Esterase-Activatable Synthetic M+/Cl? Channel Induces Apoptosis and Disrupts Autophagy in Cancer Cells

Article abstract of DOI:10.1002/chem.202002964

The formation of a supramolecular synthetic M⁺/Cl^? channel in the membrane phospholipid bilayer has been reported upon activation of a methyl pivalate-linked N¹,N³-dialkyl-2-hydroxyisophthalamide by esterases. The channel formation induces apoptosis in cancer cells via the intrinsic pathway. Interestingly, the supramolecular channel was also shown to disrupt autophagy in cancer cells by causing alkalization of lysosomes – a feature that has been confirmed at the cellular and protein level.

Full text of DOI:10.1002/chem.202002964

Accepted Article
Accepted Article
Title: Esterase Activatable Synthetic M+/Cl‒ Channel Induces
Apoptosis and Disrupts Autophagy in Cancer Cells
Authors: Javid Ahmad Malla, Virender Kumar Sharma, Mayurika Lahiri,
and Pinaki Talukdar
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202002964
Link to VoR: https://doi.org/10.1002/chem.202002964
01/2020

10.1002/chem.202002964
Chemistry - A European Journal
COMMUNICATION
Esterase Activatable Synthetic M+/Cl‒ Channel Induces Apoptosis and
Disrupts Autophagy in Cancer Cells
Javid Ahmad Malla,[a] Virender Kumar Sharma,[b] Mayurika Lahiri[b] and Pinaki Talukdar[*,a]
Abstract: The supramolecular esterase activatable synthetic M+/Cl‒
ion channel is reported which can form self-assembled rosette ion
channel in the bilayer membrane by the action of esterase enzymes.
The channel formation switches the cancer cells to apoptosis via an
intrinsic pathway. The more interesting feature is that along with
apoptosis this system disrupts autophagy in cancer cells via
alkalization of lysosomes, which was confirmed through cell imaging
and western blot analysis.
important in the development of biomedicine.[12] According to
them, the extracellular activation is important when cell
impermeable substrates are used as prodrugs and are activated
by extracellular machinery. In contrast, intracellular activation is
required when intracellular machinery is targeted for activation.
Jeong and coworkers reported a series of glycoside tethered
urea molecules as enzyme-responsive procarriers.[10] However,
the crucial limitation of these systems is associated with their
choice of enzymes for activation. It is well-known that the levels
of intracellular glycosidases are higher compared to their
Cell death plays
a
pivotal role in the normal
extracellular levels.[13] As
a result, the release of active
pathophysiological processes in the biological systems.
Dysregulation of cell death results in pathogenesis of a wide
range of diseases like neurodegenerative diseases and
cancer.[1] Three classical forms of cell death include necrosis,
apoptosis, and autophagy. The necrosis is often considered as
accidental cell death while as apoptosis is a well-known
programmed pathway to regulate normal functioning by wiping
out the damaged cells.[2] In contrary autophagic cell death (ACD)
refers to cell death that is inhibited via specific blockage of the
autophagic pathway. The cancer cells have decreased
apoptosis rate and high autophagy rate than normal cells,
helping cancer cells to proliferate at high rate. The anticancer
treatments engage autophagy as a cytoprotective answer in
response to toxic dose to mitigate cellular stress, whereas in
some cases the results are opposite. So, the apoptosis-inducing
agents such as e.g. obatoclax, cannabinoids, etc which target
autophagy have great applications to evade cancer-related
problems.[3],[4] Obatoclax is an analog of prodigiosin, and its
mechanism of action is linked to the alkalization of lysosomes,
thereby, disturbing the autophagy process.[5] The synthetic ion
transporters have apoptosis inducing activity in cancer cells.[2b, 6]
However, synthetic transporters that play a dual role in inducing
apoptosis and disrupting autophagy are very rare.[2b, 7]
transporters was done by adding the respective enzymes in the
extracellular media. Moreover, if applied in vivo these water
soluble protransporters are expected to be excreted easily by
the kidney.
