A Vinyl Sulfone-Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

Article abstract of DOI:10.1002/anie.201710856

Chemical probes are powerful tools for interrogating small molecule-target interactions. With additional fluorescence Turn-ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS-pE (and its terminal alkyne-containing version DNS-pE2) is the first small molecule that can selectively label endogenous 3-phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence-quenching properties, DNS-pE/DNS-pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro K_i=7.4 μm) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live-cell settings.

Full text of DOI:10.1002/anie.201710856

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A Vinyl Sulfone-Based Fluorogenic Probe Capable of Selective
Labelling of PHGDH in Live Mammalian Cells
Authors: Sijun Pan, Se-Young Jang, Si Si Liew, Jiaqi Fu, Danyang
Wang, Jun-Seok Lee, and Shao Q. Yao
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201710856
Angew. Chem. 10.1002/ange.201710856
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10.1002/anie.201710856
Angewandte Chemie International Edition
COMMUNICATION
A Vinyl Sulfone-Based Fluorogenic Probe Capable of Selective
Labelling of PHGDH in Live Mammalian Cells
Sijun Pan, Se-Young Jang, Si Si Liew, Jiaqi Fu, Danyang Wang, Jun-Seok Lee,* and Shao Q. Yao*
Abstract: Chemical probes are powerful tools for interrogating small
Cys295
molecule-target interactions. With additional fluorescence Turn-ON
functionality, such probes might enable direct measurements of target
engagement in live mammalian cells. Herein, we report DNS-pE (and
its terminal alkyne-containing version DNS-pE2) as the first small
molecule that can selectively label endogenous 3-phosphoglycerate
Cys234
dehydrogenase (PHGDH) from various mammalian cells. Endowed
with an electrophilic vinyl sulfone moiety that possesses fluorescence-
Cys281
quenching property, DNS-pE/DNS-pE2 became highly fluorescent
only upon irreversible covalent modification of PHGDH. With inhibitory
property (in vitro Ki = 7.4 M) comparable to that of known PHGDH
inhibitors, our probes thus offer
a promising approach to
simultaneously image endogenous PHGDH activities and study its
target engagement in live-cell settings.
3-Phosphoglycerate dehydrogenase (PHGDH), a key enzyme
in the biosynthesis of serine, is up-regulated in many rapidly
proliferating cancer cells.[1,2] Therefore it is a potential anticancer
target. To date, only several small-molecule PHGDH inhibitors,
e.g. NCT-503 and CBR-5884, are reported in the literature (Figure
S1).[3] Both were serendipitously discovered from high-throughput
screening (HTS) assays, and possess moderate micromolar in
vitro and cellular inhibitory activities against PHGDH. While useful
for studying serine metabolism, these two compounds are not
“tractable” in situ and cannot be used to study direct PHGDH-
ligand engagement. In fact, despite remarkable advances in
screening technologies,[4] very few assays can reveal direct small
molecule-target interactions in intact cellular environments.
Activity-based protein profiling offers a versatile approach to
interrogate small molecule-protein interaction at a proteome-wide
scale, and could be made live-cell compatible with cell-permeable
small molecule probes.[5] Recently, this approach has been
combined with fragment-based screening to discover ligandable
cysteines targeted by small-molecule covalent modifiers in live-
cell settings.[6] Inspired by this study, we have taken a step further
to develop chemical probes capable of covalently and selectively
labelling of PHGDH, with novel “reaction-based” fluorescence
Turn-ON properties that enabled, for the first time, simultaneous
imaging of endogenous PHGDH activities and studying its target
engagement in live mammalian cells (Figure 1).
Figure 1. (A) Molecular docking of PVS (cyan), PVSN (pink) and DNS-pE
(yellow) in the active site of PHGDH (PDB ID: 2G76), where the nucleotide- and
substrate- binding domains are shown in slate and green, respectively. The
structures of the compounds are shown on the left, with vinyl sulfone highlighted
in blue. (B) Chemical structures of small-molecule probes designed and tested
in this study. Sulfonyl fluoride (SF) controls are boxed in pink. (C) Proposed
fluorescence Turn-ON mechanism of the dual-purpose probe, DNS-pE2, upon
covalent labelling with PHGDH, leading to subsequent live-cell imaging and in
situ target identification.
reporter with significant Turn-ON fluorescence upon covalent
reaction with PHGDH, and finally 4) a chemically tractable tag (i.e.
