Tetrazole-Based Probes for Integrated Phenotypic Screening, Affinity-Based Proteome Profiling, and Sensitive Detection of a Cancer Biomarker

Article abstract of DOI:10.1002/anie.201709584

Target-identification phenotypic screening has been a powerful approach in drug discovery; however, it is hindered by difficulties in identifying the underlying cellular targets. To address this challenge, we have combined phenotypic screening of a fully functionalized small-molecule library with competitive affinity-based proteome profiling to map and functionally characterize the targets of screening hits. Using this approach, we identified ANXA2, PDIA3/4, FLAD1, and NOS2 as primary cellular targets of two bioactive molecules that inhibit cancer cell proliferation. We further demonstrated that a panel of probes can label and/or image annexin A2 (a cancer biomarker) from different cancer cell lines, thus providing opportunities for potential cancer diagnosis and therapy.

Full text of DOI:10.1002/anie.201709584

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Tetrazole-Based Probes for Integrated Phenotypic Screening,
Affinity-Based Proteome Profiling and Sensitive Detection of a
Cancer Biomarker
Authors: Zhengqiu Li, Ke Cheng, Jun-Seok Lee, Shao Q Yao, and Ke
Ding
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201709584
Angew. Chem. 10.1002/ange.201709584
Link to VoR: http://dx.doi.org/10.1002/anie.201709584
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10.1002/anie.201709584
Angewandte Chemie International Edition
DOI: 10.1002/anie.200((will be filled in by the editorial staff))
Target identification
Tetrazole-Based Probes for Integrated Phenotypic Screening, Affinity-
Based Proteome Profiling and Sensitive Detection of a Cancer Biomarker
Ke Cheng, Jun-Seok Lee, Piliang Hao, Shao Q. Yao, Ke Ding* and Zhengqiu Li*
Abstract: Phenotypic screening has been a powerful approach in
drug discovery, however it is hindered by difficulties in identifying
the underlying cellular targets. To address this challenge, we have
combined phenotypic screening of a fully functionalized small-
molecule library with competitive affinity-based proteome profiling
to map and functionally characterize the targets of screening hits.
Using this approach, we identified ANXA2, PDIA3/4, FLAD1 and
NOS2 as primary cellular targets of two bioactive molecules that
inhibit cancer cell proliferation. We further demonstrated that a
panel of probes can label and/or imagine annexin A2 (a cancer
biomarker) from different cancer cell lines, thus providing
opportunities for potential cancer diagnosis and therapy.
photo-crosslinking yield.[6] To alleviate these issues, competitive
proteome profiling is usually employed to distinguish between
background labeling and specific crosslinking.[7] On the other hand,
several groups have comprehensively studied the background
inventory of different photo-crosslinkers, which can help to
eliminate nonspecifically labeled proteins from target candidates.[8]
Moreover, we have recently developed novel diaryltetrazole-based
photo-crosslinkers, which possess superior photo-crosslinking
efficiency and one-/two-photon fluorescence turn-on properties, and
are suitable for simultaneous in situ proteome profiling and no-wash
real-time imaging.[9] Owing to their unique crosslinking mechanism
(Figure 1A), the photo-induced nitrile imines generated from these
photo-crosslinkers are capable of undergoing rapid reactions with a
variety of biological nucleophiles (i.e. acids, thiols, amines, etc) and
electrophiles (i.e. electron-deficient alkenes), but have shown
preferential labeling of proteins at their aspartic acid (Asp)/glutamic
acid (Glu) residues with reduced photo-crosslinker-associated
nonspecific background labeling. Despite frequent occurrence in a
protein (> 12% with combined proportion of Asp and Glu),[10] few
carboxylate-selective protein labeling chemistries are currently
available.[11] Our findings were further confirmed by a follow-up
work from Lin’s group which showed 2-aryl-5-carboxytetrazole
(ACT) can serve as an excellent photo-crosslinker in AfBP
studies.[12]