Therefore, we considered a strategy for activating amphiphillic
protransporters by intracellular enzyme, and selected esterase
enzyme for activating the synthetic channel-mediated ion
transport. This enzyme is overexpressed in cancer cells,[14] and
therefore, applied significantly in the activation of prodrugs and
drug delivery.[15] The inactive form 1 of the channel forming
molecule was designed by connecting methyl pivalate at the C-2
position of N1,N3-dihexyl-2-hydroxyisophthalamide 2 so that the
system fails to proper self-assembly (Figure 1). We have
recently demonstrated the ion channel formation by 2 activated
by glutathione.[11a] The ion channel activation by glutathione was
confirmed using MCF-7 and INS-1E cells. The detailed
mechanistic study corroborated to the apoptosis of MCF-7 cells
due to the disruption of ion homeostasis. Therefore, we realized
that upon reaction with intracellular esterases, methyl pivalate
moiety of compound 1 would be cleaved[16] releasing 2 that
would form self-assembled M+/Cl‒ ion channels in the membrane
resulting in cancer cell death via the perturbation of ion
homeostasis. Therefore, the strategy would
Very recently, considerable efforts have been invested in
the design of stimuli-responsive ion transporters aiming to target
cancer cells selectively. In this regard pH,[8] light,[9] enzymes,[10]
glutathione,[11] etc. have drawn significant attention. Rotello and
coworkers reported that the site of activation of prodrugs is very
[a] J. A. Malla, Prof. P. Talukdar
Department of Chemistry
Indian Institute of Science Education and Research Pune
Dr. Homi Bhabha Road, Pashan, Pune 411008, Maharashtra (India)
E-mail: ptalukdar@iiserpune.ac.in
[b] V. K. Sharma, Dr. M. Lahiri
Department of Biology
Indian Institute of Science Education and Research Pune
Dr. Homi Bhabha Road, Pashan, Pune 411008, Maharashtra (India)
Supporting information for this article is given via a link at the end of the
document
Figure 1. Representation of esterase activatable M+/Cl− channel (A). Structure
of inactive ester form 1 (T-Est) and channel forming form 2 (T) (B).
This article is protected by copyright. All rights reserved.

10.1002/chem.202002964
Chemistry - A European Journal
COMMUNICATION
provide a new being higher in lysosome compared to the
cytoplasm,[17] an activation of such ion transport across
lysosomal membranes would also perturb the functions of these
organelles. So the channel can act as a double-edged sword for
inducing apoptosis and disrupting autophagy in cancer cells.
compound (IC50 value of 15 µM for MCF-7 cells) compared to
our previously reported glutathione activatable compound (IC50
value of 1 µM for MCF-7 cells)[11a] may be corroborated to (i)
better permeability of the 2,4-dinitrobenzenesulfonyl (DNS)
linked protransporter and (ii) better cleavability of DNS group
(i.e., faster activation. The effect of in situ generated by-products
(formaldehyde and pivalic acid as 4-methoxyphenol is proved to
be non-toxic to cells)[20] on cell viability was evaluated
independently in MCF-7 cells and the compound 6 showed
negligible cytotoxicity compared to 1 (Figure S2).
The synthesis of 1 was carried out by reaction of compound 2
with iodimethyl pivalate presence of triethylamine[18] (Scheme
S1, ESI). The compound was characterized by 1H NMR, 13C
NMR, and high-resolution mass spectrometry.
After the successful synthesis of compound 1, the release of
compound 2 from 1 upon enzymatic cleavage was studied using
1H NMR studies. The compound 1 (1 mM) in phosphate buffer
saline (PBS) was treated with 4 U/mL of porcine liver esterase
enzyme (Sigma Aldrich) prepared in PBS at pH 7.2 for different
time intervals. The disappearance of the peak for Hbʹ at d = 5.6
ppm, the shift of peak for Ha at d = 8.0 ppm to 7.98 ppm as well
as the reappearance of the peak for Hc around 14.4 ppm was
monitored and it was found that almost 95% of the cleavage
takes place within three hours of reaction time (Figure 2). These
results suggest the release of 2 from compound 1 is a very
feasible process when incubated in cells due to higher
expression of esterases.
