A mechanism-based fluorescent probe for labeling O6- methylguanine-DNA methyltransferase in live cells

Article abstract of DOI:10.1039/c2ob25231g

A mechanism-based small molecular fluorescent probe has been developed to label active O⁶-methylguanine-DNA methyltransferase in live cells.

Full text of DOI:10.1039/c2ob25231g

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 3189
www.rsc.org/obc
COMMUNICATION
A mechanism-based fluorescent probe for labeling O6-methylguanine-DNA
methyltransferase in live cells†
Xin Li,a Shijing Qian,b Lin Zheng,b Bo Yang,b Qiaojun Heb and Yongzhou Hu*a
Received 31st January 2012, Accepted 1st March 2012
DOI: 10.1039/c2ob25231g
In our continuous work on the development of specific fluor-
escent probes for biomedical research,8 we report herein the
design, synthesis and biological application of a mechanism-
based probe, 1, which enables direct live cell labeling and
imaging of MGMT as well as subsequent fluorogenic analysis.
Probe 1 was designed on the basis of O6-benzylguanine (O6-BG,
2) (Fig. 1), a potent MGMT substrate which has been shown
unequivocally to inactivate MGMT effectively, resulting in the
formation of S-benzylcysteine in the protein and the stoichio-
metric production of guanine.9 Based on this inactivation mech-
anism, probe 1 was designed by attaching a fluorescent moiety
onto the ‘meta’ position of the benzyl ring in 2, a position which
A mechanism-based small molecular fluorescent probe has
been developed to label active O6-methylguanine-DNA
methyltransferase in live cells.
Many alkylating antitumor agents, such as 1,3-bis(2-chloro-
ethyl)-1-nitrosourea (BCNU) and temozolomide, provoke cyto-
toxic effects by targeting the O6-position of guanine in DNA.1
However, the therapeutic efficacy of these drugs can be signifi-
cantly attenuated by the repair capacity of O6-methylguanine-
DNA methyltransferase (MGMT), which restores DNA with O6-
alkylguanine lesions by transferring the alkyl groups to an
internal cysteine residue via an irreversible and stoichiometric
reaction.2 A number of in vitro and in vivo studies have estab-
lished a negatively correlated relationship between the levels of
MGMT and the sensitivity to guanine O6-targeting drugs.3
MGMT is therefore considered a crucial biomarker for individual
susceptibility to alkylating agents. Apart from its importance in
clinical resistance to alkylating drugs, MGMT is also of great
interest because, although it is not essential for DNA replication
or cell survival, it is expressed in normal cells at levels that far
exceed its need to repair endogenous DNA lesions and the
underlying role remains an enigma.4
has been revealed to tolerate even quite large substituents.10
A
poly(ethylene glycol) (PEG) chain was employed to link the
fluorophore with the O6-benzylguanine scaffold. This chain acts
to increase the probe’s solubility and decreases its steric hin-
drance which may exclude access to the active site of MGMT.
The well known 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY) was selected as the fluorescent moiety because of its
MGMT is highly variably expressed in tumors.5 There are
various methods available for MGMT determination, including
the complex and laborious assays based on radioactive DNA
substrates, and the measurement of immunoblots by anti-MGMT
antibodies, etc.6 However, all these techniques are limited to cell
lysates and require cell homogenation, a process that disrupts
cellular organization and may destroy the balance between active
MGMT and its endogenous inactivated state.7 Thus, the results
obtained may not represent the actual situation of MGMT in a
living cell. Moreover, the employment of radioactive isotopes in
these assays requires more care in handling in a laboratory
setting. A simple, nonradioactive and cell permeable probe to
detect MGMT in live cells would prove more relevant for the
research on the protein’s role in pathophysiology and physiology.
aZJU-ENS Joint Laboratory of Medicinal Chemistry, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058,
China. E-mail: huyz@zju.edu.cn; Fax: +086-571-88208460
bInstitute of Pharmacology and Toxicology, College of Pharmaceutical
Sciences, Zhejiang University, China
Fig. 1 Structure of probe 1 and O6-benzylguanine (2). The structure
template of 1 consists of the O6-BG moiety for binding and labeling
MGMT, a PEG linker to minimize the steric hindrance, and a BODIPY
fluorophore for visualization and fluorogenic analysis of the labeled
protein.
†Electronic supplementary information (ESI) available: Synthesis,
spectra data. See DOI: 10.1039/c2ob25231g
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3189–3191 | 3189