Phenotypic screening plays a pivotal role in the discovery of novel
bioactive compounds.[1] However, the utility of phenotypic
screening is limited by its difficulties in identifying the underlying
cellular targets.[2] Common approaches for target identication, such
as in vitro-based assay and affinity chromatography, mostly rely on
recombinant proteins or crude cell lysates, rendering them
unsuitable for reporting genuine drug-protein interactions under
native environments.[3] To address these challenges, affinity-based
proteome profiling (AfBP), in which photoprobes capable of
recapitulating drug-protein interactions in situ leading to subsequent
protein enrichment and large-scale proteome-wide target
identification, has been developed as a powerful approach for in situ
target identification during the past decade.[4] In order to further
improve this approach, we have developed three types of
“minimalist” bioorthogonal handle-containing photo-crosslinkers
(L3-6) capable of facilitating the synthesis of affinity-based probes
(AfBPs) and enabling simultaneous proteome profiling and live-cell
imaging.[5]
Recently, Cravatt et al. reported that phenotypic screening and
target identification can be integrated by using affinity-based probe
compound libraries, so as to accelerate the mechanistic
characterization of screening hits.[13] Inspired by these elegant
(A)
As a widely applied approach for target identification, AfBP still
suffers from two key issues - nonspecific labeling and unsatisfactory
(B)
[1]
K. Cheng, Prof. K. Ding, Prof. Z. Li
School of Pharmacy, Jinan University
601 Huangpu Avenue West, Guangzhou, China 510632
Email: pharmlzq@jnu.edu.cn, dingke@jnu.edu.cn
Prof. Shao Q. Yao
Department of Chemistry, National University of Singapore
Prof. J.-S. Lee
Molecular Recognition Research Center, Korea Institute of
Science and Technology (KIST), and Department of Biological
Chemistry, University of Science & Technology (Republic of
Korea)
Prof. P. Hao, School of Life Science and Technology,
ShanghaiTech University, China
[2]
[3]
(E)
Tz5
(C)
UV time/min
0.3
(D)
Tz5
Tz1 Tz2 Tz3 Tz4
0
1
5
Tz1 Tz2 Tz3 Tz4
kDa
Tz1
Tz2
72
55
Tz3
Tz4
43
34
[4]
[] Funding was provided by National Natural Science Foundation
of China (21602079), Science and Technology Program of
Guangdong province (2017A050506028), Science and
Technology Program of Guangzhou (201704030060), KIST
(2E26632/2E26110, CAP- 16-02-KIST) and the Bio & Medical
Technology Development Program of the National Research
Foundation (NRF- 2016M3A9B6902060) from Ministry of
Science, Korea. We thank Dr. W. Lorenzo (JNU), Dr. L. Gao
(IBN) for the invaluable suggestions on this work.
Tz5
FL
FL
CBB
FL
Figure 1. (A) The carboxylate-selective photo-crosslinking
mechanism between a tetrazole and a protein. (B) Structures of
compounds Tz1-5. (C) Time-dependent labeling of BSA (2 μg)
with Tz1-5 (1 μM) in PBS buffer. Proteome reactivity profiles of
cell lysates (D) and live MCF-7 cells (E) treated with Tz1-5. FL =
in-gel fluorescence scanning. CBB = Coomassie gel.
1
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10.1002/anie.201709584
Angewandte Chemie International Edition
(B)
Tz6 Tz7 Tz8 Tz9 Tz10 Tz11 Tz12 Tz13 Tz14
(C)
(D)
kDa
MCF-7
HepG2
MDA-MB-231
1.0
95
FL
55
0.5
0.0
43
34
CBB
Figure 2. (A) Stuctures of the probes Tz6-22 (red-coloured probes were used in the following biological studies). (B) Proteome reactivity
profiles of live MCF-7 cells treated with Tz6-14 (5 μM final concentration, 10 min of UV irradiation). (C) Inhibition of cancer cell viability
of Tz6-22 determined by CCK8 assay. (D) IC50 values of Tz6/10/21/22 against MCF-7 cells.
strategies, we endeavored to incorporate a diaryltetrazole, a group
Tz4-treated sample showed notable labeling bands (Figure 1D, lane
can simultaneously act as a photo-crosslinker and a bioactive moiety, 4). These results indicate that 2,5-diphenyltetrazole (Tz4) is an
and an alkyne handle into a 20-membered small molecule library,
with the aim to discover bioactive tetrazole-based molecules
followed by identifying the interacting cellular targets.
efficient photo-crosslinker with superior protein crosslinking ability.