Figure 3. Schematic representation of assay (A) and comparison of Cl- ion
influx into EYPC-LUVsÉlucigenin by compounds 2 and 1 treated with esterase
enzyme for specific time intervals (B).
The above results encouraged us to further evaluate the
mechanism of cell death i.e., whether cell death is being
mediated by necrosis or apoptosis. We evaluated the apoptotic
pathway starting with monitoring the mitochondrial membrane
potential (MMP) change on treated cells using JC-1 dye.[21] The
enhanced green fluorescence in treated cells compared to
control cells indicated that the MMP is damaged (Figure 5A, B,
Figure S3). This change in MMP leads to disturbances in the
electron transport chain in the mitochondrial respiratory cycles,
which ultimately results in the generation of reactive oxygen
species (ROS).[22] Therefore, we monitored the ROS produced in
the mitochondria using mitosox[23] (Figure S4) and overall
cellular ROS using H2DCFDA probes[24] (Figure S5, S6). These
results indicated the production of ROS in the cells treated with
compound 1.
Figure 2. Esterase mediated release of 2 from 1 monitored by 1H NMR
experiments.
The ion transport properties of 1 and 2 were evaluated using
lucigenin based fluorescence assay using large unilamellar
vesicles (LUVs) composed of egg-yolk phosphatidylcholine
(EYPC).[19] The compound 1 was found to be almost inactive
than compound 2 for ion transport at identical concentrations
(Figure 3). However, upon treatment with esterase enzyme, the
transport activity was regained showing the efficient release of
the active channel forming compound 2 from 1. These studies
encouraged us to further carry out the cellular experimentations.
The compound 1 was incubated in the human epithelial breast
cancer cells MCF-7, for 30 min and then the cells were observed
under
a confocal microscope (Leica SP8). The results
suggested that there is an enhancement of blue fluorescence in
the cells (in the cytoplasm and near the membrane) incubated
with 1 compared to control (Figure 4) suggesting that compound
2 gets released from 1 by intracellular esterases.
Figure 4. Live cell imaging of MCF-7 cells upon incubation with 0 μM (A) and
15 μM (B) of 1. Dose-dependent viability of MCF-7 and HeLa cells incubated
with 1 evaluated by MTT assay (C).
The results of the cell-imaging assay encouraged us to
evaluate the cell viability of cancer cells in the presence of
compound 1. MCF-7 cells and HeLa cells were incubated with
compound 1 in a dose-dependent manner for 24 hours and then
the cell viability was evaluated by MTT assay.[6b] The results
suggested that compound 1 displayed enhanced cytotoxicity
with increase in concentration of the compound giving IC50
values of 15 µM and 20 µM for MCF-7 and HeLa cells
respectively. The higher IC50 value of the esterase activatable
It is reported that the intrinsic apoptotic pathway initiates from
the mitochondria and releases cytochrome c which binds to the
Apaf-1 to form an apoptosome.[6b] The apoptosome activates the
caspase 9 pathway, which switches the cells to apoptosis.
Therefore, to confirm the apoptotic pathway is functional we
This article is protected by copyright. All rights reserved.

10.1002/chem.202002964
Chemistry - A European Journal
COMMUNICATION
intracellular esterase enzymes. The successful activation of ion
channel inside cells leads to ion transport across the plasma
membrane, which switches the cells to apoptosis through the
mitochondrial pathway. Furthermore, this transport can dissipate
the pH gradient of the lysosomes, which leads to lysosomal
dysfunctions, and ultimately leads to autophagy disruption in
MCF 7 cells.
probed the activation of the caspase
9 pathway using
immunoblot analysis of MCF-7 cells upon treatment with
compound 1 (15 µM). GAPDH was used as the loading control.
