Cellular target identification of Withangulatin A using fluorescent analogues and subsequent chemical proteomics

Article abstract of DOI:10.1039/c9cc03653a

Withangulatin A (WA) has been reported to exhibit potent antitumor activity. However, its possible mechanism and direct proteomic targets remain unknown. Herein we report the subcellular localization of WA by designing and synthesizing its fluorescent analogues with coumarin moieties. Furthermore, sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 was identified as the potential target of WA for its antitumor activity by chemical proteomics.

Full text of DOI:10.1039/c9cc03653a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: T. Zhu, C. Chen,
S. Wang, Y. Zhang, D. Zhu, L. Li, J. Luo and L. Kong, Chem. Commun., 2019, DOI: 10.1039/C9CC03653A.
Volume 52 Number
1
4
January 2016 Pages 1–216
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Chemical Communications
www.rsc.org/chemcomm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
first methanofullerene derivatives of Sc
3
N@C2n (2n
=
68 and 80). Synthesis of the
3
N@D5h-C80
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 5
ChemComm
View Article Online
DOI: 10.1039/C9CC03653A
Journal Name
COMMUNICATION
Cellular target identification of Withangulatin A using fluorescent
analogues and subsequent chemical proteomics
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
Tianyu Zhu, Chen Chen, Sisi Wang, Yi Zhang, Dongrong Zhu, Lingnan Li, Jianguang
a
a
Luo* and Lingyi Kong*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Withangulatin A(WA) has been reported to possess potent
antitumor activity. However, its possible mechanism and
direct proteomic targets remain unknown. Herein we report
the subcellular localization of WA by designing and
synthesizing its fluorescent analogues with coumarin
moieties. Furthermore, sarco/endoplasmic reticulum
calcium-ATPase (SERCA)2 was identified as the potential
target of WA for its antitumor activity by chemical
proteomics.
As an important source for anticancer drug discovery, in the last
few decades, natural products and their derivatives have gained
much attention.1 Physalis angulata L., known as a traditional
Chinese medicine, has been used for treatment of diseases such as
tumors, hepatitis, rheumatism and nephronia for a long time.
Various withanolides that have great bio-activities have been
-3
phenomenon at many levels.12, 13 Whereas, a major drawback of
this method is that it is
Fig. 1 Chemical structures of (A) WA; (B) XDS-1; (C) WA-XDS-1, WA-XDS-2
and (D) WA-XDS-3, WA-XDS-4; (E) Excitation (a) and emission (b) spectra of
WA-XDS-1 in dichloromethane (DCM) and PBS.
4
5
, 6
isolated from this species.
Withangulatin A(WA) is one of the
7
withanolides isolated from P. angulata L and exhibits potent
antitumor, anti-inflammatory and immunosuppressive activity.
8
-10
hard for fluorescent imaging alone to detail the direct target and
explain the deeper behaviour of bioactive molecules. At the same
time, activity-based protein profiling (ABPP) and affinity-based
In spite of numerous studies have been undertaken to elucidate the
anticancer mechanisms of WA, such as changing cellular
1
1
morphology, inhibiting topoisomerase II activities and suppressing
1
4, 15
7
probe (AƒBP) has been widely applied in target profiling,
and
general protein synthesis, its molecular targets and detailed
clickable probes are widely preferred to discover biological target of
bio-active molecules as it can make probes more cell-permeable
with less perturbation to the native binding and can accurately
mechanisms remain elusive, which largely hampered the further
development of WA for cancer therapy.
To overcome these obstacles, recently a number of chemical
biology approaches have been developed to tackle these
challenges. Among these methods, fluorescent probe that
combined natural products and fluorescent organic dyes has
become a powerful tool for visualizing the subcellular location,
monitoring changes in cellular morphology and exploring biological
1
6,
reflect the drug’s actions in the in vivo physiological environment.
1
7
However, the toxicity of copper(I) hindered the usage of this
1
8
method in living system. Furthermore, sometimes it is hard for us
to accurately identify the exact protein that can clarify the
mechanism of active natural products from hundreds of proteins. In
order to identify the proteomic target of WA more efficiently,
herein we first synthesized a series of new fluorescent analogues
based on WA to explore its subcellular location. Subsequently WA
was conjugated with Epoxy-activated Sepharose 6B beads to pull
down the target protein of WA. With the help of fluorescent WA
probes, we can rule out non-specific binding proteins and improve
a. State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, Nanjing 210009, China.
