Identification of a class of novel tubulin inhibitors

Article abstract of DOI:10.1021/jm300100d

We previously developed a series of anticancer agents based on cyclooxygenase-2 (COX-2) inhibitor nimesulide as a lead compound. However, the molecular targets of these agents still remain unclear. In this study, we synthesized a biotinylated probe based on a representative molecule of the compound library and performed protein pull-down assays to purify the anticancer targets of the compound. Via proteomic approaches, the major proteins bound to the probe were identified to be tubulin and heat shock protein 27 (Hsp27), and the compound inhibited tubulin polymerization by binding at the colchicine domain. However, the tubulin inhibitory effect of the compound activated the Hsp27 phosphorylation and possibly overrode the direct Hsp27 inhibitory effects, which made it difficult to solely validate the Hsp27 target. Taken together, the compound was a dual ligand of tubulin and Hsp27, inhibited tubulin polymerization, and had the potential to be a class of new chemotherapeutic agents.

Full text of DOI:10.1021/jm300100d

Article
pubs.acs.org/jmc
Identification of a Class of Novel Tubulin Inhibitors
Xin Yi,† Bo Zhong,† Kerri M. Smith,† Werner J. Geldenhuys,§ Ye Feng,† John J. Pink,∥ Afshin Dowlati,⊥
Yan Xu,† Aimin Zhou,†,‡ and Bin Su*,†,‡
‡
†Department of Chemistry and Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions,
Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115, United States
§Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, Ohio 44272,
United States
⊥
∥Division of General Medical SciencesOncology and Division of Hematology and Oncology, Case Western Reserve University
School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States
ABSTRACT: We previously developed a series of anticancer agents
based on cyclooxygenase-2 (COX-2) inhibitor nimesulide as a lead
compound. However, the molecular targets of these agents still remain
unclear. In this study, we synthesized a biotinylated probe based on a
representative molecule of the compound library and performed pro-
tein pull-down assays to purify the anticancer targets of the com-
pound. Via proteomic approaches, the major proteins bound to the
probe were identified to be tubulin and heat shock protein 27 (Hsp27),
and the compound inhibited tubulin polymerization by binding at the
colchicine domain. However, the tubulin inhibitory effect of the
compound activated the Hsp27 phosphorylation and possibly overrode
the direct Hsp27 inhibitory effects, which made it difficult to solely
validate the Hsp27 target. Taken together, the compound was a dual
ligand of tubulin and Hsp27, inhibited tubulin polymerization, and had
the potential to be a class of new chemotherapeutic agents.
and survival of cancer patients.4 In addition to the resistance
issue, neurotoxicity is one of the major side effects of the tubulin
1. INTRODUCTION
Tubulin-containing structures are important for many
inhibitors derived from complex natural products, which affects
important cellular functions, including chromosome segrega-
the quality of life of cancer patients.7−9 Furthermore, low oral-
tion during cell division, intracellular transport, development,
bioavailability limits the convenient oral drug administration.10
and maintenance of cell shape, cell motility, and distribution of
molecules on cell membranes.1 The rapid growth of cancer cells
There is an urgent need to develop new tubulin inhibitors with
fewer side effects and with good oral bioavailability and that are
less prone to the development of resistance.
leads to their high dependence on tubulin polymerization/
depolymerization, which makes tubulin a good target for
anticancer drug development. Paclitaxel (Taxol), the represen-
tative tubulin inhibitor approved by the Food and Drug
Administration (FDA) in 1992 for cancer treatment, is one of
the most powerful chemotherapeutic agents currently in use.
Paclitaxel binds to tubulin and results in its precipitation and
sequestration, which interrupt many important biological
functions of cancer cells that depend on a dynamic tubulin
polymerization and depolymerization process.2 This explains
the high potency and efficacy of paclitaxel in fighting cancer.
Besides paclitaxel, FDA also approved other tubulin inhibitors
with complex structures for cancer treatment including
epothilone analogues, vinca alkaloid analogues, and halichondrin
analogues (Figure 1). Treatment with tubulin inhibitors has led
to improvement in the duration and quality of life for many
cancer patients.3 However, most of them eventually develop
progressive disease after initially responding to the treatment.4−6
Drug resistance of most tubulin inhibitors represents a major
obstacle to overcome in order to improve the long-term response
There are three well-documented binding domains of
tubulin, which can be utilized to design new tubulin inhibitors.
The first binding domain is the pocket for taxanes and
epothilones.11,12 The second domain is the pocket for vinca
alkaloids and halichondrins.13,14 FDA approved tubulin
inhibitors binding to these two domains all are derivatives of
complex natural products (Figure 1). These compounds have very
diverse structures but share the same characteristics: high
bulkiness, hydrophobicity, and complicated structure. In addition,
they tend to be easily recognized by P-glycoprotein and pumped
out of cancer cells, which cause drug resistance.3−5,15 The potential
to develop better tubulin inhibitors binding at these two domains
therefore appears to be limited.
The third binding domain of tubulin is the pocket for
colchicine, a small natural product with a relatively simple
Received: January 23, 2012
Published: March 21, 2012
© 2012 American Chemical Society
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Figure 1. Structures of the classic tubulin inhibitors.