The results showed an increase in expression of cleaved
caspase 9 (Figure 5E), which confirms the activation of the
intrinsic pathway of apoptosis. To further support, the expression
of cleaved poly(ADP-ribose) polymerase (PARP) was also
monitored, which showed a significant amount of degradation of
full-length PARP-1 (116 KDa) with a concomitant increase of
cleaved PARP-1 (86 kDa)[25] (Figure 5F) supporting activation of
the caspase 9 pathway of apoptosis.
Acknowledgements
The chloride concentration in lysosomes (80 mM) maintains
the lysosomal pH (4.5-5.0),[17] and any disturbance in this
chloride concentration leads to a change in the lysosomal pH,
which causes the disturbance in the activity of the lysosomal
enzymes, leading to autophagy disruption.[26] Therefore, to
evaluate the effect of compound 1 on lysosomes, we first
monitored the pH of lysosomes of treated cells using acridine
We are grateful to SERB, Govt. of India (EMR/2016/001897 to
P.T.; EMR/2016/001974 to M.L.), the Department of Science
and Technology (DST), Govt. of India (DST/INT/RUS/RSF/P-
24/C), and the Department of Biotechnology (DBT), Govt. of
India (BT/HRD/NBA/36/06/2018) for funding support.
orange (AO) dye.[27] AO is
a cell-permeable dye which
Keywords: Supramolecular chemistry • Ion Channel • Enzyme
accumulates in acidic compartments, such as lysosomes, and
shows a characteristic orange fluorescence emission while as
emits green fluorescence at higher pH, such as in the cytosol.
Upon treatment with 1 followed by AO staining the MCF-7 cells
showed complete loss of granular orange fluorescence (Figure
5G) compared to control (Figure 5F), indicating the alkalization
of lysosomes.[28]
The alkalization is known to be associated with a decrease in
the activity of lysosomal enzymes, hence, autophagy.[7]
Therefore, we probed the effect of compound 1 on autophagy
using immunoblot analysis. One of the markers of autophagy is
Lamin B1, which decreases during autophagy.[29] The treatment
of MCF-7 cells with 1 (15 µM) for 24 hours showed a marked
decrease in the expression of Lamin B1 (Figure 5H) compared
to control, indicating that autophagy is being induced. To further
corroborate this data, the expression of microtubule-associated
protein 1 light chain 3 (LC3) was monitored. Typically increased
expression of LC3 indicate the induction of autophagy in cells.[30]
responsive • Apoptosis • Autophagy
REFERENCES
[1]
[2]
Q. Chen, J. Kang, C. Fu, Signal Transduct. Tar. 2018, 3, 18.
(a) D. D. Newmeyer, S. Ferguson-Miller, Cell 2003, 112, 481-490; (b) N.
Busschaert, S.-H. Park, K.-H. Baek, Y. P. Choi, J. Park, E. N. W. Howe,
J. R. Hiscock, L. E. Karagiannidis, I. Marques, V. Félix, W. Namkung, J.
L. Sessler, P. A. Gale, I. Shin, Nat. Chem. 2017, 9, 667-675.
Z. Huang, Chem. Biol. 2002, 9, 1059-1072.
[3]
[4]
[5]
S. Fulda, Front. Oncol. 2017, 7, 128-128.
V. Kaushik, J. S. Yakisich, A. Kumar, N. Azad, A. K. V. Iyer, Cancers
2018, 10, 360.
[6]
(a) C. Ren, X. Ding, A. Roy, J. Shen, S. Zhou, F. Chen, S. F. Yau Li, H.
Ren, Y. Y. Yang, H. Zeng, Chem. Sci. 2018, 9, 4044-4051; (b)T. Saha,
A. Gautam, A. Mukherjee, M. Lahiri, P. Talukdar, J. Am.Chem. Soc.
2016, 138, 16443-16451; (c) J. A. Malla, R. M. Umesh, A. Vijay, A.
