Imaging farnesyl protein transferase using a topologically activated probe

Article abstract of DOI:10.1021/ja063599x

We report on the development and application of a "smart" activatable peptide-based probe for detection of Ras-related farnesyl protein transferase (FPT). Upon farnesylation by FPT, the probe was brought close to a hydrophobic milieu and as a consequence

Full text of DOI:10.1021/ja063599x

Published on Web 08/18/2006
Imaging Farnesyl Protein Transferase Using a Topologically Activated Probe
Wellington Pham,† Pamela Pantazopoulos, and Anna Moore*
Molecular Imaging Laboratory, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology,
Massachusetts General Hospital, HarVard Medical School, Room 2301, Building 149, 13th Street,
Charlestown, Massachusetts 02129
Received May 23, 2006; E-mail: amoore@helix.mgh.harvard.edu
Scheme 1. Topologically Activated Probe
Ras proteins play important roles in cell signal transduction,
including the cell division cycle, programmed cell death, and
differentiation.1 In addition, Ras proteins are also responsible for
about 30% of all human carcinomas,2 including 90% of pancreatic,
50% of colon,3 and 30% of lung4 and breast cancers.5 The proteins
must be localized to the inner surface of the plasma membrane to
be biologically active in all of these events. This first and most
critical step in the post-translational modification is mediated by
farnesyl protein transferase (FPT), which transfers a lipid farnesyl
group from farnesyl pyrophosphate (FPP) to the cysteine sulfhydryl
of Ras which ends with a CA1A2X motif at its carboxyl terminal.6
Positions A1 and A2 are occupied by aliphatic amino acids, while
X is held by either methionine or serine.7 Since the relocation of
Ras proteins from the cytoplasm to the plasma membrane upon
farnesylation is crucial for tumor growth and survival, detection of
FPT activity using molecular imaging probes based on a signal
transduction platform is of considerable biomedical significance.
We report herein on the synthesis, characterization, and applica-
tion of a fluorescent reporter probe to detect FPT in a cell-based
assay. The underlying principle was to label the CA1A2X sequence
with an environmentally sensitive fluorescent dye. Upon farnesy-
lation by FPT, this sequence will be relocated from a hydrophilic
(cytoplasm) to a hydrophobic (plasma membrane) environment. We
expect the environmentally sensitive dye at this point to switch
from a quenched to an enhanced fluorescent status. While screening
a set of dyes for this purpose, we learned that dansyl and dapoxyl
dyes are very environmentally sensitive. The fluorescence emissions
of these dyes are strong in organic solvents and very weak in water.
Detection of FPT by dansyl dye in a continuous assay has been
pursued previously.8 From the imaging point of view, dapoxyl dye
has a number of distinct advantages over dansyl dye. First, dapoxyl
dye has a high intensity absorption with a molar extinction of 26 000
M-1 cm-1 compared to 4000 M-1 cm-1 for dansyl dye. Second,
dapoxyl dye could fluoresce with a λmax at 600 nm, which may
reduce background fluorescence from a biological environment.
Third, dapoxyl dye has a very large Stokes shift; therefore, cross-
talk resulting from scattered light is greatly reduced (optical spectra
are available in Figure S1). Employing this environmentally
activated dye in the system eventually offers a much more compact
version compared to the previously reported fluorescence resonance
energy transfer (FRET) probes9 since only a single dye is employed,
thus better enabling it for prying targets with minimum steric
hindrance. We have assessed the topological effect of dapoxyl dye
by imaging the signal enhancement in a hydrophobic environment
using octanol (Figure S2).
Ile-Met (CVIM) via a â-alanine linker, while the thiol group on
cysteine was protected by a t-buthiol moiety (Scheme 1). The
stability of this protecting group over a mildly acidic environment
enabled us to deprotect 2-chlorotrityl chloride (2-cTC) resin 1 in
5% TFA. To increase the efficiency of the synthesis, resin 1 was
swelled carefully in DCM before coupling with the Fmoc-
methionine manually. The product of methionylated 2-cTC 2
commenced the solid phase synthesis using a peptide synthesizer.
After the synthesis, the Fmoc group was selectively deprotected
from the N-terminal of â-alanine to provide 3. The resin 1 was
detached from the peptide in the presence of 5% TFA in DCM to
provide 4. After labeling the peptide with a dapoxyl dye, the
t-buthiol group on compound 5 was deprotected by dithiothreitol
(DTT) to provide 6. The progress of the reaction was conveniently
monitored by TLC in 9.5:0.5 of methylene chloride and methanol.
The Rf spot could be visualized using an optical imaging system.
In addition to this probe, we synthesized a native CVIM peptide 7,
which serves as a control using Wang resin (Supporting Informa-
tion). To test the specificity of the labeled probe 6 compared to
that of 7 toward FPT, we performed an FPT activity assay based
on a reported procedure.10,11 Furthermore, we used probe 5, in which
the farnesylation site was blocked by a t-buthiol protecting moiety
as an additional control. As shown in Figure 1, FPT activity was
reduced 7-fold in the labeled probe 6 (IC50 ) 1.2 µM) compared
to the unlabeled native CVIM 7 (IC50 ) 0.17 µM). Meanwhile,
farnesylation was abolished when the thiol moiety was blocked in
