the Modfit
LT software (Becton Dickinson).
Tubulin Polymerization Assays. HTS-tubulin polymerization
assay kit (cat. No. BK004/ CDS01, Cytoskeleton, USA) was used.
All components were added to microwells held at 0 °C, and base-
lines were established at 340 nm. Assembly was followed sequen-
tially at 0 °C and 37 °C, and tubulin assembly was measured
turbidometrically in Molecular Device Spectra Max Plus model
(Molecular Devices, USA), equipped with electronic temperature
controllers.
(17) Shin, E. C.; Shin, J. S.; Park, J. H.; Kim, J. J.; Kim, H.; Kim, S. J.
Expression of Fas-related genes in human hepatocellular carcinomas.
Cancer Lett. 1998, 134, 155-162.
(18) Park, J. G.; Lee, J. H.; Kang, M. S.; Park, K. J.; Jeon, Y. M.; Lee,
H. J.; Kwon, H. S.; Park, H. S.; Yeo, K. S.; Lee, K. U. Characteriza-
tion of cell lines established from human hepatocellular carcinoma.
Int. J. Cancer 1995, 62, 276-282.
(19) AutoDock, version 3.0.5; http://www.scripps.edu/mb/olson/doc/au-
todock/.
(20) Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.;
Sobel, A.; Knossow, M. Insight into tubulin regulation from a
complex with colchicine and a stathmin-like domain. Nature 2004,
428, 198-202.
(21) Gaussian 98 User Guide; Gaussian, Inc.: Pittsburgh, PA, 1998.
(22) Lawrence, N. J.; McGown, A. T. The chemistry and biology of
antimitotic chalcones and related enone systems. Curr. Pharm. Des.
2005, 11, 1679-1693.
(23) Robinson, T. P.; Ehlers, T.; Hubbard, R. B., IV; Bai, X.; Arbiser, J.
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Acknowledgment. This work was supported by the Micro-
bial Genomics Frontier R&D Grant from the Ministry of Science
and Technology of Korean Government.
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of paclitaxel resistance in human hepatocellular carcinoma cells.
Biochem. Biophys. Res. Commun. 2004, 12, 771-779.
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