Mukherjee, M. Lahiri, P. Talukdar, Chem. Sci. 2020; (d) S.-K. Ko, S. K.
Kim, A. Share, V. M. Lynch, J. Park, W. Namkung, W. Van Rossom, N.
Busschaert, P. A. Gale, J. L. Sessler, I. Shin, Nat. Chem. 2014, 6, 885-
892.
The treatment of MCF-7 cells with
1 showed increased
expression of LC3 (Figure 5I) compared to control. Together,
these results suggest the activation of the autophagy process
upon treatment with compound 1.
[7]
S.-H. Park, S.-H. Park, E. N. W. Howe, J. Y. Hyun, L.-J. Chen, I. Hwang,
G. Vargas-Zuñiga, N. Busschaert, P. A. Gale, J. L. Sessler, I. Shin,
Chem 2019, 5, 2079-2098.
[8]
[9]
S. V. Shinde, P. Talukdar, Angew. Chem. Int. Ed. 2017, 56, 4238-4242;
Angew. Chem. 129, 4302-4306.
S. B. Salunke, J. A. Malla, P. Talukdar, Angew. Chem. Int. Ed. 2019, 58,
5354-5358; Angew. Chem. 131, 5408-5412.
[10] Y. R. Choi, B. Lee, J. Park, W. Namkung, K.-S. Jeong, J. Am.Chem.
Soc. 2016, 138, 15319-15322.
[11] (a) J. A. Malla, R. M. Umesh, S. Yousf, S. Mane, S. Sharma, M. Lahiri,
P. Talukdar, Angew. Chem. Int. Ed., 2020, 59, 7944-7952; Angew.
Chem. 132, 8018-8026 (b) N. Akhtar, N. Pradhan, A. Saha, V. Kumar,
O. Biswas, S. Dey, M. Shah, S. Kumar, D. Manna, Chem.Commun.
2019, 55, 8482-8485; (c) C. Lang, X. Zhang, Z. Dong, Q. Luo, S. Qiao,
Z. Huang, X. Fan, J. Xu, J. Liu, Nanoscale 2016, 8, 2960-2966.
[12] R. Das, R. F. Landis, G. Y. Tonga, R. Cao-Milán, D. C. Luther, V. M.
Rotello, ACS Nano 2019, 13, 229-235.
[13] M. J. Niedbala, R. Madiyalakan, K. Matta, K. Crickard, M. Sharma, R. J.
Bernacki, Cancer Res. 1987, 47, 4634-4641.
Figure 5. Live cell imaging of MCF7 cells upon treatment with 0 μM (A) and 15
μM (B) of 1 for 24 h followed by staining with JC-1 dye. Red and green
channel images were merged to generate the displayed image. Immunoblot
assay for PARP cleavage (C) in MCF7 cells, and active caspase 9 (D) after 24
h incubation with 0 μM and 15 μM of 1 with GAPDH as a loading control (E).
Live cell imaging of MCF 7 cells incubated with 1 at 0 μM (F), and 15 μM (G)
concentrations followed by staining with acridine orange. Immunoblot assay
for a decrease in L amin B1 expression (H) and an increase in LC3 expression
(I) in MCF 7 cells, with GAPDH as a loading control (J).
[14] C. A. McGoldrick, Y.-L. Jiang, V. Paromov, M. Brannon, K. Krishnan, W.
L. Stone, BMC Cancer 2014, 14, 77.
[15] (a) J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T.
Järvinen, J. Savolainen, Nat. Rev. Drug Discov. 2008, 7, 255-270; (b) K.
M. Huttunen, H. Raunio, J. Rautio, Pharmacol. Rev. 2011, 63, 750-771;
(c) J. B. Zawilska, J. Wojcieszak, A. B. Olejniczak, Pharmacol. Rep.
2013, 65, 1-14; (d) D. H. Jornada, G. F. Dos Santos Fernandes, D. E.
Chiba, T. R. F. De Melo, J. L. Dos Santos, M. C. Chung, Molecules
2016, 21, 42.
