A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase - A dual-specific protein tyrosine phosphatase

Article abstract of DOI:10.1039/c2cc31377d

We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).

Full text of DOI:10.1039/c2cc31377d

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A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related
phosphatase—a dual-specific protein tyrosine phosphatasew
a
b
Myeong Seon Jeong,z Eunha Kim,z Hyo Jin Kang,a Eun Joung Choi,b Alvin R. Cho,yb
Sang J. Chung*a and Seung Bum Park*bc
Received 7th January 2012, Accepted 4th May 2012
DOI: 10.1039/c2cc31377d
We report a Seoul-Fluor-based bioprobe, SfBP, for selective
monitoring of protein tyrosine phosphatases (PTPs). A rational
design based on the structures at the active site of dual-specific
PTPs can enable SfBP to selectively monitor the activity of
these PTPs with a 93-fold change in brightness. Moreover,
screening results of SfBP against 30 classical PTPs and 35
dual-specific PTPs show that it is selective toward vaccinia
H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).
development of selective inhibitors for specific PTPs.4 In this
regard, selective PTP probes can be useful tools to monitor the
activity of specific PTPs, which define the structural difference
and similarity at the active sites of various PTPs.5 Class I
human PTPs consist of classical PTPs and dual-specific PTPs
(DUSPs). While classical PTPs have narrow and deep active
sites (depth B10 A) to accommodate phosphotyrosine, DUSPs
have wide and shallow active sites (depth = 4.5–6 A).6 On the
basis of this structural information, we aimed to design and
synthesize selective fluorescent probes for a single DUSP or a
group of DUSPs employing ortho-substituted phenyl phosphate.7
Herein, we report a highly selective fluorescent probe called SfBP
(Seoul-Fluor-based Bioprobe for specific PTP) for vaccinia
H1-related (VHR) phosphatase.
Protein phosphorylation is one of the major post-translational
modification mechanisms that nature utilizes to control various
signal transduction pathways. Specifically, many essential
regulatory processes in signal cascades are controlled by
tyrosine phosphorylation, whose homeostasis is modulated
by the interplay between protein tyrosine phosphatases (PTPs)
and protein tyrosine kinases (PTKs).1 As observed in PTKs,
the malfunction of PTPs has a serious correlation with many
human diseases including cancers, diabetes, rheumatoid arthritis,
and hypertension.2 For instance, PTP1B is a major negative
regulator of insulin signaling in muscle and liver, and the loss
of PTP1B activity leads to enhanced insulin sensitivity and
resistance to weight gain in mice, which implies that the
development of selective inhibitors of PTP1B can provide a
potential treatment for type 2 diabetes and/or obesity.3 Therefore,
the discovery of novel and specific small-molecule regulators of
certain PTPs with distinct modes of action can provide an
essential tool for the understanding of the molecular basis of
PTP catalysis and substrate specificity. Despite their importance,
the high degree of structural similarity between PTPs, which
constitute a large family of enzymes, has hampered the
Previously, we reported the discovery of a tunable and
predictable fluorescent core skeleton, namely Seoul-Fluor,8
and its subsequent application as a fluorescent bioprobe.9
Because the photophysical properties of Seoul-Fluor have a
significant correlation with the electron density of its substi-
tuents, we can rationally design sensitive bioprobes for specific
biological events using this molecular frame. In other words,
a BioNanotechnology Research Center, KRIBB and NanoBio Major,
UST, 111 Kwahangno, Yuseong, Daejeon 305-806, Korea.
E-mail: sjchung@kribb.re.kr; Fax: +82 42 879 8594;
Tel: +82 42 879 8433
b Department of Chemistry, Seoul National University, Seoul, Korea
c Department of Biophysics and Chemical Biology/BioMAX Institute,
Seoul National University, Seoul 151-747, Korea.
E-mail: sbpark@snu.ac.kr; Fax: +82 2 884 4025;
Tel: +82 2 880 9090
w Electronic supplementary information (ESI) available: Detailed
synthetic procedures, photophysical property data, enzyme profiling
and spectroscopic data of the compounds. See DOI: 10.1039/c2cc31377d
z These authors equally contributed to this work.
y Current address: Department of Pharmacology, University of
Washington, Seattle, WA 98195, USA.
Fig. 1 (A) Designing principles of Seoul-Fluor-based bioprobes for
specific PTPs (SfBP). (B) Schematic representation of the assay system
used to monitor PTPs activity via the PeT-based sensing mechanism.
c
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Chem. Commun., 2012, 48, 6553–6555 6553

