Re-evaluation of the mechanism of cytotoxicity of dialkylated lariat ether compounds

Article abstract of DOI:10.1039/d0ra08494h

The cytotoxicity of dialkylated lariat ethers has been previously attributed to their ionophoric properties. Herein, we provide evidence that these effects are due to loss of membrane integrity rather than ion transport, a finding with important implications for the future design of synthetic ionophores.

Full text of DOI:10.1039/d0ra08494h

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Re-evaluation of the mechanism of cytotoxicity of
dialkylated lariat ether compounds†
Cite this: RSC Adv., 2020, 10, 40391
a
b
b
b
Willy Carrasquel-Ursulaez, Ryan D. Reeves, Mahzad Dehghany,
Corey Jones,
b
a
Jennifer M. Schomaker * and Baron Chanda*
Received 6th October 2020
Accepted 30th October 2020
The cytotoxicity of dialkylated lariat ethers has been previously attributed to their ionophoric properties.
Herein, we provide evidence that these effects are due to loss of membrane integrity rather than ion
transport, a finding with important implications for the future design of synthetic ionophores.
DOI: 10.1039/d0ra08494h
rsc.li/rsc-advances
1,2
Since the rst report of their synthesis, crown ethers and their diaza(18-crown-6) ethers, a subset of the lariat class, have been
derivatives have generated wide interest due to their ability to reported to have toxic activity towards prokaryotic and eukary-
2
20
form stable complexes with cations. These properties have otic cells. Evidence from toxicity and depolarization assays
been successfully exploited in ion transport through bulk liquid initially suggested that these compounds behave as ion
3,4
5
20
membranes as well as in sensors and scaffolds for materials,
carriers. However, experiments in asolectin bilayers revealed
developments which prompted their examination as biologi- that dioctylated and diundecylated lariat ethers elicit discrete
6–8
21
cally relevant ionophores.
The transport of ions through increases in membrane conductance, a result typical of ion
biological membranes underlies many key physiological channels, as opposed to ion carriers. Moreover, the effect of the
9–18
processes
and understanding the complexities of this alkyl chain lengths on the toxicity and transport implied that
phenomenon continues to be an area of active research. Crown the interaction of these compounds with a bilayer membrane
ethers are potentially powerful tools in this pursuit, due to their differs from their behaviour in a bulk liquid membrane. In
binding properties and highly customizable structures. Indeed, general, the literature suggests that hydrophobic lariat ethers
19
crown ether derivatives, such as monoalkylated and dialky- that bear longer alkyl chains function as more efficient cation
2
0,21
20,30
lated lariat ethers,
tives,
amphiphilic benzo(crown) ether deriva- carriers.
In contrast, dialkylated lariat ethers show peak
22
23
24
hydraphiles,
and ion shuttles
have been activity when a 10 carbon chain is present on the core, with the
20
demonstrated to function as ionophores. Despite these activity diminishing with increasing chain length. This
successful examples, our understanding of the mechanisms of observation prompted Leevy and co-workers to propose that
ion transport by these crown ether derivatives remains dialkylated lariat ethers require a minimum hydrophobicity to
extremely limited. An improved understanding of these act as ion carriers, but when the alkyl chains are too long, the
processes will provide critical insights that will both advance molecules are able to nest inertly within the membrane. In
fundamental knowledge about ion transport mechanisms and order to test this mechanism and establish a deeper under-
provide a framework for the rational design of synthetic iono- standing of their transport behavior in membranes, we focused
20
phores with well-dened properties.
on a representative set of lariat ethers bearing dialkylated tails
Towards this aim, we identied previously reported dialky- ranging from 6 to 14 carbons.
20,21
lated diaza crown ethers
as an ideal starting point. Lariat
The series of dialkylated diaza(18-crown-6) ether were
ethers are crown ether analogues with one or more sidearms prepared by a simple one-step reductive amination of 4,13-
25
attached to the macrocyclic core structure. The possibility of diaza(18-crown-6) and the appropriate aldehyde (Fig. 1;
adding customized pendant groups to the crown ether core Methods in ESI†), as opposed to previous two-step proce-
2
0,21
allows a high degree of selective modication to their physical dures.
n
The notation LEC refers to the dialkylated lariat
25
parameters. Lariat ethers have also been reported to bind ether substituted with an alkyl chains of n carbons. The cyto-
alkali cations and behave as ionophores in bulk liquid toxicity was determined by measuring the minimum inhibitory
2
6–29
membranes and ion-selective electrodes.
