Proteome interrogation using nanoprobes to identify targets of a cancer-killing molecule

Article abstract of DOI:10.1021/ja111137n

We report a generic approach for identification of target proteins of therapeutic molecules using nanoprobes. Nanoprobes verify the integrity of nanoparticle- bound ligands in live cells and pull down target proteins from the cellular proteome, providing very important information on drug targets and mechanisms of action. As an example, target proteins as R-tubulin and HSP 90 have been identified and validated.

Full text of DOI:10.1021/ja111137n

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pubs.acs.org/JACS
Proteome Interrogation Using Nanoprobes To Identify Targets of a
Cancer-Killing Molecule
Liwen Li,† Qiu Zhang,† Aifeng Liu,† Xiue Li,† Hongyu Zhou,‡ Yin Liu,† and Bing Yan*,†,‡
†Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, China
‡Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, United States
S Supporting Information
b
ABSTRACT: We report a generic approach for identifica-
tion of target proteins of therapeutic molecules using
nanoprobes. Nanoprobes verify the integrity of nanoparti-
cle-bound ligands in live cells and pull down target proteins
from the cellular proteome, providing very important in-
formation on drug targets and mechanisms of action. As an
example, target proteins as R-tubulin and HSP 90 have been
identified and validated.
evastating diseases such as central nervous system disorders
and cancer are complex diseases that can result from mul-
D
tiple genetic mutations. In the search for new treatments, simple
approaches such as single-target protein screenings often fail to
yield innovative drugs. In contrast, multitargeting molecules are
often advantageous in the treatment of these complex diseases
because they affect multiple pathways.1,2 Drug resistance is also
less likely to develop with these molecules.1 However, multi-
targeting drugs are difficult to discover through rational drug
design or single-protein screening approaches. Phenotype screen-
ing and optimization are promising methods for developing such
therapeutics, provided that the targets and mechanisms of
action can be elucidated to show the therapeutic and side
effects of the drugs and develop second-generation drugs with
a better profile.
Figure 1. Structure of 1and 3, their derivative 2and 4, GNP-2and GNP-4.
analogue of compound 1, it was much less toxic to H460 (EC50
53.7 μM).
=
Previous structureÀactivity relationship studies showed that
rings B and C in 1 are stereochemically confined, while ring A is
much more tolerant of structural modifications. We modified
ring A [Scheme S1 in the Supporting Information (SI)] by
attaching a flexible and biocompatible linker to give compound 2,
and a similar modification of 3gave 4. GNP-2and GNP-4(Figure1)
were then synthesized in situ using a reported method8 (see the
SI for detailed syntheses and characterizations of GNP-2 and
GNP-4). GNP-4 derived from inactive compound 3 was used as
a control (Figure 1) for target validation.
High-resolution transmission electron microscopy (TEM)
analysis (Figure 2a,b) showed that the average diameter of the
GNP-2 particles was 2.5 nm (Figure 2b). The GNP-linked
ligands were analyzed by high-performance liquid chromatogra-
phy/mass spectrometry (HPLC/MS) after cleavage with I2. The
chromatogram (Figure 2c) and the associated electrospray
ionization MS (ESI-MS) spectrum (Figure 2d) showed that
the peak at 6.6 min corresponded to compound 2 (MW 672.25).
Affinity methods, which are often used in target identification,
use a solid support that attaches active compounds to pull down
proteins after incubation with cell lysate.3À6 Bound proteins are
separated by sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE), and then their identities are determined.
However, a major drawback of these methods is that it is not pos-
sible to confirm whether the target-binding specificity is altered
by chemical modification and the linkage to the solid support
because of the large size of testing beads.