terminal alkyne shown in DNS-pE2; Figure 1C) for in situ target
identification and enrichment.[7] Previously, quenched activity-
based probes were designed based on similar criteria, but were
only applicable to hydrolytic enzymes.[8] Recent advances in the
development of reaction-based small molecule biosensors
provided a clue to design the corresponding “reaction-based”
PHGDH sensor,[9] whose fluorescence would be turned on only
upon covalent reaction with PHGDH (Figure 1C). Based on the
available X-ray structure of PHGDH (PDB ID: 2G76) and a
previous report,[3a] several cysteine residues located within the
Such so-called dual-purpose probes should contain several
essential components: 1) a PHGDH active site-binding group, 2)
an electrophilic moiety for covalent labelling of PHGDH, 3) a
PHGDH active site, i.e. Cys234
,
Cys281 and Cys295 (Figure 1A)
could be potentially targeted by electrophilic compounds. Indeed,
some of these residues were reported as hyper-reactive cysteines
that were readily labelled by iodoacetamide (IA, a common
cysteine-alkylating agent),[6] and Cys234 was previously reported
to be important for NCT-503/PHGDH interaction.[3a] Gratifyingly,
[*]
[*]
Dr. S. Pan, S. S. Liew, J. Fu, D. Wang, Prof. Dr. S. Q. Yao
Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmyaosq@nus.edu.sg
S.-Y. Jang, Prof. Dr. J.-S. Lee
Molecular Recognition Research Center, Bio-Med Program of KIST-
School UST, Korea Institute of Science & Technology,
Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, 136791 (Korea)
E-mail: junseoklee@kist.re.kr
upon carrying out
a preliminary docking experiment, we
discovered commercially available phenyl vinyl sulfone (PVS) and
phenyl vinyl sulfonate (PVSN), two neutral and electrophilic
phosphotyrosine mimics,[10] could fit snugly into the PHGDH
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.201710856
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active site with predicted binding affinities (pKd) of -5.3 and -6.3
kcal/mol respectively (Figure 1A); all three cysteine residues were
shown in close proximity to the docked ligands (< 10 Å). We
therefore hypothesized that both PVS and PVSN, as previously
reported mechanism-based inhibitors of protein tyrosine
phosphatases (PTPs),[10] might also be electrophilic irreversible
inhibitors of PHGDH (a phosphate-binding protein). Since both
molecules contain a vinyl sulfone, we envisaged this group could
be used as both an electrophile and a fluorescence quencher in
our probe design (Figure 1B). Several cysteine-reactive,
electrophilic α,β-unsaturated moieties, including ketones, amides
and vinyl sulfonamides, are known fluorescence quenchers in
suitably designed small molecule fluorophores.[11-13] We took note
of the structural and electronic similarities between vinyl sulfone
and sulfonamide, an acceptor moiety commonly used in
push−pull fluorophores, including 2,6-dansyl amide, dansyl amide
and 4-sulfamoyl-7-aminobenzoxadiazole (Figure S1).[12b,14] By
replacing sulfonamide in these fluorophores with vinyl sulfone
(giving DNS-pE, DNS-E and SBD-E, respectively; Figure 1B), our
newly designed probes might provide a unique starting point to
develop novel fluorogenic PHGDH probes. We further noted that
our probes are also structural analogs of two well-known thiol-
reactive fluorogenic small molecules, i.e. acrylodan and DNS-D
(Figure S1).[11,12b] As controls, probes containing a sulfonyl
fluoride (SF) were also examined (PSF in Figure 1A, DNS-
pF/DNS-F/SBD-F in Figure 1B); with numerous reports on
biocompatible SF-based compounds as emerging electrophilic
probes capable of modifying different nucleophilic residues in
proteins,[15] these probes may help to expand the potential
ligandable region around the PHGDH active site. Docking
experiments were again carried out amongst these newly
designed PHGDH probes, and DNS-pE emerged as the best
PHGDH binder with an improved pKd value of -6.9 kcal/mol and a
potential interaction between DNS-pE and Cys234 (Figure 1A).
Finally, a terminal alkyne was installed in this probe, providing the
dual-purpose DNS-pE2 (Figure 1C).