Figure 2. From COX-2 inhibitor nimesulide to potent anticancer agent 2.
structure (Figure 1). So far, there are no FDA approved
anticancer drugs targeting this domain.3 Colchicine is clinically
used as a monotherapy for the treatment of familial
Mediterranean fever and acute gout flares but not as a cancer
therapy because of its high toxicity. Efforts have been made to
develop less toxic candidates targeting the colchicine binding
domain. Some synthetic small molecules targeting this domain
such as compound 1, N-[2-[(4-hydroxyphenyl)amino]-3-
pyridinyl]-4-methoxybenzenesulfonamide (ABT-751)3 (Figure 1),
are in clinical trials as cancer treatment.3,16−18 Their unique
capability to inhibit the growth of multiple drug resistant
(MDR) tumor cell lines, lower neurotoxicity, and good oral
bioavailability are their primary advantages, compared to the
currently FDA approved tubulin inhibitors.16−18 Drug develop-
ment of compound 1 demonstrated the potential of colchicine
domain binders as better tubulin inhibitors.16−18
To develop new tubulin inhibitors, it traditionally starts with
a lead compound binding to a certain domain of tubulin but
with relatively weak activity. Structural optimization will be
performed to increase the binding affinity and the anticancer
activity of the lead compound. In the current study, we describe
the development of a novel tubulin inhibitor via a different
approach to the traditional drug development. We synthesized
a class of anticancer agents without knowing their molecular
targets. However, the antiproliferation activity guided drug
optimization dramatically improved the cell growth inhibitory
activity of the lead compound, a COX-2 inhibitor nimesu-
lide.19,20 A very potent compound 2, benzo[1,3]dioxole-5-carboxylic
acid [3-(2,5-dimethylbenzyloxy)-4-(methanesulfonylmethylamino)-
phenyl]amide (NSC751382)20 developed in our previous study,
showed very promising anticancer activity and suppressed the
growth of various cancer cell lines with IC50s of 0.1−0.5 μM.20
In the current study, we identified the molecular targets of
compound 2 as tubulin and heat shock protein 27 (Hsp27) via
proteomic and chemical biology approaches. The compound
was proved to be a colchicine domain binder and exhibited
potent tubulin polymerization inhibitory activity. The direct
biological response of Hsp27 after the compound treatment
was difficult to determine because of the unclear downstream
cellular signals and also the indirect effects of tubulin inhibition
on Hsp27. Nevertheless, there is a great potential to develop a
class of novel tubulin inhibitors based on our discovery. Our
drug discovery process also demonstrates that ligand based lead
optimization via traditional medicinal chemistry is an effective
strategy to improve the cell proliferation inhibitory activity of
the lead compound. More potent derivatives make the target
identification easier, since they should have a much higher
binding affinity with their target(s). Further optimization based
on the identified targets could involve computational chemistry
if the binding domain is well-defined and could greatly speed
up the drug development.
2. RESULTS AND DISCUSSION
2.1. Origin of Compound 2 and the Construction of
Its Biotinylated Probe. Compound 2 is a potent anticancer
agent structurally derived from the COX-2 inhibitor nimesulide
(N-(2-phenoxy-4-nitrophenyl)methanesulfonamide).20 Numerous
studies have demonstrated the anticancer activity of nimesulide.
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Figure 3. Structure of compound 2 probe.
a
Scheme 1
a
Regents and conditions: (a) 2,5-dimethylbenzyl bromide, K2CO3, DMF; (b) (1) MsCl, NaH, DMF; (2) NaOH, MeOH; (c) (1) FeCl3, Zn, DMF/
H2O; (2) piperonyloyl chloride, K2CO3, 1,4-dioxane; (d) 6-bromo-1-hexanol, K2CO3, DMF; (e) (1) MsCl, Et3N, DCM; (2) NH3·H2O, EtOH;
(f) (+)-D-biotin, PyBOP, Et3N, DMF.
However, the nimesulide concentrations used in these studies
ranged from 200 to 500 μM, which greatly exceeded the
concentration necessary to inhibit COX-2 activity.21−26 This
line of reasoning suggests that nimesulide targeted other
pathways in order to achieve anticancer activity, and blockage
of these pathways required much higher concentrations. This
supported the hypothesis that nimesulide inhibited cancer cell
growth and induced apoptosis independent of its effects on
COX-2.22,23,27 Structurally, the ionization of the sulfonamide
group of nimesulide was critical for its COX-2 inhibition
(Figure 2).28,29 The N-methylation of nimesulide blocked the
ionization of the sulfonamide group, which abolished the COX-
2 inhibitory activity.28,29 Nimesulide showed minor hepatotox-
icity after long-term usage because of the multistep nitro-
reductive bioactivation that produced the hazardous nitro anion
radical and nitroso intermediate of the nitro group.30 The
conversion of the nitro group could erase the potential
hepatotoxicity of the new analogues. These two critical
modifications eliminated the COX-2 inhibitory activity and
the hepatotoxicity, but it was not clear if the anticancer activity
would be improved. After extensive investigations, we obtained
our first generation non-COX-2 active nimesulide anticancer
derivative 3, N-(3-(2,5-dimethylbenzyloxy)-4-(methylmethyl-
sulfonamido)phenyl)cyclohexanecarboxamide (JCC76)
(Figure 2).19,31,32 It inhibited SKBR-3 breast cancer cell growth
with an IC50 of 1.38 μM, which was about 100-fold more active
than nimesulide. Furthermore, compound 2 was recently
developed based on compound 3 and has demonstrated greater
anticancer potency,20 with IC50 of 0.1−0.5 μM to inhibit cancer
cell growth. However, the specific molecular targets of
compounds 3 and 2 were still unclear.
