In situ monitoring of bindings between dasatinib and its target protein kinases using magnetic nanoparticles in live cells.

Subscriber access provided by Vanderbilt Libraries
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Communication
In Situ Monitoring of Bindings between Dasatinib and Its Target
Protein Kinases Using Magnetic Nanoparticles in Live Cells
Dae-Joong Kim, Yong-Weon Yi, and Jin Hwan Kim
J. Am. Chem. Soc., Article ASAP • DOI: 10.1021/ja8063843
Downloaded from http://pubs.acs.org on November 18, 2008
More About This Article
Additional resources and features associated with this article are available within the HTML version:
• Supporting Information
• Access to high resolution figures
• Links to articles and content related to this article
• Copyright permission to reproduce figures and/or text from this article

In Situ Monitoring of Bindings between Dasatinib and Its Target Protein
Kinases Using Magnetic Nanoparticles in Live Cells
Dae-Joong Kim, Yong-Weon Yi, and Jin Hwan Kim*
CGK Co., Ltd., Daejeon BioVenture Town, 461-8, Jeonmin-Dong, Yuseong-Gu, Daejeon, 305-811, Korea
Received August 12, 2008; E-mail: jinhkim1@gmail.com
A thorough understanding of the binding characteristics of a small
molecule against its target proteins in live cells is of utmost
importance in designing therapeutic agents with improved selectivity
and specificity.1 Cell-based assays using yeast or mammalian three
hybrid systems have been used to study the binding characteristics
of a small molecule kinase inhibitor and its target protein kinases.2
Although these three hybrid assays can be performed in a cellular
context, the binding characteristics found are related with indirect
reporter activities through a cascade of transcriptional and trans-
lational events, rather than direct interactions between a small
molecule and its target proteins.2 Here, we have developed a new
methodology, called In Cell Interaction Trap (InCell IT), which
allows real time monitoring of the direct molecular interactions
between a small molecule and its target proteins in live cells using
magnetic nanoparticles (MNPs) (Scheme 1). MNPs with a bio-
compatible coating are widely used for cell labeling in laboratories
and MRI contrasting in clinics.3
For InCell IT, first, MNPs were coated with biocompatible
materials attached to streptavidin, followed by mixing with the
biotinylated small molecule of interest. Then, these MNPs were
transferred by using a protein transduction domain (PTD; Support-
ing Information) into living cells, and their behaviors within the
cells were being monitored under magnetic fields by differential
interference contrast (DIC) images with a microscope (Figure 1).
After incubation with MNPs, the dark vesicles were observed inside
the cells (Figure 1), and iron-specific Prussian blue stain4 confirmed
that these vesicles contained the internalized MNPs (Figure S1).
In the absence of magnetic fields, these vesicles containing MNPs
showed punctuated localization at the nuclear periphery (Figure 1,
left). Applying the magnetic fields in a horizontal direction resulted
in the linear alignment of the vesicles to the same direction of the
magnetic fields (Figure 1, right and Figure S2), which could be
redirected by altering the directions of the magnetic fields (Figure
S3). For visualization of interactions between the small molecule
on MNPs and its target proteins, the cells expressing the target
proteins fused with the EGFP (enhanced green fluorescence protein)
tag were used.
The protein kinase family has more than 500 members, and
designing selective inhibitors against specific protein kinase iso-
forms becomes critical in new drug discovery, to improve its
efficacy and reduce toxicity.1 We chose dasatinib (Sprycel, BMS-
354825, Bristol-Myers Squibb)5 as a model compound to evaluate
our InCell IT for in situ monitoring of its binding to target protein
kinases, SRC, ABL1, and CSK, in live HeLa cells. Biotinylated
dasatinib (Figure S4) showed inhibitory effects against purified
human SRC protein kinase in Vitro on a concentration-dependent
manner (Figure S5). The in Vitro affinity purification studies also
demonstrated that the biotinylated dasatinib binds specifically to
EGFP-tagged human SRC (SRC-EGFP) or CSK (CSK-EGFP)
protein kinases (Figure S6).
The streptavidin-attached MNPs were mixed with biotinylated
dasatinib (dasatinib-MNPs) and transferred by using PTD into HeLa
cells expressing CSK-EGFP. Upon applying magnetic fields, the
vesicles containing the internalized dasatinib-MNPs became aligned
to the same direction of the magnetic fields however, the fluores-
cence signals of CSK-EGFP did not colocalize with the dasatinib-
MNPs in the cells (Figure 2, left, and Figure S7). These dark
vesicular compartments were also detected, when CSK-EGFP
expressing cells were transferred with biotin-only coated MNPs
(biotin-MNP; Figure S7). On the contrary, no such vesicular
compartments were observed in the cells expressing CSK-EGFP
without treatment of MNPs (Figure S7). These results suggested
that the internalization of MNPs into the cells occurred via
endocytosis and the dasatinib-MNPs were further entrapped within
membranous vesicles, keeping them from binding with CSK-EGFP
in cytoplasm.
Several attempts to release or expose the contents of vesicular
compartments to cytoplasm of cells were reported.6 Among them,
we found that incubation of cells with a nordihydroguaiaretic acid
(NDGA)6b markedly enhanced the cytoplasmic exposure of dasa-
tinib-MNPs entrapped within these vesicles (Figure 2, right). Prior
to the addition of NDGA, CSK-EGFP signals were detected
separated from the dasatinib-MNP-containing vesicles within the
cells (Figure 2, left). Incubation of cells with NDGA, however,
enabled CSK-EGFP signals concentrating around dasatinib-MNPs,
resulting in linear alignments of CSK-EGFP signals in the same
Scheme 1. Behavior of Vesicles Containing MNPs in a Live Cell in
the Absence or Presence of Magnetic Fields
Figure 1. Differential interference contrast (DIC) images showing dark
vesicles containing the internalized MNPs and their behaviors in the absence
or presence of magnetic fields in the cell. Blue and red lines indicate the
boundary of nucleus and cell, respectively.
10.1021/ja8063843 CCC: $40.75  XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A