In summary, we have reported the novel concept of enzyme
activatable synthetic ion channel which can be activated by
This article is protected by copyright. All rights reserved.

10.1002/chem.202002964
Chemistry - A European Journal
COMMUNICATION
[16] P. Chauhan, P. Bora, G. Ravikumar, S. Jos, H. Chakrapani, Org. Lett.
2017, 19, 62-65.
[17] T. Stauber, T. J. Jentsch, Annu. Rev. Physiol. 2013, 75, 453-477.
[18] G. Ravikumar, M. Bagheri, D. K. Saini, H. Chakrapani, ChemBioChem.
2017, 18, 1529-1534
[19] J. A. Malla, A. Roy, P. Talukdar, Org. Lett. 2018, 20, 5991-5994.
[20] K. A. Pardeshi, G. Ravikumar, H. Chakrapani, Org. Lett. 2018, 20, 4-7.
[21] S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W.
Smith, G. D. Steele, L. B. Chen, Proc. Nat. Acad. Sci. 1991, 88, 3671-
3675.
[22] J. S. Armstrong, K. K. Steinauer, B. Hornung, J. M. Irish, P. Lecane, G.
W. Birrell, D. M. Peehl, S. J. Knox, Cell Death Differ. 2002, 9, 252-263.
[23] S. Yousf, D. M. Sardesai, A. B. Mathew, R. Khandelwal, J. D. Acharya,
S. Sharma, J. Chugh, Metabolomics 2019, 15, 55.
[24] S. Patil, M. M. Kuman, S. Palvai, P. Sengupta, S. Basu, ACS Omega
2018, 3, 1470-1481.
[25] A. H. Boulares, A. G. Yakovlev, V. Ivanova, B. A. Stoica, G. Wang, S.
Iyer, M. Smulson, J. Biol. Chem. 1999, 274, 22932-22940.
[26] A. Di, M. E. Brown, L. V. Deriy, C. Li, F. L. Szeto, Y. Chen, P. Huang, J.
Tong, A. P. Naren, V. Bindokas, H. C. Palfrey, D. J. Nelson, Nat. cell
Biol. 2006, 8, 933-944.
[27] V. Soto-Cerrato, P. Manuel-Manresa, E. Hernando, S. Calabuig-
Fariñas, A. Martínez-Romero, V. Fernández-Dueñas, K. Sahlholm, T.
Knöpfel, M. García-Valverde, A. M. Rodilla, E. Jantus-Lewintre, R.
Farràs, F. Ciruela, R. Pérez-Tomás, R. Quesada, J. Am. Chem. Soc.
2015, 137, 15892-15898.
[28] M. G. Palmgren, Anal. Biochem.1991, 192, 316-321.
[29] Z. Dou, C. Xu, G. Donahue, T. Shimi, J. A. Pan, J. Zhu, A. Ivanov, B. C.
Capell, A. M. Drake, P. P. Shah, J. M. Catanzaro, M. D. Ricketts, T.
Lamark, S. A. Adam, R. Marmorstein, W. X. Zong, T. Johansen, R. D.
Goldman, P. D. Adams, S. L. Berger, Nature 2015, 527, 105-109.
[30] K.-H. Baek, J. Park, I. Shin, Chem. Soc. Rev.2012, 41, 3245-3263.
This article is protected by copyright. All rights reserved.

10.1002/chem.202002964
Chemistry - A European Journal
COMMUNICATION
COMMUNICATION
Esterase cleavage of a
Javid Ahmad Malla,a Virender Kumar
Sharma,b Mayurika Lahirib and Pinaki
Talukdar*,a
protransporter leads to ion channel
formation in membrane. The activation
causes apoptosis and disrupts
autophagy in cancer cells.
Page No. – Page No.
Title: Esterase Activatable Synthetic
M+/Cl‒ Channel Induces Apoptosis
and Disrupts Autophagy in Cancer
Cells
This article is protected by copyright. All rights reserved.