Target identification represents a major challenge in
anticancer drug development. The generally used method is
to immobilize the compound via a chemical linker on a solid-
phase support, followed by affinity purification of the cellular
targets (Figure 3).33 Therefore, we designed and synthesized
biotin-linked compound 2 as a probe for the target
identification (Scheme 1).34 Biotinylated compound 2 showed
acceptable antiproliferative activity on SKBR-3 breast cancer
cells (IC50 = 2 μM), although the anticancer activity is lower
than that of the parental compound with an IC50 of 0.2 μM.20
2.2. Affinity Purification of Compound 2-Bound
Proteins. We hypothesized that compound 2 bound to certain
proteins to achieve its anticancer activity. We mixed compound
2 probe with SKBR-3 breast cancer cell lysate in order to allow
specific binding proteins to attach to the compound 2 moiety of
the probe. For the protein isolation, the biotin moiety of the
probe was bound to the neutravidin resin to immobilize the
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Figure 4. Affinity isolation of 2-binding proteins. (A) Procedure for the purification of compound 2 binding proteins. (B) SKBR-3 breast cancer cell
lysate exposed to the probe (final probe concentration in the lysate was 25 μM) was incubated for 1 h at room temperature and then incubated with
neutravidin resin for 30 min. The mixture was loaded on a separation column and centrifuged. The resin was washed with binding buffer 5 times.
The last wash elution solution was kept. The resin was further washed twice with buffer containing different concentrations of compound 2. The last
buffer without drug wash solution (lane 3), two times wash solution with compound 2 (lane 5 and 6), and the resin (lane 4) were denatured in SDS
sample buffer and separated by SDS−PAGE. Lane 1 represents the molecular weight markers, and lane 2 is the whole cell lysate. Visualization of the
separated proteins with silver stain showed the specific binding proteins with compound 2.
probe. After extensive washing with binding buffer, the
nonbinding proteins were eluted. Then compound 2 was
used as a competing agent to wash the immobilized probe, and
the proteins specifically binding to the probe at the compound
2 domain were pulled out. The main procedure is described in
Figure 4A. We next examined the molecule weight of the
binding proteins using sodium dodecyl sulfate−polyacrylamide
gel electrophoresis (SDS−PAGE) and stained the gel with
silver staining reagent (Figure 4B). Lane 2 is the cell lysate, and
lane 3 is the final elution solution. Lane 5 is the binding buffer
with compound 2 (25 μM) as elution solution. Lane 6 is
binding buffer with compound 2 (50 μM) as elution solution,
and lane 4 is the resin boiled with SDS buffer. The results
suggest that compound 2 was not competitive enough to pull
all the proteins out of the probe, and the resin with the probe
still held a good amount of proteins. It is also possible that the
competition experiment was finished on the resin packed
column and only lasted 30 min, which led to less effective
competition. There are seven visible protein bands that can be
identified on the gel. Protein a and b have molecular weights
above 110 kDa, protein c has a molecular weight about 80 kDa,
proteins d, e, and f have molecular weights about 55−60 kDa,
and protein g has a molecular weight about 28 kDa. Apparently,
d and e are the major proteins bound to compound 2; the other
proteins are relatively minor. However, it is too early to
speculate which protein(s) is (are) the role player(s) at this
stage. Minor attached proteins are not less important than the
highly binding proteins. We will identify all the visible proteins
on the gel in next step.
trypsin in the digestion step, and band a itself did not give any
countable signals. Bands b, c, and f were keratin proteins with
peptide mass fingerprint matching scores below 10%. These
low binding, and without clear functions related to cell growth
proteins are not listed in Table 1. The major bands d and e
were identified to be tubulin α and tubulin β, respectively. Band
g was identified to be Hsp27. Both proteins are very critical for
cancer cell proliferation.35 To confirm the protein identity, we
repeated the SDS−PAGE assay combined with Western blot
(Figure 5A). The binding proteins were confirmed by using the
corresponding antibodies. Tubulin and Hsp27 as molecular
targets are new observations for the non-COX-2 active
nimesulide anticancer derivatives. It was reported that tubulin
has relatively strong interaction with Hsp27, and the two
proteins could be co-precipitated in the immunoprecipitation
experiment.36 In our study, both proteins were pulled out by
the probe, which could be due to the interaction between the
two proteins. The probe might only bind either to tubulin or to
Hsp27, but the interaction between the two proteins could have
caused them to both be retained by the probe. To rule out the
possibility of co-precipitation, we used tubulin and Hsp27 pure
proteins and our probe to repeat the binding experiments
individually. The protein binding ability was determined with
Western analysis (Figure 5B). After 5 times extensive washing
with binding buffer, the extra proteins were all eluted and there
were no proteins detected in the fifth elution solution.
However, tubulin and Hsp27 were further eluted after 2 was
added in the elution solution. The results indicated that both
proteins could specifically bind to the probe and could be
eluted with buffer containing compound 2, suggesting that
compound 2 was the ligand for both proteins. Tubulin inhibitor
1 with a sulfonamide moiety is a colchicine domain binder.18
Because of the structure similarity between compound 2 and 1
(Figure 1), we speculated that compound 2 might also be a
colchicine domain binder. To prove our hypothesis, we
preincubated tubulin protein with an equivalent amount of
colchicine for 1 h and then repeated the experiment. We found
that the probe did not hold any tubulin protein (Figure 5B),
suggesting that compound 2 and colchicine share same binding
site and that colchicine has higher binding affinity in the
2.3. Identification of the Binding Proteins. Compound
2 binding proteins were separated by SDS−PAGE and
collected by cutting the gel with the visible protein bands.