Encouraged by the excellent in vitro cross-linking efficiency of Tz4,
we sought to assess its in situ labeling properties. Upon incubation
of Tz1-5 with live MCF-7 cells for 5 h, the cells were irradiated
with UV light and then lysed; the resulting cell lysates were
conjugated with TAMRA-N3, separated by SDS-PAGE followed by
in-gel fluorescence scanning (Figure 1E); only Tz4-treated samples
gave fluorescently labeled bands, highlighting the efficient labeling
ability of 2,5-diphenyltetrazole in situ. To identify the crosslinked
targets in cell lysates and live cells, labeled proteins under both
settings were clicked with TAMRA-Biotin-N3 and then enriched
using avidin-agarose beads, separated on SDS-PAGE, followed by
LC-MS/MS analysis. As listed in Table S1, a series of high-
abundance proteins, such as CD9, CD44, PRC1 and CC2D1A, were
positively identified. Compared with previously reported
background inventory of three common photocrosslinkers (diazirine,
benzophenone and arylazide),[8] the background proteins of
diaryltetrazole include unique and common proteins, which can be
useful references for AfBP, especially in cases where diaryltetrazole
is chosen as a photocrosslinker in the probe design.
After confirming that 2,5-diphenyltetrazole is an efficient photo-
crosslinker under both in vitro and in situ settings, we next set out to
prepare a photoprobe library by incorporating different substituents
at the 2- and 5- positions (Tz6-20, Figure 2A). The synthesis was
largely based on published methods (Scheme S3-6). Two
nonclickable analogues of Tz6 and Tz10, Tz21 and Tz22, were
prepared accordingly as competitors (Scheme S7). With these
probes in hand, we first evaluated the labeling performance of the
library in mammalian cells (5 μM final probe concentration, 10 min
of UV irradiation). As shown in Figure 2B, individual probes
showed markedly distinct protein labeling profiles, which indicates
the diversity elements introduced into the probe library were
Given that the effects of different substituents on the photolysis
of tetrazole have not been well studied, we synthesized five probes
(Tz1-5) bearing methyl, ester, phenyl, 2-pyridyl and 2-pyrimidyl
groups, respectively, at the 5- position, following the typical
synthetic methods of tetrazoles (Figure 1B, and Schemes S1 &
S2);[14] an alkyne handle was embedded into the probes for
visualization, enrichment, and identification of interacting proteins.
We first evaluated the rate of photolysis of Tz1-5 by HPLC analysis
(Figure S1); upon exposure of each of the probes (dissolved in PBS
buffer) to 302 nm UV light for different periods of time followed by
HPLC separation of the reaction products, complete photolysis of
Tz1 and Tz2 was observed within 5 and 2 min, respectively,
indicating that the electron-withdrawing ester group in Tz2 was
beneficial for the photolysis. Interestingly, Tz3/4/5 appeared to be
more efficient than Tz1/2 and their complete photolysis could be
observed in as little as 1 min. Next, time-dependent labeling with
bovine serum albumin (BSA) was carried out to assess the photo-
crosslinking ability of these probes. Upon incubation of BSA with
each of the probes for 10 min in PBS buffer, the resulting mixtures
were exposed to UV light (302 nm) for different periods of time,
clicked with TAMRA-N3 and separated by SDS-PAGE.