E-mail: luojg@cpu.edu.cn, lykong@cpu.edu.cn
†
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any accuracy in the proteome-wide identification of molecular targets.
supplementary information available should be included here]. See
It is important for fluorescent probes not to affect the activity
DOI: 10.1039/x0xx00000x
when natural product is linked to fluorophore. Like many bioactive
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
COMMUNICATION
Journal Name
withanolides, WA has an α,β-unsaturated ketone group in ring A Fig. 2 (A) Uptake of probes in MDA-MB-231 cells: (a) 5 μM WVAie-wXDArSt-ic1lefoOrn1linhe;
DOI: 10.1039/C9CC03653A
(
Fig. 1A), which is considered as an essential group to maintain its (b) 5 μM WA-XDS-4 for 1h; (c) 100 μM XDS-1 for 1h. (B) Time- and
1
9
activity and the modification on 4-position hydroxyl of WA did not concentration-dependent uptake of WA-XDS-1 in MDA-MB-231 cells.
2
0, 21
diminish or even improve the anticancer activity.
With this in with maximum excitation at 410nm and maximum emission at
mind, fluorescent probes were designed and synthesized by 470nm, and the influence of solvent on fluorescence spectra was
connecting 4-hydroxyl group of WA with a coumarin moiety obvious. Compared with the excitation and emission spectra of
through linkers of various lengths. Specifically, we chose high fluorescent probes in dichloromethane, the aqueous PBS solution
fluorescent N,N-dialkyl-7-aminocoumarin (Scheme S1) as
a
quenched the fluorescence gravely (Fig. 1E and Fig. S10), indicating
fluorescent moiety because it is relatively small and has no that these fluorescent conjugates were suitable to detect the
2
2
toxicity. Furthermore, as an environment-sensitive fluorophore change in micro-environmental polarity with a turn-on property
with “off-on” mechanism, N,N-dialkyl-7-aminocoumarin is suitable when the probes were interacting with target proteins.
of detecting hydrophobic interaction between WA and its target
proteins.
The uptake properties of four fluorescent derivatives and XDS-1
were detected by MDA-MB-231 cells (Fig. 2A and Fig. S11). Bright
2
3
Fluorescent probes were synthesized from WA in five or six steps. fluorescence inside MDA-MB-231 cells was observed when probes
3
-azido-7-diethylaminocoumarin was synthesized first based on were incubated with 5 μM of WA-XDS-1 for 1h. Affected by the long
2
4
previously published procedures. Then in order to identify the linker which decreased the cytotoxic activity, WA-XDS-4 showed the
influence of linkers to the activity of WA, four different lengths of weakest fluorescence among four probes. Moreover, naked
linkers were designed to combine WA with the coumarin moiety by coumarin dye (XDS-1) showed no significant cellular location even
click reactions between acetylenic and azide groups. Finally, four at a concentration of 100 μM after 2h staining, verifying that it was
WA-based fluorescent probes were synthesized (Fig. 1C and Fig. 1D) WA moiety that led the uptake and localization of fluorescent
and XDS-1, a negative control probe without WA moiety, was also probes in MDA-MB-231 cells. Then the uptake of WA-XDS-1 was
synthesized to exclude the possibility that the uptake and investigated based on time and concentration to optimize
localization of the probes were driven by fluorescent moiety itself experimental condition. It was best for MDA-MB-231 cells to be
(Fig. 1B). The detailed synthetic procedures are provided in incubated with 2.5 μM of WA-XDS-1 for 1h, the higher
Schemes S1-S4 and Fig.S1-S9. Upon finishing of synthesis, we concentration or longer time was harmful to cells and may change
investigated the cytotoxic activity of four new fluorescent probes cellular morphology (Fig. 2B).
and XDS-1 against several human cancer cell lines using Cell
To further detail the subcellular localization of WA, we co-stained
Counting Kit-8 (CCK-8) assay. WA was also tested for comparison fluorescent derivatives trained MDA-MB-231 cells with a panel of
and the data were summarized in Table S1. WA-XDS-1 had the organelle-specific dyes (Fig. 3A-3C and Fig. S12-S14). Compared
greatest cytotoxic activity versus WA and the anticancer activity of with MitoTracker (mitochondria specific dye) and LysoTracker
WA-XDS-4 was weaken, indicating a long linker was harmful to (lysosomes specific dye), the co-localization of probes with ER-
maintaining the cytotoxic activity. Meanwhile, XDS-1 had no Tracker (endoplasmic reticulum specific dye) matched much better,
cytotoxic activity until 100 μM. Then the environment-sensitive which indicated ER was the main organelle that WA worked in,
property of these fluorescent probes was tested. Spectral analysis
showed that all the probes showed obvious stokes shift (~60nm)
Fig. 3 (A) Co-stain of WA-XDS-1 with LysoTracker in MDA-MB-231 cells: (a)
2
.5 μM of WA-XDS-1; (b) 50 nM of LysoTracker; (c) Reddot (nuclei specific
dye); (d)overlay of three images. (B) Co-stain of WA-XDS-1 with MitoTracker
in MDA-MB-231 cells: (a) 2.5 μM of WA-XDS-1; (b) 100 nM of MitoTracker;
(c) Reddot; d) overlay of three images. (C) Co-stain of WA-XDS-1 with ER-
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 5
ChemComm
Journal Name
COMMUNICATION
Tracker in MDA-MB-231 cells: (a) 2.5 μM of WA-XDS-1; (b) 1 μM of ER-
View Article Online
DOI: 10.1039/C9CC03653A
Tracker; (c) Reddot; (d) overlay of three images.