After silver-staining agent was removed, the protein bands were
subjected to tryptic digestion in situ. The resulting peptide
mixture was identified with mass spectrometry. The molecular
weight of the peptide mass fingerprint was used to identify the
protein identity via Mascot database (Matrix Science Mascot,
Boston, MA). The proteins with the best score (highest
possibility) are listed in Table 1. Band a was identified to be the
trypsin residue, which is possibly the signal of the leftover
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Figure 5. Protein identity and binding specificity confirmed with
antibodies and pure tubulin and Hsp27. (A) Protein identity was
further confirmed via Western blot with tubulin and Hsp27 antibodies.
SDS−PAGE assay in Figure 4 was repeated, and the protein was
transferred to a PVDF membrane and probed with tubulin and Hsp27
antibodies. (B) Binding of biotinylated compound 2 with pure Hsp27,
tubulin, colchicine preincubated tubulin, and paclitaxel preincubated
tubulin. The proteins (separated experiments) were exposed to the
probe (final probe concentration in the protein solution was 2 μM)
and incubated for 1 h at room temperature and then incubated with
neutravidin resin for 30 min. The mixture was loaded on a separation
column and centrifuged. The resin was washed with binding buffer 5
times and then further washed twice with buffer containing different
concentrations of compound 2 (25, 50 μM). All the elution solutions
and the resin were denatured with SDS buffer and then separated by
SDS−PAGE, transferred to a PVDF membrane, and probed with
tubulin and Hsp27 antibodies.
domain than compound 2. However, preincubation tubulin
with paclitaxel did not affect the binding ability of the probe
with the tubulin. These data are consistent with our hypothesis
that compound 2 binds to the colchicine binding domain on
tubulin. Preincubating tubulin with colchicine saturated the
binding domain and blocked the probe to attach to the protein,
but paclitaxel with different binding domain did not affect the
binding.
2.4. Biological Activity of Compound 2 to Tubulin and
Hsp27. We showed here that compound 2 binds to tubulin;
however, it is still not clear if the compound can interfere with
tubulin function. Tubulin can be affected in two manners.
Taxanes and epothilones stabilize tubulin polymerization,
whereas vinca alkaloids, halichondrins, and colchicine inhibit
tubulin polymerization.12,13,37,38 Although binding differently,
colchicine and vinca alkaloids show the same mechanism of
inhibition of tubulin. Compound 2, a relatively smaller and
nonchiral molecule compared to the bulky and bearing multiple
chiral centers natural product tubulin interfering agents,
inhibited tubulin polymerization dose-dependently (Figure 6A).
Nocodazole, a well-known tubulin inhibitor, was used as a positive
control for the assay. Compound 3, the first generation nimesulide
anticancer derivative, showed moderate tubulin polymerization
inhibitory activity. In addition, the tubulin polymerization
inhibitory effect was observed in cancer cells (Figure 6B). After
12 h of treatment with compound 2 at 0.5 and 1 μM, the
microtubules were disorganized and their density was significantly
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Figure 6. Effect of compound 2 on the organization of the microtubule cytoskeleton in vivo and in vitro. (A) The tubulin polymerization was done
using bovine brain tubulin with an in vitro assay. MAP-rich tubulin was incubated at 37 °C in the presence of vehicle (DMSO), 3 μM nocodazole,
0.5 and 1 μM 2, and 5 μM 3. Absorbance at 340 nm was measured for 20 min and presented as the increased polymerized microtubule. The data
were from one of two independent experiments with similar results. (B) SKBR-3 cells were incubated with 0.1% DMSO, 0.05 μM nocodazole, and
0.5 and 1 μM 2 for 12 h and 24 h. Cells were fixed, labeled with Alexa Fluor 488 antitubulin antibody, and observed with a Leica TCS SP2
fluorescence microscope.
reduced in SKBR-3 breast cancer cells. After 24 h of treatment, the
effect was more pronounced. The results indicate that compound
2 could inhibit tubulin polymerization in an assay with purified
protein and in SKBR-3 cells. The results also are in agreement
with the cell cycle arrest studies of compound 2 in previous
studies.20
Hsp27 is a chaperone of the small heat shock protein (sHsp)
family. The common functions of small heat shock proteins are
chaperone activity, thermotolerance, inhibition of apoptosis,
regulation of cell development, and cell differentiation. They
are also partially involved in cell signal transduction.35,39−42
Compound 2 showed significant anticancer activity in our
previous studies.20 Tubulin inhibitory activity was responsible
for the cancer cell toxic effect of the compound. Did Hsp27
play any role during the cell death since compound 2 bound to
this protein? The phosphorylation of Hsp27 is the key step for
Hsp27 to participate in the cell signal transduction process.36,40
We checked the levels of phosphorylated Hsp27 and total
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Hsp27 in SKBR-3 breast cancer cells after compound 2
treatment (Figure 7). The compound significantly increased
with Tyr224 (α-tubulin). The 2, 5-dimethylbenzyl moiety made
hydrophobic contacts with the Tyr224 and Ser178 residues
located on the α-tubulin (Figure 8B). The piperonylic amide
moiety formed a hydrophobic interaction with Leu248
(β-tubulin). The three interactions contributed to the strong
binding affinity of the compound to tubulin and subsequently
led to the tubulin polymerization inhibition activity. Negative
compound 4 without the 2,5-dimethyl groups on the benzyl
ring lost the three interactions (Figure 8C). A new hydrophobic
interaction (Asn 258 interacted with the central benzene ring)
was formed because of the molecule shifting from the pocket
(Figure 8D), but the interaction was weak compared to the
three interactions of compound 2. Compound 4 inhibited
SKBR-3 breast cancer cell growth with an IC50 of 62.9 μM. In
addition, negative compound 5 with a benzylamide moiety
instead of piperonylic amide moiety only interacted with
Ser178, even though the dimethyl group was maintained in the
structure (Figure 8E). The longer side chain of the amide
moiety pushed the 2,5-dimethylbenzyl moiety away from the
pocket and forced the molecule to rotate in the binding
domain. Compared with 2, the benzyl and the sulfonamide
moieties of compound 5 switched (Figure 8F), and therefore,
the molecule lost its interaction with TyrA224 and Leu248. The
only interaction between the compound and the binding pocket
was the central benzene ring with Ser178, which led to a weaker
tubulin inhibitor. Compound 5 inhibited SKBR-3 breast cancer
cell growth with an IC50 of 120.8 μM. These results further
demonstrated that compound 2 bound at the colchicine
binding site of tubulin and that the dimethylbenzyl and the
benzamide moieties of the compound were very important for
anticancer activity.