Fluorescently labeled BSA was finally visualized and quantified by
in-gel fluorescence scanning (Figure 1C); we observed strong
fluorescent bands of Tz4/BSA labeling in as little as 20 s with 1 μM
of the probe. On the contrary, the labeling profiles of Tz1/2/3/5
appeared to be significantly weaker under the same labeling
conditions across all UV irradiation time. Further, we carried out
labeling profiles of MCF-7 lysates with Tz1-5 under the same
labeling conditions as BSA labeling, which also appeared that only
2
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10.1002/anie.201709584
Angewandte Chemie International Edition
(A)
(B)
(lane 5), which again confirmed the low
background labeling property of tetrazole
photo-crosslinkers in general as earlier
discussed (e.g. Figure 1), the Tz6-treated
Tz21/22
(10×)
kDa
Tz6
Tz6 + Tz21 (10×)
Tz10 + Tz22 (10×)
Tz10
+
+
95
Probe
sample showed
a highly selective probe
72
55
43
labeling profile, with a strong fluorescently
labeled band predominantly at ~37 kDa and
several much fainter bands at ~72 kDa (lane
1), indicating excellent target selectivity. In
the presence of excessive Tz21 (10 x) as a
competitor, all fluorescent bands including the
37-kDa band were abolished (lane 2),
indicating they were potential Tz21-targeted
proteins and not background proteins caused
by nonspecific photo-crosslinking. In sharp
contrast, the Tz10-treated sample showed a
significantly less selective, albeit still Tz22-
competitive, probe labeling profiles, resulting
in strong fluorescently labeled bands
throughout the whole lane (lane 3). To track
the subcellular probe localization, cellular
imaging of live MCF-7 cells with Tz6/10 was
next carried out. The cells were first treated
with each probe followed by UV irradiation to
initiate photo-crosslinking. Subsequently,
cells were fixed, permeabilized and clicked
with TAMRA-N3 under previously optimized
click chemistry conditions,[5b,5c,9] then imaged.
Strong fluorescence signals were observed in
the cell membrane and nucleus from Tz6-
34
Probe
/NU
(C)
(D)
LFQ intensity ratio
Gene
name
MW/
kDa
Tz6/
(Tz6 +
Tz6/
Tz10/
(Tz10 +
10×
Tz10/
Tz4
Tz10/
Tz11
Tz4
Tz21
+
(10×)
10× Tz21)
Tz22)
Anti-ANXA2
38 kDa
ANXA2
CKAP4
PDIA4
NOS2
FLAD1
MRC2
TPP1
38
66
72.9
131
65
0
0
0
0
0
0
0
∞
2.5
∞
∞
∞
0
0
0
∞
0
Tz22
(10×)
+
∞
∞
∞
2.6
∞
∞
∞
∞
∞
∞
5.1
Anti-NOS2
Anti-FLAD
131 kDa
0
0
0
0
0
0
0
0
166
60.4
75 kDa
∞
∞
Pull-down/WB
MLEC
32.2
2.7
∞
Figure 3. (A) Proteome reactivity profiles of live MCF-7 cells treated with Tz4/6/10 in
the presence or absence of corresponding competitors (2 μM final concentration, 10
min of UV irradiation). (B) Live cell imaging of MCF-7 cells with Tz6/10 (2 μM) in
the presence or absence of corresponding competitors. Sacle bar = 10 μm. (C) High-
confidence protein hits of Tz6/10, “∞” means infinity, “0” means not appearing. (D)
Validation of three protein hits by pull-down/WB. WB = Western blotting.
sufficient to direct preferential bindings of different probes to
treated cells, while Tz10 mainly located outside of the nucleus.
Consistently, the fluorescence signals of both probes were abolished
in the presence of excessive competitors (Figure 3B). Control
imaging experiments with DMSO under similar conditions gave
minimal background fluorescence compared to labeled cells (Figure
S5). Results from these competitive in situ labeling and live-cell
imaging experiments thus indicate that both Tz6 and T10 were able
to efficiently capture their intended cellular targets, and Tz6
appeared to have highly selective cellular targets.
We next proceeded to identify potential cellular targets of Tz6/10
by large-scale chemoproteomics experiments. Similar to the
procedures described above, probe-labeled proteins were affinity-
purified and identified by LC-MS/MS analysis following in situ
proteome labeling and click chemistry with TAMAR-Biotin-N3.
Control experiments were done concurrently with DMSO- and Tz4-
treated control samples, which served to filter off “false hits”
various sub-proteomes in the cells. Interestingly, these in situ
proteome reactivity profiles were different from the corresponding
profiles obtained under in vitro conditions (Figure S3A), indicating
that the probes interacted with different sets of proteins between live
cells and cell lysates.