Fig. 4 (A) Proteomic analysis of proteins interact specifically with WA-conjugated beads. (B) WA-conjugated beads were incubated with MDA-MB-231 cell
lysates in the absence or presence of WA with different concentrations at 4 °C for 12 h, then the proteins bound to WA beads were detected by
immunoblotting. (C) The co-localization of WA-XDS-1 and SERCA2 in MDA-MB-231 cells: (a) 2.5μM of WA-XDS-1; (b) immunofluorescence against SERCA2; (c)
5
00nM of PI (nuclei specific dye); (d) overlay of three images. (D) MDA-MB-231 cell lysates were pre-incubated with WA beads for 2 h and then further
incubated with or without 20μM of WA at 4 °C for 12 h for competitive binding. (E) WA conjugated beads were pre-incubated with or without 40μM of DTT
for 2 h and then further incubated with MDA-MB-231 cell lysates at 4 °C for 12 h. (F) MDA-MB-231 cells were incubated with WA of various concentrations
2
+
and thapsigargin for 24h and lysed, then measured the activities of Ca -ATPase. (G) MDA-MB-231 cells were incubated with various concentrations of WA
for 24h and lysed, then the protein levels of GRP78, phosphorylated eIF2α, ATF4, CHOP and Cleaved PARP were detected by immunoblotting.
revealing a great possibility that the target protein of WA was was obvious to discover SERCA2 was pulled down by WA beads and
located in ER.
it can be reversed by excess amount of WA concentration-
Upon the identification of the subcellular location of WA, next we dependently. Immunofluorescence experiment was also performed
performed pull-down/LC-MS experiments using affinity-based and the co-localization matched well (Fig. 4C and Fig.S16). To
profiling to identify the targets of WA. WA-conjugated sepharose further confirm the interaction between WA and SERCA2. in vitro
beads were prepared as affinity reagent based on previous report25 pull-down assay and biolayer interferometry (BLI) test with
and MDA-MB-231 cell lysates were incubated with the beads to recombinant protein were then carried out. Consistent with the
capture the target proteins, then the complex was resolved by result of initial pull-down assay, WA-beads successfully co-
boiling and the proteins interacted with WA-beads were resolved precipitated with purified SERCA2 protein and excess amount of
by SDS/PAGE followed by silver staining and further identified using WA weaken the interaction (Fig.S17). Moreover, WA showed direct
LC-MS/MS analysis(Fig.S15). Fig. 4A represents different proteins binding with the recombinant SERCA2 protein in BLI analysis with
found to interact specifically with WA-conjugated sepharose.
According to the discovery of fluorescent probes that ER was the
K
D
= 10.3μM (Fig.S18).
Considering the α, β-unsaturated ketone group in ring A of WA is
main organelle that WA played a role in, we concentrated on ER a bioactive group, which is easy to act with sulfhydryl in cysteine via
located proteins which could possibly clarify the anticancer the Michael addition reaction, whether WA could covalently bind to
mechanism of WA. As the first protein that located in ER with the SERCA2 was further investigated. WA-conjugated sepharose beads
highest score of peptide spectrum matches (PSMs), SERCA2, known were pre-incubated with MDA-MB-231 cell lysates, and then the
to play an important role in calcium transport, was finally identified lysates were further treated with an excess amount of WA as
as a potential target of WA.
competition. Post-competition of WA failed to prevent the binding
To verify the interaction between WA and SERCA2, we incubated between WA beads and SERCA2 (Fig. 4C), which meant an
WA beads with MDA-MB-231 cell lysates in the absence or irreversible interaction between WA and SERCA2. Then we used
presence of different concentrations of WA as competition dithiothreitol (DTT), which also have sulfhydryl, to detect its
substance and the protein was detected by Western blot (Fig. 4B). It influences on the association of SERCA2 with WA-conjugated
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
7
8
.