Upon successful synthesis of these probes (Scheme S1), we
first investigated their thiol-dependent fluorescence properties. As
shown in Figure 2A, DNS-pE showed a significant fluorescence
increase upon incubation with cysteine but not any other
nucleophilic amino acids (i.e. lysine, serine or tyrosine). Its
fluorescence Turn-ON ratio was comparable to that of acrylodan
(21x vs 14x), but markedly higher than that of DNS-D (6x), under
identical conditions. In contrast, negligible cysteine-responsive
fluorescence changes were observed with other probes, including
DNS-E and SBD-E, as well as all three SF-based control probes
(DNS-pF, DNS-F and SBD-F). These results offered the first clue
that vinyl sulfone could indeed act as a fluorescence quencher
when properly positioned in a suitable fluorophore (compare
DNS-pE vs DNS-E). Even more significant Turn-ON fluorescence
was observed when DNS-pE or DNS-pE2 was reacted with 2-
mercaptoethanol (BME) under physiological conditions (Figures
2B & S2); 215- and 185-fold increases in fluorescence were
quantitatively recorded at the probe’s absorption/emission
maxima (λabs/λem = 333/453 nm and 324/437 nm, respectively).
The complete Michael addition reaction (DNS-pE2 + BME) was
unequivocally confirmed by LCMS (Figure S2B). For DNS-pE2,
both the quantum yield (ΦF) and extinction coefficient (ε) went
from 0.0073 and 3900 M- 1cm1 in the “OFF” state to 0.88 and 6000
M-1cm-1 in the “ON” state. Similar increases were recorded for
(DNS-pE + BME) (Figure S2A). Since acrylodan is a well-known
polarity-sensitive dye that can label cysteines in a protein,[16] the
emission spectra of cysteine-reacted acrylodan and DNS-pE
were further investigated in different solvents (Figure S3A-C);
unlike the fluorescence of the acrylodan adduct which was highly
solvent polarity-dependent, fluorescence of the DNS-pE adduct
was only marginally solvent-sensitive. To gain better insights into
the fluorescence Turn-ON property of DNS-pE (and DNS-pE2),
–
(A) 20000
(B)
(C)
(D)
14 x
20 min
1 h
3 h
4.0×106
3 μM
2.0×106 1 μM
2×106
1×106
Probe only
*
185-fold
(DNS-pE2)
Probe + Cysteine
21 x
10000
2.0×106
y
= (2.129x - 0.017)×10
R² = 0.998
6
1.0×106
0
0.5
1 3
215-fold
(DNS-pE)
1 x
[PHGDH] (µM)
0
6 x
0.8 x
0.8 x
2 x 1 x
0
0
370
430
490
550
0
370
430
490
550
Wavelength (nm)
Wavelength (nm)
DNS-pE2 (1 µM)
(E)
(F)
(G)
DNS-pE2 (µM)
Competitor
conc. (µM)
CBR-5884
0.1 0.3
NCT-503
10 10 30
100
100
-
1
3
-
0.1 0.3
1
180 –
180 –
130 –
95 –
72 –
55 –
43 –
80
60
80
60
40
20
130 –
95 –
[PHGDH]
1.5 μM
30
[DNS-pE2]
1 μM
10
72 –
55 –
43 –
20
*
*
y
= 36.248x + 4.8664
R² = 0.997
40
20
0
10
kchem = 0.21 min-1
Ki = 7.4 µM
34 –
26 –
34 –
26 –
0
0.8
1/[PHGDH] (µM-1
60
)
0
0
30
60
0
30
90
Time (min)
Time (min)
Figure 2. (A) Turn-ON fluorescence ability of each probe (10 µM) in the presence of cysteine (100 µM) for 1 h at RT (50 mM HEPES, pH 7.4). The Turn-ON ratio
of each probe was indicated on top of the bars. (B) Turned-ON emission spectra of DNS-pE or DNS-pE2 (1 µM) upon addition of 2-mercaptoethanol in HEPES
buffer (50 mM, pH 7.4). (C) Western blotting analysis of endogenous tyrosine phosphorylation levels (anti-pTyr) of COS-7 cells upon treatment with 200 µM of probe
at 37 °C (20 min, 1 h, 3 h). The 170-kDa band (*) was determined to be from EGFR phosphorylation at Tyr1068 (Figure S4F), consistent with previous report that PTP
inhibitors up-regulated EGFR phosphorylation.[10] (Bottom gel): GAPDH loading control. (D) Fluorescence emission spectra (ex = 324 nm) of DNS-pE2 (1 µM) with
increasing concentrations of purified PHGDH (0 to 3 µM) in HEPES buffer. (Inset): fitted graph showing a linear relationship between 0 to 1 M protein concentration.