Subsequently, we tested the inhibitory activities of the probe
library against three common cancer cell lines (MCF-7, HepG2 and
MDA-MB-231; Figures 2C/D & S2). Gratifyingly, two probes, Tz6
and Tz10, exhibited potent antiproliferative effects under normal
cell culture conditions with IC50 values of 4.1 and 0.69 μM,
respectively. Remarkably, the cytotoxicity of Tz10 was comparable
to doxorubicin (a well-known anticancer drug). The corresponding
alkyne handle-free competitors, Tz21/22, displayed comparable
antiproliferative activities in MCF-7 cells (Figure 2C/D). We found
that the structures of Tz6/10 differ from the other molecules only in
the amide moiety, suggesting that this part is essential for
maintaining the compounds’ observed bioactivities. Similar to
previously reported bioactive molecules,[13a] probes bearing a t-
butoxycarbamoylpiperazine carbonyl group (i.e. Tz6/9/14) also
exhibited moderate antiproliferative effects in MCF-7 cells (Figure
2C).
To identify the cellular targets of the two most potent probes,
Tz6/10, chemoproteomic experiments and live-cell bioimaging were
next carried out concurrently. As shown in Figure 3A, the in situ
proteome labeling was performed by following the procedures
described above with the inactive Tz4 probe as a control (lane 5),
but at a lower probe concentration (2 μM) in order to reduce
nonspecific binding. As shown in Figure 3A, while the Tz4-treated
sample showed an overall negligible, nonspecific labeling profile
identified from the LC-MS/MS experiments as
a result of
background photo-crosslinking and/or nonspecific protein binding.
In addition, only proteins that appeared in duplicated runs and
whose LFQ intensity[15] ratios from probe-treated and competitive
labeling experiments (i.e. Tz6 vs (Tz6 + 10× Tz21), or Tz10 vs
(Tz10 + 10× Tz22) of greater than 2 were considered further.
Finally, a comparison of the proteomic targets of Tz10 versus Tz11,
an inactive analogue differs only in the position of piperidine, was
made to reveal proteins relevant for antiproliferative activity of
Tz10 (LFQ ratio > 2). As listed in Figure 3C and Table S3/4, 35 and
60 protein hits met these criteria for Tz6 and Tz10, respectively.
Further analysis of these protein hits revealed that annexin A2
(ANXA2) could match the ~37-kDa major band detected in Tz6-
labeled proteomes (i.e. Figure 3A, * in lane 1), while flavin adenine
3
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10.1002/anie.201709584
Angewandte Chemie International Edition
dinucleotide synthetase 1 (FLAD1) and nitric oxide synthase (NOS2)
could match the labeling bands at ~70 kDa and ~130 kDa,
respectively, in Tz10-treated sample (Figure 3A, * in lane 3). These
three target candidates were further validated by pull-down/Western
blotting (WB) with the corresponding antibodies (Figure 3D).
Moreover, we found that the locations of most proteins hits
coincided well with the imaging results (Figure 3B);[16] for example,
the protein hits of Tz6, ANXA2, VDAC and CKAP4, are known to
be mainly located in the cell membrane, and NOS2, FLAD1 of
Tz10-identified proteins are predominantly cytosolic proteins. We
further verified ANXA2 and NOS2 by the immunofluorescence
experiments, following our previous protocols (Figure S5).[5b,5c]
These lines of evidence underscored the high reliability of these
protein hits, and it is possible that Tz6/10 produce their
antiproliferative effects through a combination of these protein
targets.
Considering annexin A2 is a cancer biomarker,[17] it is of great
importance to develop suitable small molecule probes that can
detect its cellular expression/activity in situ. Herein, we further
evaluated the labeling sensitivity of Tz6 and other probes toward
annexin A2 by concentration- and time-dependent labeling
experiments. As shown in Figure 4A, Tz6/8/12 can successfully
label ANXA2 at 2 μM probe concentration, which was confirmed
by pull-down/WB (Figure 4A, bottom gel). An even lower
concentration of the probes (1 μM) was sufficient to produce
prominent corresponding bands in live MCF-7 cells from
concentration-dependent labeling experiments (Figure S3C, SI).