.
C. M. Chen, Z. T. Chen, C. H. Hsieh, W. S. Li and S. Y. WeVnie,wHeAtrteicrloecOycnlleinse,
990, 31, 1371-1375.
W. C. Lee, K. Y. Lin, C. M. Chen, Z. T. Chen, H. J. Liu and Y. K. Lai, J. Cell.
Physiol., 1991, 149, 66-76.
sepharose beads. DTT remarkably abolished the interaction
between beads and SERCA2 (Fig. 4D), suggesting that WA might
covalently bind to the sulfhydryl of cysteine on SERCA2.
1
DOI: 10.1039/C9CC03653A
As the main pump that transports Ca2+ from cytoplasm to ER, 9. L. Sun, J. Liu, D. Cui, J. Li, Y. Yu, L. Ma and L. Hu, J. Cell. Biochem., 2010,
SERCA dependent calcium transport is important to maintain the
109, 532-541.
2
+
10. L. Sun, J. Liu, P. Liu, Y. Yu, L. Ma and L. Hu, Process Biochem., 2011, 46,
calcium homeostasis and keep the concentration of Ca in ER four
4
82-488.
2
6
orders of magnitude higher than that in cytosol. Various cancer
1
1. W. C. Lee, Y. C. Lee, M. D. Perng, C. M. Chen and Y. K. Lai, J. Cell.
Biochem., 1993, 52, 253-265.
2
+
27
cells exhibit dysregulated Ca signaling and overexpression of
SERCA activates cell survival pathways through the effect on cellular 12. S. M. DeGuire, D. C. Earl, Y. Du, B. A. Crews, A. T. Jacobs, A. Ustione, C.
2
+
28-30
Daniel, K. M. Chong, L. J. Marnett, D. W. Piston, B. O. Bachmann and G.
A. Sulikowski, Angew. Chem. Int. Ed., 2015, 54, 961-964.
3. C. Li, T. Dong, Q. Li and X. Lei, Angew. Chem. Int. Ed., 2014, 53, 12111-
Ca peak and oscillations in various cancers,
which make it a
3
1
potential target for antitumor therapy. To test the effects of WA
on SERCA activity, MDA-MB-231 cells were incubated with WA and
the Ca -ATPase activity was measured using Ca -ATPase kit. The 14. H. Shi, C.-J. Zhang, G. Y. J. Chen and S. Q. Yao, J. Am. Chem. Soc., 2012,
result indicated WA significantly inhibited the Ca -ATPase activity
in a dose-dependent manner (Fig. 4E), and 8 μM of WA inhibited
1
1
2115.
2
+
2+
2
+
134, 3001-3014.
1
5. Z. Li, P. Hao, L. Li, C. Y. J. Tan, X. Cheng, G. Y. J. Chen, S. K. Sze, H.-M.
Shen and S. Q. Yao, Angewandte Chemie-International Edition, 2013, 52,
2
+
the Ca -ATPase activity by 62%.
8
551-8556.
It is reported the inhibition of SERCA activity often results in an 16. S. Pan, H. Zhang, C. Wang, S. C. L. Yao and S. Q. Yao, Natural product
imbalance of calcium levels in cancer cells, which causes unfolded
protein response (UPR), ER stress and eventually apoptosis.32 Thus
the effects of WA were tested on the ER stress-associated markers
and apoptotic signalling pathway in MDA-MB-231 cells. WA
reports, 2016, 33, 612-620.
17. E. M. Sletten and C. R. Bertozzi, Angewandte Chemie-International
Edition, 2009, 48, 6974-6998.
1
8. F. Wolbers, P. ter Braak, S. Le Gac, R. Luttge, H. Andersson, I. Vermes
and A. van den Berg, Electrophoresis, 2006, 27, 5073-5080.
treatment significantly caused a markedly enhanced expression of 19. Y.-M. Xu, A. D. Brooks, E. M. K. Wijeratne, C. J. Henrich, P. Tewary, T. J.
Sayers and A. A. L. Gunatilaka, J. Med. Chem., 2017, 60, 3039-3051.
GRP78, ATF4, P-eIF2α and CHOP, as well as the cleavage of PARP in
2
0. E. M. K. Wijeratne, Y.-M. Xu, R. Scherz-Shouval, M. T. Marron, D. D.
Rocha, M. X. Liu, L. V. Costa-Lotufo, S. Santagata, S. Lindquist, L.