Figure 7. Effect of compound 2 on Hsp27. SKBR-3 breast cancer cells
were treated with DMSO, 2, nocodozole, and an Hsp27 inhibitor with
various concentrations for 24 h. Total cell lysates were extracted using
M-PER reagent (Pierce). Equal amounts of proteins (50 μg) were
separated by SDS−PAGE and transferred onto PVDF membrane. The
membranes were blocked for 1 h, followed by being probed with the
respective primary antibodies (anti-Hsp27, anti-pSer78 Hsp27, and
antiactin) and secondary antibody.
pHsp27 levels, which was similar to the response after tubulin
inhibitor nocodazole treatment. Cells use pHsp27 as a
protective molecule when any damage happens to the
cells.35,42 It is very common that cells express a higher level
of pHsp27 after being treated with cytotoxic agents.43
Compound 2 inhibited tubulin polymerization and induced
cell death, which is very likely to promote the activation of
pHsp27 in the cells. KRIBB3,44 an Hsp27 binder, also retained
Hsp27 protein via its biotinylated probe in a protein pull-down
assay. It was used as a control, and it significantly inhibited
Hsp27 phosphorylation (Figure 7). It seemed that compound 2
showed a different manner to this known Hsp27 binder
regarding Hsp27 inhibition, suggesting that compound 2 might
bind to a different domain of Hsp27 and that this domain was
not involved in Hsp27 phosphorylation. It was also possible
that the tubulin inhibitory activity of compound 2 dramatically
induced Hsp27 phosphorylation and overrode the direct Hsp27
inhibitory effect of the compound. Tubulin function was
relatively easier to check with a polymerization assay. However,
it was difficult to elucidate whether Hsp27 was an anticancer
target for compound 2. Besides Hsp27 phosphorylation, the
other downstream molecular and cellular consequences of
Hsp27 inhibition were not well-defined. The tubulin inhibition
could lead to Hsp27 phosphorylation, as indicated by pure
tubulin inhibitor nocodazole in Figure 7, suggesting that tubulin
function had a close correlation to Hsp27 function. It also made
it difficult to solely investigate the Hsp27 effects of the dual
targets compound 2. More medicinal chemistry effort is needed
to develop more specific ligands and dissociate the two targets
of compound 2.
3. CONCLUSION
A class of new anticancer agents were developed based on the
COX-2 inhibitor nimesulide as a lead compound.20 To identify
the molecular targets of the agents and elucidate the anticancer
mechanism, we designed and synthesized the biotinylated
nimesulide analogue 2 as a probe. The proteins binding with
the compound were isolated and subjected to mass
spectrometric identification, and the most prevalent binding
proteins were determined to be tubulin and Hsp27. Both
proteins are very important for cancer cell proliferation. Further
investigation revealed that the compound bound at colchicine
binding domain on tubulin and interrupted the polymerization
of tubulin. However, the biological activity of compound 2 on
Hsp27 was difficult to determine because the tubulin inhibitory
activity of compound 2 stimulated the activation of Hsp27
signals. The direct Hsp27 inhibitory effects of compound 2
could be overridden by the tubulin inhibition consequences.
Studies are now underway to further optimize compound 2 in
order to generate more potent derivatives. In addition, more
specific ligands, which have dissociated targets, i.e., pure tubulin
inhibitor or pure Hsp27 inhibitor, might be identified from the
pool of new compounds. The new sole ligands will allow us to
further investigate the consequences of Hsp27 inhibition.
2.5. Docking with Tubulin. Compound 2 bound to
tubulin and inhibited polymerization, which led to abnormal
cell function and ultimately cell death. Our studies also suggest
that compound 2 is a colchicine domain binder on tubulin. To
further elucidate the binding characters of the compound with
tubulin, we performed docking studies with compound 2 at the
colchicine binding pocket (Figure 8A). It has been reported
that colchicine mainly interacts with β-tubulin.42,45 Our docking
investigation suggests that compound 2 interacted with both
α- and β-tubulin in the colchicine pocket. The sulfonamide
group of the compound formed a dipole−dipole interaction
4. EXPERIMENTAL SECTION
4.1. Biotinylation of Compound 2. Chemicals were commer-
cially available and used as received without further purification unless
otherwise noted. Moisture sensitive reactions were carried out under a
dry argon atmosphere in flame-dried glassware. Solvents were distilled
before use under argon. Thin-layer chromatography was performed on
precoated silica gel F254 plates (Whatman). Silica gel column
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Figure 8. (A) Compound 2 (orange) and colchicine (Green) share the same binding site on tubulin. The left part of the imagine is β-tubulin, and
the right part is α-tubulin. (B) There are several interactions between 2 and the binding pocket including the 2,5-dimethylbenzyl moiety interacting
with Ser178 and Tyr224 (α-tubulin), the sulfonamide group interacting with Tyr224, and the piperonylic amide group interacting with Leu 248
located on β-tubulin (all three green residues). (C) Negative compound 4 (inhibiting SKBR-3 cell growth with an IC50 of 62.9 μM) does not have
the 2,5-dimethyl group on the benzyl moiety. The compound does not interact with the three sites identified with 2. It only interacts with Asn 258
(magenta), which makes it loosely sits in the pocket. (D) Comparison of 2 and negative 4. Without the dimethyl groups on the benzyl ring, the
moiety does not interact with Ser178, and the whole molecule (cyan) slightly shifts away from the 2 (orange) binding site. From the SAR generated
in the previous studies on this series of compounds, the dimethyl group is very critical for anticancer activity. It matches this docking result. (E)
Negative compound 5 (inhibiting SKBR-3 cell growth with an IC50 of 120.8 μM) has a benzylamide moiety instead of piperonylic amide moiety.