(E) Kinetics of the emergence of fluorescence upon DNS-pE2 (1 µM) labelling of PHGDH at different concentrations (1.5, 3.8, 7.5, 15, 30 µM) in HEPES buffer.
(Inset): fitted graph showing a linear relationship at tested concentrations. (F) Same as (E) except that PHGDH (5 M) was incubated with different concentrations
of DNS-pE2 (1, 2, 4, 5, 7.5, 10 µM). (G) Probe concentration-dependent (left; 0-1 M DNS-pE2) and competitive (right) in vitro labelling of PHGDH (*) overexpressed
in bacterial lysates with DNS-pE2 (1 µM) at RT for 1 h, followed by click chemistry with Rh-N3, separation and visualization with in-gel fluorescence scanning.
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10.1002/anie.201710856
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time-dependent density functional theory (TD-DFT) calculations
were conducted (Figure S3D/E), which suggested that DNS-pE
was likely quenched by intramolecular charge transfer (ICT) from
vinyl sulfone. Upon thiol addition, the resulting adduct was able to
effectively eliminate such an ICT state and attained a planarized
conformation capable of emitting strong fluorescence.[17] Thus,
both DNS-pE and DNS-pE2 are reaction-based biosensors.[9]
Since PVS/PVSN are reported irreversible PTP inhibitors,[10]
we next evaluated the potential cross-reactivity of our probes
toward PTPs. By using PTP1B as a model,[10] we carried out
PTP1B inactivation assay and in vitro protein labelling (Figure
S4A-D). Interestingly, while all probes showed similar PTP1B
inactivation profiles, those except SF-based probes could
covalently label PTP1B in an activity-dependent manner. The
inability of SF-based probes to label PTP1B suggests such
probes might not form stable covalent complexes with the protein
under SDS-PAGE conditions. Next, by using the ‘clickable” DNS-
pE2 to more quantitatively compare its PTP class selectivity with
various PTPs,[18] we observed some preferences toward labelling
of PTP1B, followed by TC-PTP and SHP1, and insignificant
labelling toward SHP2, LMW-PTP and MPtpB. In addition, we
observed only a moderate 4.4-fold increase in fluorescence when
DNS-pE (1 M) was incubated with purified PTP1B (3 µM) (Figure
S4E). In a cell-based assay, unlike relatively strong effects from
both PVSN and acrylodan in inhibiting endogenous PTP activities,
the effect from DNS-pE-treated cells was minimal (Figure 2C).[10]
These results indicated that despite noticeable in vitro cross-
reactivity of DNS-pE toward some PTPs, they might not be its
major cellular targets. Remarkably, when the same amount of
DNS-pE2 (1 M) was incubated with different concentrations of
purified PHGDH (0 to 3 M), very strong fluorescence increases
with peaked emission at 420 nm were observed (Figure 2D),
which followed a linear relationship with increased PHGDH
concentrations that reached saturation at 1 M (1:1 protein/probe;
inset), resulting in an 50-fold increase in Turn-ON fluorescence.
This indicates DNS-pE2 only labelled a specific cysteine in
PHGDH. Such linearity can be used to quantify the labelling
kinetics between DNS-pE2 and PHGDH.[13] By using a fixed
concentration of DNS-pE2 (1 μM) and an excessive amount of
PHGDH, time-course experiments were performed to measure
the emergence of fluorescence upon mixing of the probe and
PHGDH (Figure 2E); the obtained curves followed single-turnover
pseudo-first-order reaction kinetics, which provided a maximum
rate constant for the chemical labelling (kchem) of 0.21 min−1 and a
concentration of PHGDH that achieved a half-maximal rate of
labelling (Ki) of 7.4 μM (inset). This value is comparable to the in
vitro IC50 of NCT-503 (2.5 μM) and CBR-5884 (33 μM).[3] On the
contrary, when a fixed concentration of PHGDH (5 μM) with
different concentrations of DNS-pE2 (1-10 μM) was used, no
apparent change in kobs was observed (Figure 2F). We further
performed time-course experiments to monitor the reaction
between DNS-pE2 (3 or 10 µM) and 5 mM of glutathione (a highly
abundant endogenous thiol), which revealed a pseudo-first-order
rate constant (k) of 0.14 M-1s-1 (Figure S4G). In comparison,
acrylodan showed a much faster kobs of 1.94 M-1s-1. We reasoned
that the conversion from enone (acrylodan) to vinyl sulfone (DNS-
pE/DNS-pE2) was able to attenuate the thiol reactivity in DNS-
pE/DNS-pE2, and with their preferential PHGDH binding, led to
selective labelling of cellular PHGDH over other endogenous thiol
sources (e.g. glutathione, PTPs and others). Indeed as shown in
Figure 2G, DNS-pE2 was able to selectively label PHGDH
overexpressed in bacterial lysates in both dose- (left gel) and
activity-dependent manner (right gel). Competitive labelling
experiments showed effective but incomplete inhibition of DNS-
pE2 in the labelling of PHGDH with CBR-5884 (an allosteric
covalent PHGDH inhibitor [3b]), while such effect was
comparatively poorer for NCT-503 (a reversible inhibitor [3a]).