Whitesell and A. A. L. Gunatilaka, J. Med. Chem., 2014, 57, 2851-2863.
a dose-dependent manner (Fig. 4F). These results indicated WA
could act against cancer cell through inhibiting SERCA activity and
inducing ER stress-associated apoptosis.
21. G. G. Llanos, L. M. Araujo, I. A. Jimenez, L. M. Moujir, J. Rodriguez, C.
In conclusion, we developed four new WA-based fluorescent
probes to explore its subcellular distribution and found ER was the
main organelle WA located in, through chemical proteomics-based
target identification and molecule/cell-based validation, we
Jimenez and I. L. Bazzocchi, Eur. J. Med. Chem., 2017, 140, 52-64.
2. S. Xu, S. Luo, H. Yao, H. Cai, X. Miao, F. Wu, D.-H. Yang, X. Wu, W. Xie, H.
Yao, Z.-S. Chen and J. Xu, J. Med. Chem., 2016, 59, 5022-5034.
2
2
3. Q. Sun, J. Qian, H. Tian, L. Duan and W. Zhang, Chem. Commun., 2014,
5
0, 8518-8521.
elucidated that WA targets SERCA2, inhibits its activity and finally 24. K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q. Wang,
Org. Lett., 2004, 6, 4603-4606.
5. L. Liu, Y. Hua, D. Wang, L. Shan, Y. Zhang, J. Zhu, H. Jin, H. Li, Z. Hu and
W. Zhang, Chem. Biol., 2014, 21, 1415-1415.
6. M. Periasamy and A. Kalyanasundaram, Muscle & Nerve, 2007, 35, 430-
442.
7. J. A. Rosado, P. C. Redondo, J. A. Pariente and G. M. Salido, Cancer
Ther., 2004, 2, 263-270.
8. B. Papp, J.-P. Brouland, A. Arbabian, P. Gelebart, T. Kovacs, R. Bobe, J.
Enouf, N. Varin-Blank and A. Apati, Biomolecules, 2012, 2, 165-186.
9. G. R. Monteith, D. McAndrew, H. M. Faddy and S. J. Roberts-Thomson,
Nat. Rev. Cancer 2007, 7, 519-530.
causes ER stress-associated apoptosis in MDA-MB-231 cells. This
2
2
2
2
2
work highlighted a new strategy to find the cellular target of
bioactive natural product more accurately through the combination
of affinity-based proteome profiling and fluorescent imaging and
could help to understand the antitumor activity of WA and the
related derivatives.
This research was financially supported by the Open Project of
State Key Laboratory of Natural Medicines (No. SKLNMZZCX
2
8
0
01815), the National Natural Science Foundation of China (No.
1872983) and the Drug Innovation Major Project (2018ZX09711- 30. L. Wang, L. Wang, R. Song, Y. Shen, Y. Sun, Y. Gu, Y. Shu and Q. Xu, Mol.
01-007).
Cancer Ther., 2011, 10, 461-471.
3
3
1. S. B. Christensen, D. M. Skytte, S. R. Denmeade, C. Dionne, J. V. Moller,
P. Nissen and J. T. Isaacs, Anti-Cancer Agent. Me., 2009, 9, 276-294.
2. S. Orrenius, B. Zhivotovsky and P. Nicotera, Nat. Rev. Mol. Cell Bio.,
Notes and references
2
003, 4, 552-565.
1
2
3
4
5
6
.
.
.
.
.
.
J. Khazir, D. L. Riley, L. A. Pilcher, P. De-Maayer and B. A. Mir, Natural
product communications, 2014, 9, 1655-1669.
M. S. Butler, A. A. Robertson and M. A. Cooper, Nat. Prod. Rep., 2014,
3
1, 1612-1661.
G.-X. Yang, G.-L. Ma, H. Li, T. Huang, J. Xiong and J.-F. Hu, Chin. J. Nat.
Medicines, 2018, 16, 881-906.
Y. S. Lin, H. C. Chiang, W. S. Kan, E. Hone, S. J. Shih and M. H. Won, Am.
J. Chin. Med., 1992, 20, 233-243.
A. G. Damu, P.-C. Kuo, C.-R. Su, T.-H. Kuo, T.-H. Chen, K. F. Bastow, K.-H.
Lee and T.-S. Wu, J. Nat. Prod., 2007, 70, 1146-1152.
H. I. F. Magalhaes, M. L. Veras, M. R. Torres, A. Alves, O. D. L. Pessoa, E.
R. Silveira, L. V. Costa-Lotufo, M. O. de Moraes and C. Pessoa, J. Pharm.
Pharmacol., 2006, 58, 235-241.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C9CC03653A
Find the target of Withangulatin A with the combination of fluorescent probes and
chemical proteomics.