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we can monitor the enzyme-mediated changes in electronic
characteristics of substituents, which might alter the photo-
induced electron transfer (PeT) process in Seoul-Fluor.10 We also
surmised that the Seoul-Fluor-based bioprobe can specifically
interact with certain enzymes due to the presence of a well-known
pharmacophore, indolizine, in the Seoul-Fluor core skeleton.11
On the basis of this hypothesis, we designed a Seoul-
Fluor-based fluorescent bioprobe for a specific PTP. As shown
in Fig. 1A, we introduced the O-phosphate moiety at the R1
position to mimic para-substituted phenyl phosphate of phos-
photyrosine and the acetyl moiety at the R2 position to ensure
good photophysical properties of bioprobes in an aqueous
environment. Upon cleavage of the P–O bond by the enzymatic
activity of a specific PTP, the liberated phenol moiety can
perturb the electronic state of SfBP, which leads to the PeT-
based reduction of the fluorescence signal. In order to maximize
the on–off amplitude of the fluorescence signal, we can basify
the resulting phenol with 1 N NaOH to form the phenoxide,
which enhances the electron density at the R1 position and
further suppresses the emission signal of the bioprobe (Fig. 1B).
Under this design hypothesis, we initiated the synthesis of SfBP
from cinnamaldehyde derivative 1 prepared by a previously
reported efficient 3-step synthesis (see Scheme 1). With an intra-
molecular 1,3-dipolar cycloaddition between olefin and azomethine
ylide as a key step, five subsequent reactions allowed for the
preparation of compound 6.8 After TBS deprotection,
compound 6 was phosphorylated with diethyl phosphoroiodidate,
generated in situ by the Arbuzov reaction of triethylphosphite with
iodine,12 to yield compound 7. The hydrolysis of the resulting
phosphodiester 7 with bromotrimethylsilane completed the
preparation of SfBP in moderate yield (63.4% over 2 steps).
For the enzyme kinetic study, the dephosphorylated form of
SfBP (compound 8) was also prepared via BOC deprotection
of compound 6 with TFA (see ESIw).
Table 1 Photophysical properties of SfBP and 8 in PBS and 1N
NaOH solution
Compound Condition labs lem ec
Fd
e ꢀ F Folde
a
b
SfBP
PBS
1 N NaOH 417 580 1.3 ꢀ 104 0.058 778.4 93
420 580 1.3 ꢀ 104 0.044 579.9 70
8
PBS
420 580 7.4 ꢀ 103 0.019 140.1 17
1 N NaOH 450 n/a 8.3 ꢀ 103 0.001
Longest wavelength absorption maximum (nm). Emission wavelength
8.3
1
a
b
c
(nm) by the excitation at the absorption maximum. Molar extinction
d
coefficient (Mꢁ1 cmꢁ1) at absorption maximum. Absolute quantum
e
yield. Fold difference of brightness (e ꢀ F). Brightness of 8 in 1 N
NaOH was normalized as 1 (see ESI).
Fig. 2 Emission spectra and photographic images of SfBP (black)
and 8 (gray) in PBS (left) and 1N NaOH (right), respectively.
group in SfBP caused dramatic effects on the fluorescence
brightness (e ꢀ F).13 As shown in Table 1 and Fig. 2, SfBP in
PBS was 4-fold brighter than 8, and, surprisingly, SfBP was
93-fold brighter in the basic environment. Therefore, this
result implies that the PeT-based fluorescence turn on–off
of SfBP is a plausible mechanism for monitoring the
activity of PTPs, and shows that SfBP is a highly sensitive
fluorescent bioprobe useful for monitoring the activity of
certain PTPs.
To test the specificity of SfBP, 65 different human PTPs,
including 30 classical PTPs and 35 dual-specific PTPs, were
individually expressed in E. coli and purified by affinity chromato-
graphy. Their activities were confirmed with 6,8-difluoro-
4-methylumbelliferyl phosphate (DiFMUP; see ESIw). As shown
in Fig. 3, DiFMUP is a general substrate for PTPs and exhibits a
The direct comparison of photophysical properties of SfBP
and 8 showed that SfBP was a highly sensitive fluorescent
probe for PTPs (Table 1 and Fig. 2). First, UV-Vis absorption
and molar absorptivity of SfBP and 8 were consistent, both in
phosphate buffered saline (PBS) and in 1 N NaOH, except for
the 30-nm bathochromic shift of absorption maximum from SfBP
to 8 in 1 N NaOH. In contrast, the existence of the phosphate
Scheme 1 Synthesis of Seoul-Fluor-based bioprobes for specific
PTPs. Reagents and conditions: (a) tert-butyl-2-aminoethylcarbamate,
AcOH, Na2SO4, DCM, rt; then NaBH4, MeOH, 0 1C; (b) bromoacetyl
bromide, TEA, DCM, ꢁ78 1C; (c) 4-acetylpyridine, DCM, 60 1C; then
DBU, toluene; (d) DDQ; (e) HF/pyridine, THF; then TMSOMe; (f) I2,
DMAP, triethylphosphite, DCM, 0 1C; (g) TFA, DCM; (h) TMSBr.
Fig. 3 Screening result of DiFMUP and SfBP against 65 different
PTPs. Individual PTPs (1 mM) were incubated with either DiFMUP or