View Article Online
Fig. 2 Schematic representation of MGMT labeling by probe 1. The
probe acts as a substrate to inactivate MGMT and labels the protein with
the benzyl group bearing a fluorophore (F).
Fig. 3 Probe 1 is a substrate of MGMT. Exposing HT 29 cells to probe
1 or 2 at 5 μM for 2 hours prior to the addition of 0–800 μM BCNU
resulted in a dramatic decrease of cell survival, implying the potent
activity of 1 to inactivate cellular MGMT (A). Treating HT29 cells with
probe 1 or 2 at 5 μM for 24 hours led to the degradation of MGMT,
while the non-treatment control remained to express MGMT at a high
level (B). All experiments were run in triplates.
inactivate MGMT. Adapted from the method reported by Pauly
et al.,12 a methylthiazolyldiphenyl-tetrazolium-bromide (MTT)
colorimetric assay was carried out to determine the inhibitory
effect of 1 on MGMT by testing the sensitization of HT 29 cells
to killing by BCNU. As shown in Fig. 3A, probe 1 alone had no
cytotoxicity at 5 μM, while it significantly improved the efficacy
of BCNU. This sensitization illustrated that probe 1 inactivated
cellular MGMT effectively and counteracted the cells’ resistance
to BCNU, an effect similar to that of O6-BG (2). To further
verify this conclusion, MGMT expression levels in HT 29 cells
were examined before/after treatment with probe 1 using an
immunoblotting technique with anti-MGMT. It turned out that
exposure of HT 29 cells to probe 1 at a concentration of 5 μM
for 24 hours led to a significant decline of MGMT level
(Fig. 3B), which is in agreement with the previous report that
inactivated MGMT was degraded slowly via the ubiquitin pro-
teolytic pathway.7 In summary, these data suggested that probe 1
retained MGMT substrate properties, confirming its validity as a
mechanism-based probe to label MGMT in live cells.
Scheme 1 Synthesis of the mechanism-based probe 1. Reagents and
conditions: (a) 4, NaH, dry THF, 0 °C–r.t., 58%; (b) (i) LiAlH4, dry
THF, reflux, 2 h; (ii) CF3COOEt, Et3N, anhydrous EtOH, r.t., 5 h, 87%
t
in two steps; (c) BuOK, 7, DMF, 0 °C–r.t., 3 h, 58%; (d) (i) K2CO3,
Probe 1 was then tested for its capacity to label MGMT in live
cells. HT 29 or HeLa S3 cells were incubated with probe 1
(5 μM) for 20 min. At the end of the incubation period, cells
were washed with phosphate buffered saline (PBS) twice and
subsequently processed for microscopy analysis using a Leica
DMI4000B fluorescence microscope. An incubation time of
20 min was established based on the observation that intracellu-
lar fluorescence was visible after an incubation time of 5 min
and reached the saturation point at around 20 min. The concen-
tration was also determined based on dose–response studies,
which showed that intracellular fluorescent labeling was detect-
able using concentrations of 2–10 μM of probe 1, but a concen-
tration of 5 μM was required to obtain consistent and
reproducible labeling. Under these conditions, cells appeared to
be stained mainly in the cytoplasm (Fig. 4A, a, c), in agreement
with a previous report on the cytoplasmic localization of MGMT
in HeLa S3 cells.14
MeOH, 60 °C, 3 h; (ii) Et3N, 9, anhydrous MeOH, 91% in two steps.
many attractive spectral characteristics and its hydrophobic
nature.11 Probe 1 is expected to be a pseudosubstrate of MGMT.
It should inactivate MGMT in the same way as compound 2, and
label MGMT, taking advantage of the transfer of its fluorescent
benzyl residue to the protein’s active site (Fig. 2).
Preparation of probe 1 is outlined in Scheme 1. Nucleophilic
substitution of ethyl 3-(bromomethyl)benzoate (3) with 2-(2-(2-
azidoethoxy)ethoxy)-ethanol (4) yielded 5, which was reduced
with LiAlH4, followed by protection of the amine group as the
trifluoroacetate 6. The potassium alkoxide of 6 was reacted with
1-(2-amino-9H-purin-6-yl)-1-methylpyrrolidinium chloride (7)12
to furnish the crucial intermediate 8. Deprotection of 8 and sub-
sequent acylation with the activated ester 9, N-hydroxysuccini-
midyl-4-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
indacene-8-yl)-butyric acid,13 yielded probe 1.
Competition experiments were also performed in parallel to
test the specificity of probe 1. For this purpose, cells were pre-
incubated with O6-BG (50 μM) for 30 min, and then were
To demonstrate the feasibility of 1 as a sensitive probe for
MGMT in live cells, it was first evaluated for its ability to
3190 | Org. Biomol. Chem., 2012, 10, 3189–3191
This journal is © The Royal Society of Chemistry 2012