The dialkylated concentrations (MIC) for this series of dialkylated lariat ethers
in the Gram-negative bacteria Escherichia coli, the Gram-positive
bacteria Bacillus subtilis, and human embryonic kidney
a
Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri
(HEK293T) cells. The results showed that B. subtilis is more
6
3110, USA. E-mail: bchanda@wustl.edu
susceptible to LEC –LEC , as judged by lower MICs; LEC is
b
6
14
10
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
the most toxic to E. coli (MIC ¼ 10 mM), while LEC10, LEC11, and
LEC12 are the most toxic to B. subtilis at concentrations as low as
2 mM (Fig. S1†), results that are in good agreement with similar
E-mail: schomakerj@chem.wisc.edu
Electronic supplementary information (ESI) available: Methods, NMR and MS
data, MIC determination, DiSC (5) release assays. See DOI: 10.1039/d0ra08494h
†
3
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ionophore-like transport mechanism. Thus, some degree of ion
26–29
selectivity is expected,
as observed in the case of valinomy-
cin, a natural carrier-type ionophore that is extremely selective
+
+
39,40
for K over Na
and does not transport N-methyl-D-gluc-
+
amine (NMDG ). When depolarization assays were performed
in the presence of different cations, valinomycin promoted
+
DiSC
3
(5) release only when K was added to the external solu-
+
+
tion, but not when Na or NMDG was added (Fig. 2a and b).
Conversely, when LEC10 was tested under the same conditions,
Fig. 1 Synthesis of dialkylated diaza(18-crown-6) ethers compounds.
no signicant differences in relative DiSC (5) release were
3
observed (Fig. 2c and d). Furthermore, our control experiments
2
0
show that even in the absence of the alkali cation (Fig. S2a–i,
studies (Fig. S1, ESI†). We also conrmed a remarkable
3
and S3;† black curves), the relative DiSC (5) release rates were
discontinuity in the toxicity between LEC12 and LEC , where
the addition of only a single methylene group to the alkyl chain
completely abrogates the toxicity towards B. subtilis from a MIC
2 mM with LEC12 to undetectable with LEC13 at concentration
as high as 400 mM. The toxicities of dialkylated lariat ethers
towards HEK293T cells were more consistent than the trends
with E. coli and B. subtilis; however, LEC10 (MIC ¼ 6 mM) proved
to be the most toxic lariat ether towards all three tested cellular
systems.
Dialkylated lariat ethers LEC
their ability to depolarize a B. subtilis membrane using the
uorescent dye 3,3-dipropylthiadicarbocyanine (DiSC (5)),
which undergoes membrane voltage-dependent partitioning
13
+
similar to those observed in the presence of K (Fig. S2a–i and
S3;† violet curve). This unexpected result forced us to consider
two possible explanations. Either the dialkylated lariat ethers
behave as non-selective ionophores that are capable of trans-
¼
+
porting large cations, such as NMDG , or their primary effect is
to disrupt membrane integrity, i.e. the DiSC (5) efflux is due to
3
the lysis of the cells, rather than ion transport across the
membrane.
+
To determine whether transport of NMDG can account for
6
–LEC14 were then tested for
3
the observed efflux of DiSC (5) from cells, we tested LEC10
activity in a cation-free dextrose solution (see Methods, ESI†). In
this experiment, the only cation in the external solution (if any)

3
31
between the intracellular and the extracellular medium. Cell
hyperpolarization (more negatively charged inside the cell)
results in an uptake of the dye, while cell membrane depolar-
ization (more positively charged inside the cell) results in
a release of the dye. The accumulation of the dye in the interior
of the cell can be detected by a decrease in uorescence due to
31
self-quenching, which enables the dye to be utilized as an
32
indirect reporter of changes in cell membrane voltage. As the
resting membrane voltage in B. subtilis is approximately
3
3
ꢀ
120 mV, DiSC (5) quickly accumulates inside intact bacteria
3
(
(
timepoint t in Fig. S2a–i†). The addition of up to 60 mM KCl
violet curve, timepoint t3 in Fig. S2a–i†) does not cause
2
substantial membrane depolarization, as determined by the
limited increase in uorescence, mainly because the endoge-
+
34–38
nous K transporters and channel activities
2
are inhibited at
ꢁ
5 C. However, upon addition of the dialkylated lariat ether
4
(timepoint t in Fig. S2a–i†), ions move down the electro-
chemical gradient and give rise to membrane depolarization, as
evidenced by an increase in uorescence. The effects of the alkyl
chain length on the relative DiSC (5) release aer 10 minutes
3
following addition of the dialkylated lariat ether (Fig. S3†) were
20
qualitatively similar to those reported previously. The dialky-
lated lariat ethers with the highest toxicities elicited faster
Fig. 2 Comparison between the selectivity of valinomycin and LEC10
:
+
DiSC
3
(5) efflux in the presence of K , suggesting they are more
(
a) normalized changes in DiSC (5) fluorescence due to the activity of 2
3
efficient at transporting cations (Fig. S2a–i and S3; † violet mM valinomycin; events in green are dye additions (t ), B. subtilis cells
1
curves). As the induction of membrane depolarization is (t
2
), concentrated salts up to 60 mM KCl (violet), NaCl (orange) or
6
0 mM NMDG-Cl (black) (t ), and valinomycin up to 2 mM (t ); (b)
3
4
consistent with an ionophoric mechanism, the next step was to
fluorescence values at the end of the experiment, as shown in panel
a (average ꢂ S.E.M); (c) normalized changes in DiSC (5) fluorescence
due to the activity of 2 mM LEC10; the experiment is identical to that in
determine the cation selectivity exhibited by this class of the
compounds.