Similar labelling experiments were done with the active-site
mutants of PHGDH (Figure S4H); significant attenuation in
labelling of C234S mutant by DNS-pE2 indicates that Cys234
might be the probe-labelled cysteine residue in PHGDH. We thus
concluded DNS-pE/DNS-pE2 were indeed suitable fluorogenic
probes of PHGDH.
To further evaluate whether DNS-pE/DNS-pE2 could
selectively label endogenous PHGDH from live mammalian cells,
we first carried our competitive proteome labelling with “clickable”
IA, previously reported to label ligandable cysteines from a variety
of endogenous proteins including PHGDH (Figure 3A);[6] live
MCF-7 cells treated with the IA probe (1 µM, 1 h) showed a large
number of labelled bands (lane 1) including a 56-kDa band whose
fluorescent intensity was progressively weakened with increasing
concentrations of competing DNS-pE (0-50 M). This band
subsequently matched a major fluorescent band from MCF-7 cells
labelled directly with DNS-pE2 (Figure 3B; labelled with *). Further
competitive labelling by pre-treatment of the cells with a
competitor followed by pull-down (PD)/Western blotting (WB) with
anti-PHGDH unequivocally confirmed that this band was indeed
covalently labelled, endogenous PHGDH. Next, the direct probe-
PHGDH engagement in MCF-7 cells was studied by using cellular
thermal shift assay (CETSA; Figure 3C);[19] both NCT-503 and
CBR-5884 induced an increase in the thermal stability of cellular
PHGDH, resulting in a positive shift in the melting temperature of
the CETSA curve (∆Tm = 1.8 and 1.5 °C, respectively). On the
contrary, DNS-pE2 induced a negative thermal shift (∆Tm = -
3.8 °C) which might have resulted from the destabilization of
cellular PHGDH.
Having confirmed that DNS-pE/DNS-pE2 were able to
covalently and selectively label endogenous PHGDH in an
activity-based manner, as exemplified by the nearly exclusive,
single-band labelling profile in lane 1 of Figure 3B, and that both
probes had excellent fluorescence Turn-ON properties, we next
investigated their ability in live-cell imaging of endogenous
PHGDH. As shown in Figure 3D, strong dansyl fluorescence
signals were detected in DNS-pE2-treated live MCF-7 cells, most
of which was retained after cell fixation and permealization
(compare panels 1 & 2), indicating successful Turned-ON
fluorescence via covalent target labelling. Highly colocalized
rhodamine fluorescence was obtained on cells further “clicked”
with Rh-N3 (Figure S5). Competitive imaging was also performed
by pre-treatment of cells with a PHGDH inhibitor (10x; 1 h) prior
to probe addition (panels
3 & 4); significantly reduced
fluorescence signals were observed in the competitor-treated
cells, especially in the nuclear region, with more pronounced
effect from CBR-5884. Immunofluorescence (IF) by using anti-
PHGDH was used to further confirm the Turned-ON fluorescence
from DNS-pE2-treated cells was indeed from the PHGDH-bound
probe (compare panels 1 & 2 in Figure 3E). Finally, in order to
unequivocally confirm the highly selective PHGDH labelling
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10.1002/anie.201710856
Angewandte Chemie International Edition
COMMUNICATION
DNS-pE2 (1 µM)
IA (1 µM)
10 20 50
DNS-pE2 (1 µM)
(B)
(A)
(C)
DNS-pE (µM)
250 –
-
1
2
5
Competitor
(10X)
-
-
1.0
0.5
0.0
180 –
130 –
180 –
130 –
95 –
130 –
100 –
70 –
95 –
72 –
72 –
DMSO
55 –
55 –
43 –
55 –
*
*
*
DNS-pE2
NCT-503
CBR-5884
43 –
35 –
25 –
34 –
26 –
PD/WB: anti-PHGDH
55 –
Input
FL
40
50
60
70
Temperature ( C)
(D)
DNS-pE2
Live cells
Fixed cells
+ NCT-503
+ CBR-5884
(F)
MCF-7
3
DNS-pE2 (µM)
SILAC PD/MS
-
0.1 0.3
1
3
10
1
180 –
130 –
95 –
72 –
PHGDH
(E)
DNS-pE2
IF (anti-PHGDH)
Merged
Bright field
55 –
43 –
34 –
*
26 –
High Contrast