Interestingly, the compound docks in the pocket in a different mode. Compared with 2, the benzyl and the sulfonamide moieties switch binding
domain. However, the compound only has an interaction with Ser178 (yellow), which leads to a weak tubulin inhibitor. (F) Comparison of 2 and
negative 5. Changing piperonylic amide moiety to benzylamide leads to a rotation of the molecule (blue) in the docking site compared to 2 (orange). The
Ser178 residue interacts with the central benzene ring of the negative compound, and this is the only interaction of the molecule with the docking site.
chromatography was performed using silica gel 60A (Merck, 230−400
mesh), and hexane/ethyl acetate was used as the elusion solvent. Mass
spectra were obtained on the Micromass quadrupole time-of-flight
(QTOF) electrospray mass spectrometer at Cleveland State University
MS Facility Center. All the NMR spectra were recorded on a Varian 400
Negative compounds 4 and 5, compounds I and II, and the
methodology of the probe synthesis were described in previous studies
(Scheme 1).20,46
Benzo[1,3]dioxole-5-carboxylic Acid [3-(2,5-Dimethylbenzyloxy)-
4-(methanesulfonylamino)phenyl]amide (III). A mixture of ferric
chloride (4 mmol, 4 equiv) and II (1 mmol, 1 equiv) was added to a
solvent mixture of DMF/water (3:1, 8 mL). It was stirred for 15 min,
1
MHz in either DMSO-d6 or CDCl3. Chemical shifts (δ) for H NMR
spectra are reported in parts per million to residual solvent protons.
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and then zinc dust (10 mmol, 10 equiv) was added slowly. The
mixture was stirred at room temperature for half an hour and then
filtered by passing it through a Celite pad. The filtrate was diluted with
water. The precipitated solid was collected by filtration, and then it was
dissolved in acetone. After filtration of the insoluble residue, the filtrate
was collected and concentrated under vacuum to afford the substituted
aniline. To the substituted aniline (1.0 mmol, 1.0 equiv) in 3 mL of
1,4-dioxane were added K2CO3 (5 mmol, 5 equiv) and piperonyloyl
chloride (1.2 mmol, 1.2 equiv), and the mixture was stirred at room
temperature overnight. Then 10 mL of H2O and 3 mL of saturated
aqueous Na2CO3 were added to the mixture, and the mixture was
stirred at room temperature overnight. The precipitated solid was
DMSO vehicle at a concentration equal to that in drug-treated cells.
The medium was removed, replaced by 200 μL of 0.5 mg/mL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in fresh
medium, and cells were incubated in the CO2 incubator at 37 °C
for 2 h. Supernatants were removed from the wells, and the reduced
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye
was solubilized in 200 μL/well DMSO. Absorbance at 570 nm was
determined on a plate reader. Statistical and graphical information was
determined using GraphPad Prism software (GraphPad Software
Incorporated) and Microsoft Excel (Microsoft Corporation). Deter-
mination of IC50 values was performed using nonlinear regression
analysis.
4.2.3. Biotin−Neutravidin Pull-Down Assay. SKBR-3 cells (1.0 ×
107) were disrupted in a NP-40 lysis buffer and sonicated with freshly
added protease inhibitor cocktail (Roche). The cell lysate was
incubated with biotin-conjugated compound 2 probe at room
temperature for 1 h. The mixture was further incubated with
equilibrated and packed neutravidin resin in columns at room
temperature for 30 min, which was followed by centrifugation and
five times repeated washing with binding buffer to wash out
nonbinding proteins. The pull-down assay was performed according
to the protocol of neutravidin (Thermofisher). The protein interacting
with the biotinylated compound 2 was cleaved from the beads by
eluting with binding buffer containing compound 2. The resin was also
collected and boiled with SDS buffer to determine the leftover
proteins. The elution solution was boiled with 1× loading buffer (100
mmol/L DTT plus bromophenol blue) for 5 min and then
electrophoresed on a 10% SDS−polyacrylamide gel. The resulting
gel was visualized with silver stain kit (for mass spectrometry,
compatible silver staining kit, Invitrogen).
4.2.4. Peptide Analysis of the 2-Binding Protein via Mass
Spectrometry. Bands visualized by silver staining were cut and
transferred to 0.65 mL siliconized tubes (National Scientific Supply,
Claremont, CA, U.S.). The silver reagent was removed with the
denatured reagents (Invitrogen). Proteins were in-gel digested by
trypsin (sequencing grade, modified; Promega, Madison, WI, U.S.).