compound 5 (IC50 ) 2.4 mM). The binding constant (Kd) of probe
6 was calculated by measuring the fluorescence intensity of a fixed
concentration of the probe while adding increasing concentrations
of FPT. The data were analyzed as specified in Supporting
Information, and the Kd was found to be 26 nM (Figure S3).
The results from the FPT assay confirm the selectivity of probe
6 and suggest the usefulness of its predecessor 5 as a control in a
cell-based assay, given the challenge to find a control cell line that
has no FPT expression. We performed a pure enzyme assay where
probes 5 and 6 were incubated with FPT and FPP in a 96-well
To synthesize the probe, dapoxyl dye was selectively conjugated
to the N-terminal of the Ki Ras-B peptide sequence of Cys-Val-
† Current address: Vanderbilt University Institute of Imaging Science, 1161
21st Avenue South, Nashville, TN 37232. E-mail: wellington.pham@
vanderbilt.edu.
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J. AM. CHEM. SOC. 2006, 128, 11736-11737
10.1021/ja063599x CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
in the pure enzyme assay format. To further confirm the cellular
activation of FPT and the potential future use of the probe in
florescence-based applications, MDA-MB-231 cells were grown
on a round cover slip to 70-80% confluency and incubated with
control 5 or probe 6 for 2 h followed by fixing with 4%
paraformaldehyde and counterstaining with DAPI for nuclear
staining. As shown in Figure S5, the fluorescent signal coming from
the cells treated with probe 6 was significantly brighter than that
coming from the cells treated with control 5 under the same
incubation conditions. To demonstrate the possibility that this
technique could be utilized for tissue imaging, we performed
imaging of a phantom imitating tumor in the tissue. To do that,
human breast cancer cells MDA-MB-231 were incubated with probe
6 and injected into the middle of a tube filled with agarose gel. A
bright fluorescent signal was detected in the middle of the tube
after positioning the phantom in the optical imaging system (Figure
2C).
In conclusion, we report on the development and application of
a novel method to image FPT in live cells using a topologically
activated probe. Our results strongly suggest that the probe has good
specificity toward FPT in in vitro assay, and farnesylation is a main
switch that turns the fluorescent signal on upon incubation of breast
cancer cells with probe 6. This technique could have wide
applications in high throughput drug screening and potentially in
the imaging of small animal models.
Figure 1. Farnesyl protein transferase activity. Value is an average of
duplicate assays.
Figure 2. Live cell imaging of probe activation. Compounds were dissolved
in DMSO and diluted 60-fold into the assay to give the final solvent
concentration of <2%.
plate, and the fluorescence signal was detected by a high throughput
plate reader. Farnesylation of probe 6 resulted in a 2-fold increase
in the fluorescence signal compared to 5 (Figure S4, part A). Despite
modest augmentation of fluorescence, we observed a significant
and easily distinguished bright image of the probe (p < 0.0001)
(Figure S4, part B). To demonstrate that farnesylation could result
in fluorescent enhancement and enable imaging in a high throughput
screening fashion in live cells, a human breast cancer cell line,
MDA-MB-231, was incubated with either control 5 or probe 6 for
2 h. The medium was aspirated, and the cells were washed,
trypsinized, and transferred to a 96-well plate for optical imaging
as described in the Supporting Information. As expected, cells
treated with probe 6 produced a remarkable fluorescence, compared
to cells treated with the control 5 (p < 0.0001) (Figure 2A). In
addition, we observed a reduced fluorescent signal when probe 6
was incubated with a 10-fold excess of peptide 7, indicating specific
interactions between probe 6 and the enzyme (Figure 2A). These
observations suggest that there are two possibilities that turn the
signal on. First, the attachment of the lipid in close proximity to
the dye changes its environment. Second, the fatty acylated peptide
could be relocalized to a hydrophobic plasma membrane. It is
possible that both scenarios could take place. Quantitatively, for
all three tested concentrations, probe 6 expressed significantly higher
fluorescence signal (up to 30-fold, p < 0.0001) over the control at
4, 8, and 12 µM (Figure 2B). Collectively, these results strongly
suggested that the fluorescent signal enhancement was indeed the
outcome of a topological shift of the probe from an aqueous to a
hydrophobic environment upon farnesylation. It is worth emphasiz-
ing that we observed a much stronger fluorescent enhancement in
the cell-based assay compared to a pure enzyme assay. This
difference could potentially arise from the availability of existing
lipophilic membrane compartments inside the cell that were absent
Acknowledgment. The authors would like to thank John Moore
for assistance in manuscript preparation.
Supporting Information Available: Experimental details for the
synthesis of probe 6 and control probe 7, HPLC profiles of probes 5
and 6, information regarding the FPT activity assays, cell-based assays,
Kd calculation, and the complete ref 4. This material is available free
of charge via the Internet at http://pubs.acs.org.
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