FL
Competition Ratio
Figure 3. (A) In situ proteome profile of IA-labelled MCF-7 cells pre-treated with different concentrations of DNS-pE (0 to 50 µM, 3 h). Upon IA probe labelling (1
µM), cells were clicked reaction with Rh-N3 followed by in-gel fluorescence scanning. The 56-kDa fluorescent band was marked with *. (B) In-gel fluorescence
scanning profile (FL; left) of live MCF-7 cells labelled with DNS-pE2 (1 µM for 3 h, ± 10× competitor), and the corresponding gel upon PD/WB analysis (right). The
56-kDa labelled endogenous PHGDH (*) was unequivocally enriched/identified. (C) CETSA curves of endogenous PHGDH from MCF-7 cells upon treatment with
an indicated compound (10 µM; 3 h). (D) Confocal fluorescence images of MCF-7 cells treated with DNS-pE2 (3 µM for 2 h, ± 10X competitor). (Panels 1/3/4): live
cells. (Panel 2): fixed cells. (Inset): merged images with bright-field. Scale bar = 10 μm. (E) Same as (D) except that immunofluorescence (IF) was performed with
the fixed cells. (F) In situ proteome profiles of MCF-7 cells labelled with DNS-pE2 (0-10 µM, 3 h). (Left): in-gel fluorescence scanning (FL; higher-contrasted lanes
1 & 3 were shown as indicated); (Right): SILAC ratio plots of DNS-pE2-labelled proteins identified from PD/MS studies of (1 µM, ± 10× DNS-pE). PHGDH band (*)
was highlighted, and shown in red in the SILAC plots.
profile by DNS-pE2 at a proteome-wide scale, in situ proteome
labelling followed by large-scale PD/quantitative mass
spectrometry (stable isotope labelling with amino acids in cell
culture, or SILAC[6,20]) was done on MCF-7, COS-7 and HepG2
cells (Figures 3F & S5); selective PHGDH labelling by the probe
was observed in all three cell lines, at 1 to 3 µM probe
concentration. As shown in Figure 1F (SILAC ratio plot), of the >
1000 proteins identified, PHGDH was the only hit that exhibited a
significant increase in SILAC ratios (H/L ratio > 5). Competitive
SILAC experiments, carried out with either DNS-pE or PVSN,
further showed that DNS-pE2 labelled endogenous PHGDH with
good selectivity from different live mammalian cells.
Our rational design of reaction-based probes has led to the
identification of DNS-pE2, the first fluorescence Turn-ON probe
capable of highly selective, covalent labelling of endogenous
PHGDH from different mammalian cells. The PHGDH labelling
was presumably through one of the key active-site cysteines in
PHGDH. With its dual-purpose feature, our probe thus offers a
promising approach to simultaneously image endogenous
PHGDH activities and study its active-site engagement in live-cell
settings.
funded by the Ministry of Science, ICT & Future Planning (NRF-
2016M3A9B6902060, NRF- 2017M3A9D8029942), Korea.
Conflict of interest
The authors declare no conflict of interest.
Keywords: fluorescent probe • chemical proteomics • live-cell
imaging • inhibitor • PHGDH
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Acknowledgements
Funding was provided by National Medical Research Council
(CBRG/0038/2013), the GSK-EDB Trust Fund (R-143-000-688-
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S. Pan, S.-Y. Jang, S. S. Liew, J. Fu, D.
Wang, J.-S. Lee,* S. Q. Yao*
Electrophilic quencher
Page No. – Page No.
A Vinyl Sulfone-Based Fluorogenic
Probe Capable of Selective Labelling
of PHGDH in Live Mammalian Cells
Alkyne tag
DNS-pE2
Turn-ON the Label: A fluorogenic probe that selectively and covalently labels
endogenous PHGDH from various live mammalian cells was developed based on a
vinyl sulfone-containing dansyl fluorophore, thus enabling simultaneous live-cell
imaging of PHGDH activities and studies of its active-site engagement for the first
time.
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