Time-dependent labeling experiments revealed that the ~37 kDa
band was visible within 1 h (Figure 4B). Importantly with Tz6, the
highly selective ANXA2 labeling profiles could be successfully
recapitulated in various cancer cell lines, including HepG2, A549,
HeLa and MDA-MB-231 (Figure 4C), suggesting that this probe has
the potential of broad applications in detecting endogenous ANXA2.
It is also noteworthy that, from the immunofluorescence (IF)
experiments performed with anti-ANXA2, the resulting
fluorescence signals appeared to overlap well with those obtained
from staining the same cells with Tz6 (Figures 4D & S5), implying
that Tz6 can report this protein’s activities by both protein labeling
and live-cell imaging experiments. In addition, labeling experiments
of recombinant annexin A2 with Tz6 revealed that the protein could
be successfully labeled by the probe at as low as 0.5 μM probe
concentration with 1 μg of the protein, and as little as 1.25 pmol of
the protein could be successfully detected by Tz6 at 1 μM
concentration (Figure 4E), proving that this probe possesses
excellent sensitivity toward annexin A2. To map the potential
labeling sites of Tz6 in annexin A2, the probe-labeled recombinant
protein was analyzed by LC-MS/MS. Asp110, a residue located in the
active region of the protein was positively identified (Figures 4F &
S6).[18] This crosslinking pattern is consistent with the results
previously reported by us and others.[9,12] Docking experiments
revealed that the distance between the tetrazole in Tz6 and Asp110
was around 7.7 Å, which is a reasonable distance for successful
photo-crosslinking. Finally, we evaluated the photo-crosslinking
efficiency of Tz6 with recombinant annexin A2 by pull-down/WB,
which appeared to be very high (Figure S7).
Figure 4. (A) Labeling profiles of MCF-7 cells with Tz6/8/12 (2
μM), in the presence or absence of competitor (Tz21). The
corresponding pull-down/Western blot (WB) are shown (bottom
gel). (B) Time-dependent in situ labeling with Tz6 (2 μM). (C)
Labeling profiles of different cancer cell lines with Tz6 (2 μM).
(D) Live-cell imaging of MCF-7 cells with Tz6 (2 μM).
Immunofluorescence (IF) staining using anti-annexin A2
antibodies. Sacle bar = 10 μm. (E) Labeling of recombinant
annexin A2 protein with Tz6 (different concentrations of Tz6 or
different amount of protein). (F) ASP-110, the binding site of Tz6
with annexin A2 identified by LC-MS/MS and docking
experiments to predict the binding mode of Tz6 with annexin A2.
compounds, Tz6/10, by affinity-based proteome profiling coupled
with live-cell bioimaging. Most protein hits are disease-related
targets. Importantly, several probes from our current study,
especially Tz6, were able to label endogenous annexin A2 with
excellent selectivity and sensitivity under highly complex native
cellular environments from different mammalian cells. With these
outstanding properties, we expect that these novel small molecule
probes could find potential applications in cancer-related diagnoses
and therapy.
Keywords: tetrazole · phenotypic screening · target identification.
affinity-based probe · cancer biomarker
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10.1002/anie.201709584
Angewandte Chemie International Edition
Target identification
Tetrazole-Based Probes for Integrated Phenotypic Screening, Affinity-based
Proteome Profiling and Sensitive Detection of a Cancer Biomarker
K. Cheng, J.-S. Lee, S. Q. Yao, K. Ding,* Z. Li, * __ Page – Page
Entry for the Table of Contents (Please choose one layout)
Phenotypic screening
Profiling and imaging a
and target identification
cancer biomarker
Screening
LC-MS/MS
Tetrazole-based probes
Imaging
SDS-PAGE
Tetrazole-based probes bearing an alkyne handle are suitable for phenotypic screening and affinity-based proteome profiling,
especially Tz6 that can sensitively detect a cancer biomarker by both protein labeling and bioimaging.
6
This article is protected by copyright. All rights reserved.