Nanoparticles with bioactive compounds on the surface can
enter live cells to confirm the desired biological activity and target-
ing specificity of the modified compound and at the same time
identify target proteins by interrogating the cellular proteome in cell
lysate. To test this hypothesis, we designed and synthesized
compound 1 (Figure 1). Compound 1 selectively killed nonsmall
cell lung cancer H460 cells with a median effective concentration
(EC50) of 0.9 μM and was much less toxic to normal human
fibroblasts (EC50 > 100 μM). However, its primary targets and
mechanisms of action are not known.7 Compound 3 is a structural
Received: December 14, 2010
Published: April 15, 2011
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2011 American Chemical Society
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Figure 4. Target identification by GNP-2. H460 cell extract (300 μL)
was incubated with (lane 3) or without (lane 4) 5 mM compound 1 for 1
h at 4 °C, after which 0.03 μmol of GNP-2 was added and the mixture
incubated for 1 h at 4 °C. As a control, cell extract (300 μL) was also
incubated with GNP-4 and treated in the same way (lane 2). Proteins
bound to GNPs were separated by 10% SDS-PAGE followed by
improved Coomassie brilliant blue G-250 staining.14 Protein bands with
lower intensities in the lane with compound 1 than in the lane without
compound 1 were identified by MALDI-TOF/TOF MS and Mascot
analysis (Table 1).
Figure 2. (a, b) Characterization of GNP-2: (a) TEM image of GNP-2.
Scale bar = 10 nm. (b) Size distribution of GNP-2 according to TEM
images. The average diameter was 2.5 nm. (c, d) Structure identification
of free ligands cleaved from GNP-2 by I2: (c) Chromatogram. The peak
at 5.3 min is I2, and the peak at 6.6 min is compound 2. (d) ESI-MS
spectrum corresponding to the peak at 6.6 min.
Table 1. Protein Identification by MALDI-TOF/TOF MS
and Mascot Analysis
protein score
band
protein
gene
CI (%)a
1
2
heat shock protein HSP 90-β
tubulin R-1 C chain
Hsp90ab1
TUBA1C
100
100
a The protein score confidence interval (CI) was calculated using the
Mascot search engine to assess the match between the experimental data
and the database sequence.
Figure 3. (a) TEM image of GNP-2 cell uptake. H460 cells were treated
with 5 μM GNP-2 for 48 h. Scale bar = 2 μm. (b) Percentage growth of
H460 cells treated with GNP-2 and GNP-4 for 48 h. The concentrations
of GNP-2 and GNP-4 were 0, 1, 2.5, 5, and 20 μM. Results represent
mean ( standard error of the mean (SEM) in triplicate. The * label
indicates that the percentage growth of the group treated with GNP-2
was significantly different (P < 0.05) from that of the group treated with
GNP-4 under the same conditions.
More than 10 protein bands ranging from 35 to 150 kDa were
observed for GNP-2 (lane 4), while fewer proteins were seen for
GNP-4 (lane 2). To ensure that only proteins with specific
binding to GNP-2 were correctly identified, we also preincubated
compound 1 with the lysate for 1 h at 4 °C and then incubated
this cell lysate with GNP-2 for an additional 1 h (lane 3). Com-
parison of lane 4 with lanes 3 and 2 revealed that only two protein
bands showed reduced intensities (Figure 4), indicating that they
might be the specific target proteins of compound 1. The
identities of these proteins were determined by matrix-
assisted laser desorption ionization tandem time-of-flight
(MALDIÀTOF/TOF) MS and data analysis using the Mascot
search engine (Table 1). Protein identification results with a
protein score confidence interval of 100% were accepted. These
proteins were heat shock protein 90-β (β-HSP 90) and tubulin
R-1C chain. The proteins R-tubulin9À11 and β-HSP 9012,13 are
validated therapeutic targets for cancer treatment. Thus, R-
tubulin and β-HSP 90 could be considered as the potential
targets of compound 1.
On average, there were ∼65 ligands on each GNP (see section 4
in the SI). Since the chemical modifications and the attachment
to a GNP might alter the target-binding specificity, it was
necessary to test whether GNP-2 could still enter and kill cancer
cells. Figure 3a shows that GNP-2 particles could readily enter
cells. Although some GNP-2 particles were found in the cyto-
plasm, most of them were in endosomes. The cytotoxicity results
(Figure 3b) showed that GNP-2 exhibited dose-dependent
toxicity toward H460 cells, but GNP-4 showed much less
toxicity. Because GNP-2 particles were mainly trapped in endo-
somes, as most nanoparticles are, the reduced cytoplasm entry
might have been responsible for a higher EC50 value than for the
free compound 1. However, GNP-2 maintained its target-bind-
ing and anticancer activities. Although the protein pulled down
with this method was eventually validated by a series of biological
studies, this nanoparticle-based prequalification assay in live cells
provided key guidance at a very early stage.