3
Binding to ionophores requires at least a partial substitution panel a, except LEC10 was added in t ; (d) fluorescence at the end of
4
the experiment as shown in panel c (average ꢂ S.E.M). A minimum of
of water molecules in the hydration sphere by ions to achieve an
three experiments for each condition were averaged in panels b and d.
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is added at timepoint t (Fig. S4a†). Addition of 2 mM LEC10
produced a large DiSC (5) release in the presence of KCl (Fig.-
3
S4a,† violet curve), but interestingly, this same effect was also
observed in the absence of any external cation (Fig. S4a and b,†
black curve; additional dextrose solution was added at time-
point t ). The independence of the DiSC (5) efflux from the
4 3
identity or the presence of the external cations is not compatible
with an ionophore-like mechanism.
Previous studies indicated that lariat ethers reported to
display the ability to collapse membrane potential in depolar-
ization assays also exhibited ion channel-like activity in bila-
yers. This suggested that we might employ similar
depolarization assays to serve as a convenient surrogate for
measuring electrical activity. We tested our most potent
compound, LEC10, for ion channel activity in planar lipid bila-
yers. As a control, unitary channels of the ionophore gramicidin
were recorded in the presence of a KCl solution (150 mM)
(Fig. S5a†). However, no ion channel activity was detected in
a similar experiment performed using 2 mM LEC10 (Fig. S5b†),
even aer one hour of recording. Variations in the LEC10
concentration (10, 100, and 200 mM), as well as the KCl
concentration (up to 500 mM), including replacing KCl with
NaCl (see Methods, ESI†), resulted in no indication of ion
Fig. 3 Lactate dehydrogenase release from B. subtilis in response to
LEC10 treatment: (a) two coupled redox reactions lead to increased
fluorescent resofurin concentration when lactate dehydrogenase
channel activity. This lack of unitary channel formation, or even (LDH) is released from lysed cells; (b) normalized time course of the
a carrier-like increase of the conductance, precludes an resofurin fluorescence; events in green are additions of resazurin (t );
), and up to 2 mM LEC10 (magenta
curve) or the same volume of TFE (control, black curve) (t ); (c)
1
2 3
B. subtilis cells (t ), diaphorase (t
ionophore-like mechanism of ion transport.
4
Given our ndings that this class of dialkylated lariat ether
derivatives do not act as typical ionophores, we next considered
the possibility that the activity of these compounds results from
disruption of membrane integrity. A well-established assay was
utilized to measure cell lysis by monitoring the release of the
enzyme lactate dehydrogenase (LDH), which is ubiquitous in
the cytoplasm of all cell types. The tetrameric active form of
LDH catalyzes the nal step of the glycolysis in B. subtilis and
normalized resofurin fluorescence at the end of the experiment as
shown in panel b (average ꢂ S.E.M).
of their behaviour as ionophores for cell membranes. The acute
release of LDH, a complete lack of ion specicity, depolarization
in the absence of extracellular ions, and a lack of discrete
changes in the conductance in the planar lipid bilayers solidly
demonstrates that biological activities of these lariat ethers are
due to their membrane lytic activity, as opposed to the expected
ion transport activity. Our ndings warrant re-evaluation of the
mechanisms of activity of many previously reported synthetic
ionophores, which have been classied as such based only on
cell survival assays and depolarization assays, without attention
to detailed electrical activity measurements. We also note that
even in cases where ionophores do not show electrical activity,
due to transport via carrier type mechanisms, our control
experiments are able to discriminate between ion carrier and
lytic activity.