The protein digest was reconstituted in 20 μL of 0.1% (v/v)
trifluoroacetic acid prior to LC-QTOF/MS analysis. Peptide
separation was carried out using a 10 μL sample injection at 50 μL/
min flow rate on a Vydac protein and peptide C18 (5 μm, 300 Å,
1 mm × 150 mm) column (Grace Discovery Sciences, Deerfield, IL,
U.S.) preceded by an inline filter (0.5 μm pore) (Upchurch Scientific,
Oak Harbor, WA, U.S.). The gradient elution profile consisted of 1%
of mobile phase A for 5 min, then brought to 60% of mobile phase B
over 90 min, and followed by 90% of mobile phase B for 8 min. The
total run time was 105 min. Mobile phase A was 0.1% (v/v) formic
acid in HPLC-grade doubly distilled H2O, and mobile phase B was
0.1% (v/v) formic acid in HPLC-grade acetonitrile. Peptide detection
was done using the positive information-dependent-acquisition (IDA)
mode of AB Sciex QStar Elite Q-TOF mass spectrometer (AB Sciex,
Foster City, CA, U.S.). Data acquisition was performed using AB Sciex
Analyst QS (version 2.0). Protein identification through peptides
matching was accomplished using Mascot MS/MS ions search
(http://www.matrixscience.com).
4.2.5. Western Blot. To confirm the identity of protein targets that
have been discovered in neutravidin resin pull-down assay, Western
blot was conducted. Protein samples were separated on 12% SDS−
polyacrylmide gel and transferred onto polyvinylidene difluoride
(PVDF) membrane (Pall Cooperation, FL). After blocking, the
membrane was incubated in PBST containing 5% BSA and primary
antibody specific to α-, β-tubulin or Hsp27 (Cell Signaling, MA)
overnight at 4 °C. HRP-conjugated anti-rabbit IgG or anti-mouse IgG
(Cell Signaling, MA) was used as secondary antibody and incubated at
room temperature for 1 h. Membrane was incubated in ECL plus
reagent (GE health) and then exposed to hyper film.
1
collected by filtration and recrystallized in ethanol. H NMR (400
MHz, DMSO-d6) δ 10.120 (1H, s), 8.915 (1H, s), 7.720 (1H, d, J =
2.4 Hz), 7.575 (1H, dd, J = 1.6, 8 Hz), 7.513 (1H, d, J = 1.6 Hz), 7.379
(2H, m), 7.207 (1H, d, J = 8.8 Hz), 7.080 (3H, m), 6.140 (2H, s),
5.064 (2H, s), 2.847 (3H, s), 2.318 (3H, s), 2.283 (3H, s).
Benzo[1,3]dioxole-5-carboxylic Acid {3-(2,5-Dimethylbenzyloxy)-
4-[(6-hydroxyhexyl)methanesulfonylamino]phenyl}amide (IV).
K2CO3 (5 mmol, 5 equiv) and 6-bromo-1-hexanol (1.2 mmol, 1.2
equiv) were successively added to a solution of III (1.0 mmol, 1.0
equiv) in 3 mL of DMF, and the mixture was stirred at room
temperature overnight. An amount of 12 mL of H2O was added, and
the precipitated solid was collected by filtration and purified by flash
1
column chromatography. H NMR (400 MHz, CDCl3) δ 8.227 (1H,
s), 7.962 (1H, d, J = 2 Hz), 7.455 (1H, dd, J = 1.6, 8 Hz), 7.388 (1H,
d, J = 1.6 Hz), 7.220 (1H, d, J = 8.4 Hz), 7.091 (3H, m), 6.853 (2H,
m), 6.047 (2H, s), 5.035 (2H, s), 3.575 (2H, t, J = 6.4 Hz), 3.515 (2H,
br), 2.714 (3H, s), 2.315 (6H, s), 1.504−1.287 (8H, m).
N-(3-(2,5-Dimethylbenzyloxy)-4-(N-(6-(5-((3aR,4R,6aS)-2-oxohex-
ahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexyl)-
methylsulfonamido)phenyl)benzo[d][1,3]dioxole-5-carboxamide
(V). To a solution of IV (2 mmol, 1 equiv) and triethylamine (10
mmol, 5 equiv) in dichloromethane (5 mL) at 0 °C was added
methylsulfonyl chloride (6 mmol, 3 equiv). The mixture was kept
stirring at room temperature. After the reaction was completed, ice
cold water was added to the solution. The organic layer was washed
twice with ice cold water and then evaporated to give mesylated IV. A
solution of this intermediate in ethanol (20 mL) and ammonium
hydroxide (20 mL) was stirred for 2 days at room temperature and
then concentrated to give the intermediate amine, which was used for
the next step directly without purification. To the mixture of amine
(2 mmol, 1 equiv) and biotin (2 mmol, 1 equiv) and PyBOP (2 mmol,
1 equiv) in 5 mL of DMF was added triethylamine (4 mmol, 2 equiv),
and the mixture was stirred at room temperature for 2 h. An amount of
15 mL of H2O was added to the mixture, and the precipitated solid
1
was filtered and purified by flash column chromatography. H NMR
(400 MHz, CDCl3) δ 8.864 (1H, s), 8.058 (1H, s), 7.526 (1H, d, J =
8 Hz), 7.457 (1H, s), 7.235 (1H, d, J = 8.4 Hz), 7.090 (3H, m), 6.976
(1H, dd, J = 2, 8.4 Hz), 6.871 (1H, d, J = 8 Hz), 6.277 (1H, s), 6.229
(1H, t, J = 5.6 Hz), 6.040 (2H, s), 5.345 (1H, s), 5.060 (2H, s), 4.455
(1H, m), 4.260 (1H, m), 3.500 (2H, br), 3.113 (3H, m), 2.866 (1H,
dd, J = 4.8, 12.8 Hz), 2.704 (4H, m), 2.322 (3H, s), 2.315 (3H, s),
2.165 (2H, t, J = 7.6 Hz), 1.692−1.253 (14H, m). ESI-MS calcd for
C40H52N5O8S2 [M + H]+ 794.3, found 794.2
4.2. Biological Studies. 4.2.1. Cell Culture. SKBR-3 cells were
obtained from ATCC (Rockville, MD). The cells were
maintained in RPMI 1640 medium supplemented with 10%
fetal bovine serum (FBS), 2 mmol/L L-glutamine, 1 mmol/L
sodium pyruvate, and 100 U/mL penicillin−streptomycin. FBS
was heat inactivated for 30 min in a 56 °C water bath before
use. Cell cultures were grown at 37 °C in a humidified
atmosphere of 5% CO2 in a Hereaus CO2 incubator.