Among target proteins of compound 1, tubulin is a key anti-
cancer target. Tubulin-binding agents can stabilize (such as pacli-
taxel) or destabilize (such as colchicine) microtubule formation,
block cell-cycle progression, and cause apoptosis. To validate
tubulin as a target for compound 1, we first investigated the
compound’s effect on tubulin polymerization in vitro and on
After they were proven to be active in live cells, GNP-2 and
GNP-4 were incubated with cancer cell lysate for 1 h before the
bound proteins were separated and analyzed by electrophoresis.
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Figure 6. Validation of HSP 90 as a target through Western blot analysis
for HSP 90 client proteins CRAF-1, ERBB2, and p-AKT in H460 cell
extracts prepared after 0, 24, and 48 h of treatment with (left) 5 μM
compound 1 and (right) 200 nM 17-DMAG, which is a known HSP 90
inhibitor.
AKT phosphorylation.21À23 We investigated whether compound
1, after binding to HSP 90, inhibited its chaperone activity and
caused degradation of CRAF-1 and ERBB2 and inhibition of
AKT phosphorylation in comparison with a known HSP 90 in-
hibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin
(17-DMAG).12 17-DMAG inhibits HSP 90, inducing proteo-
some-dependent degradation of CRAF-1 and ERBB2 and in-
hibiting phosphorylation of AKT (Figure 6, right panels). Similar
protein degradation patterns (CRAF-1 and ERBB2) were also
observed for compound 1. It also induced a time-dependent
inhibition of AKT phosphorylation. These results reveal that the
anticancer mechanism of action of compound 1 partly involves
inhibition of HSP 90 function.
After the discovery and validation that compound 1 specifi-
cally inhibits tubulin and HSP 90, the mechanism of action of this
compound was further substantiated by its role in inducing G2/M
cell-cycle arrest and apoptosis (Figure S5 in the SI).
In summary, we have used nanoprobes to identify dual targets
for compound 1 in this work by first validating its anticancer
activity in live cells and then interrogating the proteome in cell
lysate. Our findings demonstrate the power of nanotechnology in
drug discovery and chemical biology research. Target identifica-
tion for therapeutic compounds has been a severely under-
developed area in drug discovery research, and the validation
of uncertain targets is tedious and expensive. Nanoprobes will
likely play a pivotal role in this area.
Figure 5. Validation of tubulin as a target. DMSO and colchicine were
used as negative and positive controls. (a) Microtubule polymerization
assay. The concentrations of compound 1 and colchicine were 10 and 5
μM, respectively. Results represent mean ( SEM from two independent
experiments. (bÀd) Microtubule immunofluorescence microscopy
images of H460 cells incubated with (b) 1 μM compound 1, (c) 1
μM colchicine, and (d) DMSO for 24 h. Microtubules were labeled by
R-tubulin antibody. Scale bar = 25 μm. (e) Western blot analysis for
p-JNK and JNK in the H460 cell extracts prepared after 0, 12, 24, and
48 h of treatment with 10 μM compound 1.
microtubule assembly/disassembly processes in live cells. Tubulin
subunits self-assemble to form cylindrical microtubules in a time-de-
pendent manner (Figure 5a). Colchicine, a microtubule depolymer-
izing agent, inhibited microtubule polymerization (Figure 5a).15,16
Compound 1 also caused microtubule depolymerizaiton in a
manner similar to that of colchicine (Figure 5a). To substantiate
this finding, compound 1’s effects on microtubule organization in
live cells were investigated using immunofluorescence micro-
scopy. The microtubule network in cells treated with 1 μM
colchicine (Figure 5c) or compound 1 (Figure 5b) was disrupted
completely. To further validate compound 1 as a microtubule-
interfering agent, we investigated compound 1-induced activi-
tion of c-Jun NH2-terminal kinase (JNK). JNK activation is a
hallmark event for microtubule-interfering agents such as pacli-
taxel, vinblastine, vincristine, docetaxel, and nocodazole.17À20
The elevated level of p-JNK by compound 1 was detected, and
compound 1-induced JNK activation peaked at 12 h (Figure 5e).
These results demonstrate that compound 1 inhibits microtubule
organization in live cells by binding to tubulin and inhibiting its
polymerization.