direction of the magnetic fields in the cells (Figure 2, right). These
alignments of CSK-EGFP signals were not observed when the cells
were incubated with biotin-MNPs (Figure S7). Binding between
dasatinib and human ABL1-EGFP or SRC-EGFP, respectively, was
also confirmed in live cells using InCell IT (Figure 3, left).
To further demonstrate the specificity of bindings between
dasatinib-MNP and its target proteins, competition studies were
performed using unbiotinylated dasatinib (Scheme 2). The extent
of the binding signals between dasatinib-MNPs and CSK-EGFPs,
SRC-EGFPs, or ABL1-EGFPs was decreased substantially by
brief coincubation of excess unbiotinylated dasatinib (10 µM)
in the same cells (Figure 3, right). Dose-dependent inhibition
of these bindings was achieved when the cells were preincubated
with unbiotinylated dasatinib for 1-2 h prior to the addition of
NDGA (Figure S8). These bindings in the cells can be preserved
by cell fixation with mild fixatives such as formaldehyde, which
is useful for visualization of the drug interactions at a particular
point in time (Figure S9).
In conclusion, for in situ monitoring of bindings between a small
molecule and its target proteins in live cells, we have developed a
new technology, InCell IT, and examined its applicability using
dasatinib, a kinase inhibitor, as a model compound. In InCell IT,
dasatinib-MNPs were transferred into HeLa cells and their specific
bindings to SRC, ABL1, or CSK with EGFP-tag could be monitored
under magnetic fields by live cell imaging with a fluorescence
microscope. The specificities of these bindings were further
confirmed in the same cells by treating unbiotinylated dasatinib
itself. InCell IT is a new method for detecting the occurrence of
small molecule-target protein interactions in live cells.
Acknowledgment. We thank Dr. Yonggil Kwon for critical
review and comments on the manuscript. We also thank Dr. Je-
Wook Lee for in Vitro affinity purification studies. This work was
supported by CGK Co., Ltd.
Supporting Information Available: Detailed Experimental Proce-
dures, Supporting Figures 1-9, complete ref 2a, and Authors’ Note
on Previous Work. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) (a) Cohen, P. Nat. ReV. Drug DiscoVery 2002, 1, 309–315. (b) Hanks, S. K.
Genome Biol. 2003, 4, 111. (c) Knight, Z. A.; Shokat, K. M. Chem. Biol.
2005, 12, 621–637. (d) Force, T.; Kuida, K.; Namchuk, M.; Parang, K.;
Kyriakis, J. M. Circulation 2004, 109, 1196–1205.
(2) (a) Becker, F.; et al. Chem. Biol. 2004, 11, 211–223. (b) Caliguiri, M.; Molz,
L.; Liu, Q.; Kaplan, F.; Xu, J. P.; Majeti, J. Z.; Ramos-Kelsey, R.; Murthi,
K.; Lievens, S.; Tavernier, J.; Kley, N. Chem. Biol. 2006, 13, 711–722.
(3) Berry, C. C.; Curtis, A. S. G. J. Phys. D: Appl. Phys. 2003, 36, R198-
R206.
(4) Frank, J. A.; Miller, B. R.; Arbab, A. S.; Zywicke, H. A.; Jordan, E. K.;
Lewis, B. K.; Bryant, L. H.; Bulte, J. W. M. Radiology 2003, 228, 480–
487.
(5) (a) Lombardo, L. J.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyer, C. L. J. Med.
Chem. 2004, 47, 6658–6661. (b) Shah, N. P.; Tran, C.; Lee, F. Y.; Chen,
P.; Norris, D.; Sawyer, C. L. Science 2004, 305, 399–401.
(6) (a) Cifci, K.; Levy, R. Int. J. Pharm. 2001, 218, 81–92. (b) Fischer, R.;
Ko¨hler, K.; Fotin-Mleczek, M.; Brock, R. J. Biol. Chem. 2004, 279,
12625–12635.
JA8063843
Figure 2. Fluorescence images of cells expressing CSK-EGFP before and
after incubation of cells with NDGA under magnetic fields (A), DIC images
showing the aligned dasatinib-MNPs in the direction of magnetic fields
(B), and the overlapped images of A and B (C).
Figure 3. Bindings between dasatinib-MNPs and CSK-, ABL1-, or SRC-
EGFPs in the absence or presence of unbiotinylated dasatinib in the same
cells.
Scheme 2. Schematic Illustrations of Competition against
Dasatinib-MNPs with Unbiotinylated Dasatinib
B J. AM. CHEM. SOC. 9 VOL. xxx, NO. xx, XXXX
C O M M U N I C A T I O N S