4.2.2. Cell Viability Analysis. The effects of compounds 4, 5, 2, and
its biotinylated probe on SKBR-3 cell viability were assessed using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
assay in six replicates. Cells were grown in RPMI 1640 medium in
96-well, flat-bottomed plates for 24 h and were exposed to various
concentrations of the compounds dissolved in DMSO (final
concentration of ≤0.1%) in medium for 48 h. Controls received
To determine the effect of compound 2 on Hsp27 phosphorylation,
SKBR-3 cells were treated with 0.2, 0.5, and 1 μM compound 2,
50 and 100 nM nocodazole (Sigma), and 0.5 and 1 μM KRIBB3
(Sigma) for 24 h. Extracted proteins from SKBR-3 cells were loaded
on 12% SDS−polyacrylamide gel. Antibody specific to phosphorylated
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Hsp27 (Ser 78, Cell Signaling, MA) was used to blot membrane, and
secondary antibody anti-rabbit IgG was blotted thereafter. Hsp27 and
actin antibodies were also used to confirm equal loading amount.
4.2.6. 2 Binding to Tubulin and Hsp27. 10 μM bovine brain
tubulin (Cytoskeleton, Denver, CO) or recombinant Hsp27 (ProSpec,
East Brunswick, NJ) was incubated with 2 μM biotinylated compound
2 in binding buffer in a total volume of 500 μL for 1 h at room
temperature before loading onto prepacked neutravidin resin column
and further incubated for 30 min. The resin was washed with 500 μL
of binding buffer 5 times and 2 times with buffer containing
compound 2. For the colchicine, paclitaxel, and compound 2 probe
competition experiments, tubulin was preincubated with 10 μM
colchicine or paclitaxel at room temperature for 1 h before the
incubation with the probe. The samples were fractionated by SDS−
PAGE and examined with Western blot assay.
4.2.7. Tubulin Polymerization Assay. Microtubule-associated
protein-rich tubulin (2 mg/mL, bovine brain, Cytoskeleton) in buffer
containing 80 mM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, and
5% glycerol was placed in cuvettes, 200 μL/assay, and incubated
respectively with DMSO, 0.5 and 1 μM compound 2, 5 μM compound
3, and 3 μM nacodazole. Polymerization was started by adding 1 mM
GTP and incubating at 37 °C, followed by absorption readings at
340 nm with a Varian Cary 50 series spectrophotometer (every 5 s/
min 0 to min 3, every 10 s/min 3 to min 5, every 30 s/min 5 to min
10, and every 60 s/min 10 to min 17).
4.2.8. Indirect Immunofluorescence Staining. SKBR-3 cells were
transferred in chamber slides and cultured to 70% confluence and then
incubated with 0.5 or 1 μM compound 2 respectively for 12 and 24 h.
In parallel, the cells without treatment were used as negative control,
and cells treated with 0.05 μM nacodazole for 12 and 24 h were used
as positive control. Cells receiving different treatments were fixed in
4% paraformaldehyde for 10 min at room temperature and then
permeabilized with 0.2% Triton X-100 for another 10 min. After
blocking with 2% goat serum for 45 min at room temperature, cells
were incubated with biotinylated anti-tubulin antibody (1:200,
Molecular Probes) overnight at 4 °C. After being washed with PBS,
cells were then stained with Alexa Fluor 488 streptavidin (1:1000,
Invitrogen) for 45 min at room temperature, which was followed by
mounting with antifade reagent (ProLong Gold antifade reagent,
Invitrogen). Fluorescently stained cells were analyzed with a Leica
TCS SP2 fluorescence microscope.
4.2.9. Docking Investigation. Docking studies were done to gain
insight into the possible mode of interaction between the compounds
and tubulin. The structures were drawn with Marvin Sketch (www.
ChemAxon.com) and energy minimized in MOE 2010 (www.
chemcomp.com) using the MMFF94s force field. The database of
compounds was then used for the docking studies. The protein crystal
structure of 11SAO.pdb was prepared for docking by adjusting the pH
of the system to pH 7.4.45 The binding site in tubulin was delineated
by colchicine. After docking, only the top docked pose of each
compound was retained for analysis.
ABBREVIATIONS USED
■
COX-2, cyclooxygenase-2; Hsp27, heat shock protein 27; sHsp,
small heat shock protein; FDA, Food and Drug Administration;
SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel
electrophoresis; QTOF, quadrupole time-of-flight; FBS, fetal
bovine serum; PVDF, polyvinylidene difluoride
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 216-687-9219. Fax: 216-687-9298. Email: B.su@
csuohio.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a startup fund from Cleveland
State University, OH. We thank Dr. Xue-long Sun (Cleveland
State University) for help on the UV analysis of tubulin
polymerization, and Jones Sidney (Department of Molecular
Cardiology, Cleveland Clinic, OH) on the cellular microtubule
images taken.
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