View Article Online
In conclusion, we have developed a mechanism-based small
molecular probe for the fluorogenic analysis of MGMT in live
cells. The probe, designed based on a mechanism-based inhibitor
of MGMT and the structure–activity relationship thereof, shows
a great deal of significance. First, it retains MGMT’s substrate
properties. Second, it is easily cell permeable and specifically
labels active MGMT but not its inactivated counterpart in intact
mammalian cells. Furthermore, the labeling of cells may be
quantitatively analyzed. Therefore, probe 1 represents a valuable
tool for live cell study of the location, function, and quantifi-
cation of MGMT in both the healthy and diseased state. Further
studies are now planned to develop an MGMT assay based on
probe 1.
Acknowledgements
This work was supported by National Natural Science Foun-
dation of China (30901858), Zhejaing Provincial Natural
Science Foundation of Chian (Y2100580), China Postdoctoral
Science Foundation (201003735), the Fundamental Research
Funds for the Central Universities, and the State Key Laboratory
of Drug Research (SIMM1004KF-13).
Notes and references
Fig. 4 (A) Fluorescent labeling studies of MGMT in HT 29 and Hela
S3 cells by probe 1 with or without 2 as a competing substrate. Pretreat-
ment of cells with 2 (b, d) significantly eliminated the fluorescent inten-
sity compared with no pretreatment (a, c). Cells were detected after
excitation (ex) at 488 nm. Scale bar represents 100 μm; (B) quantifi-
cation of the fluorescent labeling events. HT 29 and Hela S3 cells were
treated with 5 μM 1 for 20 min, followed by twice of wash with PBS.
The mean fluorescence intensity was then recorded (arbitary units, A.U.)
and expressed as means SEM from three independent experiments.
The significant levels (**) represent p < 0.005; (C) HT 29, Hela S3, and
Hela cells express MGMT at different levels, as shown by immunoblot
assay with anti-MGMT antibody.
1 A. Loveless, Nature, 1969, 223, 206; R. Saffhill, G. P. Margison and P.
J. O’Connor, Biochim. Biophys. Acta, 1985, 823, 111.
2 A. E. Pegg and T. L. Byers, FASEB J., 1992, 6, 2302; A. E. Pegg, M.
E. Dolan and R. C. Moschel, Prog. Nucleic Acid Res. Mol. Biol., 1995,
51, 167.
3 S. L. Gerson, J. Clin. Oncol., 2002, 20, 2388; K. Ishiguro, K. Shyam, P.
G. Penketh and A. C. Sartorelli, Mol. Cancer Ther., 2005, 4, 1755.
4 S. L. Gerson, Nat. Rev. Cancer, 2004, 4, 296.
5 G. P. Margison, A. C. Povey, B. Kaina and M. F. Santibanez Koref, Car-
cinogenesis, 2003, 24, 625.
6 I. Preuss, I. Eberhagen, S. Haas, R. H. Eibl, M. Kaufmann, G. Von Min-
ckwitz and B. Kaina, Int. J. Cancer, 1995, 61, 321; B. Y. Daniel and N.
Y. Merrick, US Pat., US005407804A, 1995; R. Damoiseaux, A. Keppler
and K. Johnsson, ChemBioChem, 2001, 4, 285; G. Nagel, W. Brenner,
K. Johnsson and B. Kaina, Anal. Biochem., 2003, 321, 38; K. Ishiguro,
K. Shyam, P. G. Penketh and A. C. Sartorelli, Anal. Biochem., 2008, 383,
44.
7 K. S. Srivenugopal, X. Yuan, H. S. Friedman and F. Ali-Osman, Bio-
chemistry, 1996, 35, 1328.
8 X. Li, S. Qian, Q. He, B. Yang, J. Li and Y. Hu, Org. Biomol. Chem.,
2010, 8, 3627; X. Li, J. Cao, Y. Li, P. Rondard, Y. Zhang, P. Yi, J. Liu
and F. Nan, J. Med. Chem., 2008, 51, 3057.
stained with probe 1 (5 μM). As expected, the fluorescent stain-
ing was significantly decreased presumably due to the pre-occu-
pation of the active site of MGMT by 2, demonstrating the
specificity of the interaction between probe 1 and MGMT
(Fig. 4A, b, d).
9 A. E. Pegg, M. Boosalis, L. Samson, R. C. Moschel, T. L. Byers,
K. Swenn and M. E. Dolan, Biochemistry, 1993, 32, 11998.
10 T. B. McMurry, DNA Repair, 2007, 6, 1161.
11 J. Karolin, L. B.-A. Johansson, L. Strandberg and T. Ny, J. Am. Chem.
Soc., 1994, 116, 7801.
12 G. T. Pauly, N. A. Loktionova, Q. Fang, S. L. Vankayala, W. C. Guida
and A. E. Pegg, J. Med. Chem., 2008, 51, 7144.
13 Z. Li, E. Mintzer and R. Bittman, J. Org. Chem., 2006, 71, 1718.
14 T. Ishibashi, Y. Nakabeppu, H. Kawate, K. Sakumi, H. Hayakawa and
M. Sekiguchi, Mutat. Res., 1994, 315, 199.
The above labeling events were also quantified by recording
the mean fluorescence intensity. It turned out that HT 29 cells
exhibited a stronger fluorescence intensity compared with Hela
S3 cells (Fig. 4B), in agreement with the results that intact HT
29 cells contain a higher concentration of MGMT as determined
by immunoblot assay with anti-MGMT antibody (Fig. 4C). This
verifies the potential of probe 1 as a tool to detect active MGMT
quantitatively in live cells.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3189–3191 | 3191