SfBP at pH 8 to measure their selectivities. Inset data represent
screening results of SfBP specificity to 3 different PTPs (DUSP3,
DUSP14 and DUSP13B) at the lower enzyme concentration (0.1 mM).
c
6554 Chem. Commun., 2012, 48, 6553–6555
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Table 2 Kinetic parameters of DUSP3, DUSP13B, and DUSP14 for
catalysis of SfBP and DiFMUP at pH 6
PTP activity. Due to its unparalleled selectivity, short detec-
tion time, over 90-fold signal enhancement, and the applic-
ability to high throughput screening (HTS), SfBP-based assays
could be a promising tool for the discovery of specific VHR
inhibitors for the identification of therapeutic agents for VHR-
related diseases, e.g., prostate and cervical cancers. The X-ray
structural analysis and development of an SfBP-based selec-
tive inhibitor for VHR are currently being investigated and
will be reported in due course.
Enzyme Km
Substrate (nM)
kcat
kcat/Km
(minꢁ1 mMꢁ1
)
Enzyme
(mM) (minꢁ1
)
DUSP3
(VHR)
DUSP14
SfBP 10
34.5 159.36
39.1 408.64
4.62
10.45
0.36
1.25
0.01
0.49
DiFMUP 0.2
SfBP 100
DiFMUP 1.0
DUSP13B SfBP 2800
DiFMUP 25
18.2
48.5
71.3
6.53
60.54
0.73
302.4 147.98
This study was supported by National Research Foundation
of Korea (NRF) grants (NRF-2011-355-C00047), the WCU
program (NRF-2009-0078236), the Biosignal Analysis Techno-
logy Innovation Program (2011-0027722), and the Bio &
Medical Technology Development Program (2011-0019464)
funded by the Korean Ministry of Education, Science, and
Technology (MEST).
negligible selectivity for neither classical nor dual-specific
PTPs. In contrast, we observed a good selectivity of SfBP
toward VHR, also known as a dual-specific PTP-3 (DUSP3),
in the initial screening and its excellent selectivity at the lower
concentration (0.1 mM) of selected DUSPs and at pH 8.0
(see the inset of Fig. 3). Although catalytic activities of DUSPs
were higher at pH 6, known as optimal pH for DUSPs,14 than
at pH 8, the selectivity was conserved at both pH values
(see Fig. S5, ESIw). Since DUSP13B and DUSP14 showed
meaningful catalytic activities to SfBP, especially at pH 6, their
substrate specificity was determined using Lineweaver–Burk
analysis and compared with that of DUSP3. Substrate speci-
Notes and references
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ficity of DUSP3 to SfBP at pH 6 (kcat/Km = 4.62 minꢁ1 mMꢁ1
)
was increased by 231 times from that at pH 8, and was 462-
and 13-times higher than those of DUSP13B and DUSP14,
respectively, at pH 6 (Table 2). Interestingly, the substrate
specificity of SfBP toward three PTPs was dictated by the
turnover number (kcat) rather than binding affinity (Km).
While the Km values are within 4-fold difference, the kcat
values showed up to 218-fold difference among three enzymes
(Table 2), which indicates that SfBP binds to DUSP3 in proper
orientation with proximity for its catalytic activity, but not to
DUSP14 and DUSP13B.
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Using in silico analysis, we proposed a plausible explanation
of this selectivity of SfBP with a good docking score at the
active site of VHR, which has a relatively shallow pocket. In
contrast, the active sites of classical PTPs contain a deep and
narrow pocket, where SfBP cannot be accommodated due to
the presence of the bulky indolizine heterocycle at the para
position of the phosphate group, thereby making itself a poor
substrate for classical PTPs (see ESIw). Although the selecti-
vity of SfBP toward VHR among 35 dual-specific PTPs is not
yet fully addressed, this notable selectivity of SfBP toward
VHR might be caused by the drug-like indolizine core
skeleton,17 which implies that pharmacophore-embedded
fluorescent compounds18 could function as powerful research
tools for providing valuable information about players in the
proteomic arena to elucidate complicated cellular processes. In
fact, VHR has been recognized as a biomarker for various
cancers including prostate15 and cervical cancers.16 Therefore,
we envision that the VHR-specific fluorescent bioprobe, SfBP,
can provide new insight into the design of potential therapeutics
for prostate and cervical cancers.
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In summary, we have developed a new fluorescent bioprobe
that is selective for a specific PTP, VHR (DUSP3), among 30
classical and 35 dual-specific PTPs, using a PeT-based turn
on–off mechanism upon dephosphorylation caused by the
18 O. N. Burchak, L. Mugherli, M. Ostuni, J. J. Lacapere and
M. Y. Balakirev, J. Am. Chem. Soc., 2011, 133, 10058–10061.
c
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Chem. Commun., 2012, 48, 6553–6555 6555