’ ASSOCIATED CONTENT
S
Supporting Information. Details of the synthesis of
b
compound 2, quantification of the ligand contents of GNP-2,
cell-cycle arrest and apoptosis induced by compound 1, experi-
mental procedures, and complete refs 3 and 5. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
dr.bingyan@gmail.com
’ ACKNOWLEDGMENT
In cancer cells, HSP 90 is overexpressed to actively assist
folding and maturation of oncogenic proteins such as CRAF-1
and ERBB2 and also to assist AKT phosphorylation. Blocking
HSP 90 leads to degradation of these proteins and inhibition of
This work was supported by the National Basic Research
Program of China (2010CB933504), the National Natural Sci-
ence Foundation of China (90913006 and 21077068), the
National Cancer Institute (P30CA027165), the American Lebanese
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Syrian Associated Charities (ALSAC), and St. Jude Children’s
Research Hospital. We thank Dr. Vishwajeeth Reddy Pagala and
Ms. Linda Mann for technical assistance.
’ REFERENCES
(1) Frantz, S. Nature 2005, 437, 942.
(2) Roth, B. L.; Sheffler, D. J.; Kroeze, W. K. Nat. Rev. Drug Discovery
2004, 3, 353.
(3) Bach, S.; et al. J. Biol. Chem. 2005, 280, 31208.
(4) Leslie, B. J.; Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347.
(5) Shimizu, N.; et al. Nat. Biotechnol. 2000, 18, 877.
(6) von Rechenberg, M.; Blake, B. K.; Ho, Y. S. J.; Zhen, Y. J.;
Chepanoske, C. L.; Richardson, B. E.; Xu, N. F.; Kery, V. Proteomics
2005, 5, 1764.
(7) Zhou, H. Y.; Wu, S. H.; Zhai, S. M.; Liu, A. F.; Sun, Y.; Li, R. S.;
Zhang, Y.; Ekins, S.; Swaan, P. W.; Fang, B. L.; Zhang, B.; Yan, B. J. Med.
Chem. 2008, 51, 1242.
(8) Zhou, H. Y.; Du, F. F.; Li, X.; Zhang, B.; Li, W.; Yan, B. J. Phys.
Chem. C 2008, 112, 19360.
(9) Chang, J.-Y.; Chang, C.-Y.; Kuo, C.-C.; Chen, L.-T.; Wein,
Y.-S.; Kuo, Y.-H. Mol. Pharmacol. 2004, 65, 77.
(10) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med.
Res. Rev. 1998, 18, 259.
(11) Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
(12) Holzbeierlein, J.; Windsperger, A.; Vielhauer, G. Curr. Oncol.
Rep. 2010, 12, 95.
(13) Workman, P.; Powers, M. V. Nat. Chem. Biol. 2007, 3, 455.
(14) Diezel, W.; Kopperschl€ager, G.; Hofmann, E. Anal. Biochem.
1972, 48, 617.
(15) Garland, D. L. Biochemistry 1978, 17, 4266.
(16) Skoufias, D. A.; Wilson, L. Biochemistry 1992, 31, 738.
(17) Lee, L.-F.; Li, G.; Templeton, D. J.; Ting, J. P.-Y. J. Biol. Chem.
1998, 273, 28253.
(18) Wang, T.-H.; Wang, H.-S.; Ichijo, H.; Giannakakou, P.; Foster,
J. S.; Fojo, T.; Wimalasena, J. J. Biol. Chem. 1998, 273, 4928.
(19) Mollinedo, F.; Gajate, C. Apoptosis 2003, 8, 413.
(20) Gajate, C.; Barasoain, I.; Andreu, J. M.; Mollinedo, F. Cancer
Res. 2000, 60, 2651.
(21) Moser, C.; Lang, S. A.; Stoeltzing, O. Anticancer Res. 2009,
29, 2031.
(22) Sain, N.; Krishnan, B.; Ormerod, M. G.; De Rienzo, A.; Liu,
W. M.; Kaye, S. B.; Workman, P.; Jackman, A. L. Mol. Cancer Ther. 2006,
5, 1197.
(23) Powers, M. V.; Workman, P. Endocr.-Relat. Cancer 2006, 13,
S125.
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