41,42
has a molecular weight of approximately 146 kDa in,
with
ꢀ
a nearly globular shape of an approximated radius of 80 A (PDB
ID: 3PQD). The release of proteins of this size demonstrates the
test compounds are likely to cause cell lysis but we cannot rule
out the possibility that they form large pores. LDH couples two
redox reactions, the rst involving the interconversion of
pyruvate (oxidized) and L-lactate (reduced) and the second, the
+
interconversion of NAD (oxidized) and NADH (reduced). The
NADH oxidation can be coupled to the diaphorase-catalyzed
oxidation of resazurin to resofurin, which is highly uorescent
+
(
Fig. 3a). When the concentrations of L-lactate, NAD , diapho-
rase, and resazurin are saturated, the rate of increase in the
resofurin uorescence is limited only by the amount of available
LDH in the medium. Our experiments show that the addition of
LEC10 to a mixture containing B. subtilis cells and the other
Funding
components necessary for the LDH assay (see Methods, ESI†) The NMR and Mass Spectrometry facilities are funded by the
causes a rapid rise in the uorescence (Fig. 3b and c), indicating NSF (CHE-1048642, CHE-0342998, CHE-9304546 and CHE-
loss of membrane integrity. In contrast, the addition of the 9208463), the UW-Madison as well as a generous gi by Paul
same amount of the triuoroethanol (TFE) solvent used to J. and Margaret M. Bender. We thank Prof. A Prindle and Peter
dissolve LEC10 did not produce any increase in the uorescence Tran (Northwestern University) for generously sharing B. subtilis
(
Fig. 3b and c).
In summary, we report one-step syntheses of dialkylated Cowgill for their generous help and training. This work was
diaza(18-crown-6) ether derivatives and an in-depth evaluation supported by funding from the UW2020 competition and the
strains. We are grateful to Dr Jiang, Dr Chowdhury and Dr
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NIGMS award 5R21GM131662 (B. C. and J. M. S.) and NINDS 22 T. Liu, C. Bao, H. Wang, Y. Lin, H. Jia and L. Zhu, Chem.
award R35NS116850 (B. C.).
Commun., 2013, 49, 10311–10313.
2
2
3 S. Negin, B. A. Smith, A. Unger, W. M. Leevy and G. W. Gokel,
Int. J. Biomed. Imaging, 2013, 2013, 1–11.
4 S. Chen, Y. Wang, T. Nie, C. Bao, C. Wang, T. Xu, Q. Lin,
D. H. Qu, X. Gong, Y. Yang, L. Zhu and H. Tian, J. Am.
Chem. Soc., 2018, 140, 17992–17998.
Conflicts of interest
There are no conicts to declare.
2
5 G. W. Gokel and L. Echegoyen, Bioorganic Chemistry
Frontiers, ed. H. Dugas, Springer Verlag Inc., 1990, pp.
Notes and references
1
2
C. J. Pedersen, J. Am. Chem. Soc., 1967, 89, 2495–2496.
C. J. Pedersen and H. K. Frensdorff, Angew. Chem., Int. Ed.,
116–141.
2
6 J. C. Ball, J. R. Allen, J. Y. Ryu, S. Vickery, L. Cullen,
P. Bukowski, T. Cynkowski, S. Daunert and L. G. Bachas,
Electroanalysis, 2002, 14, 186–191.
1972, 11, 16–25.
3
4
5
6
7
8
9
M. Shamsipur, R. Davarkhah and A. R. Khanchi, Sep. Purif.
Technol., 2010, 71, 63–69.
T. B. Stolwijk, E. J. Sudhoelter and D. N. Reinhoudt, J. Am.
Chem. Soc., 1987, 109, 7042–7047.
2
2
2
3
3
3
3
3
3
7 P. B u¨ hlmann, E. Pretsch and E. Bakker, Chem. Rev., 1998, 98,
1593–1688.
8 D. M. Dishong, C. J. Diamond, M. I. Cinoman and
G. W. Gokel, J. Am. Chem. Soc., 1983, 105, 586–593.
9 T. Mizuno, Y. Nakatsuji, S. Yanagida and M. Okahara, Bull.
Chem. Soc. Jpn., 1980, 53, 481–484.
0 J. C. Hernandez, J. E. Traon and G. W. Gokel, Tetrahedron
Lett., 1991, 32, 6269–6272.
1 P. J. Sims, A. S. Waggoner, C. H. Wang and J. F. Hoffman,
Biochemistry, 1974, 13, 3315–3330.
2 M. Wu, E. Maier, R. Benz and R. E. Hancock, Biochemistry,
999, 38, 7235–7242.
3 A. Zaritsky, M. Kihara and R. M. Macnab, J. Membr. Biol.,
981, 63, 215–231.
4 M. Fujisawa, Y. Wada and M. Ito, FEMS Microbiol. Lett., 2004,
31, 211–217.
5 J. Gundlach, C. Herzberg, V. Kaever, K. Gunka, T. Hoffmann,
M. Weiß, J. Gibhardt, A. Th u¨ rmer, D. Hertel, R. Daniel,
E. Bremer, F. M. Commichau and J. St u¨ lke, Sci. Signaling,
G. W. Gokel, W. M. Leevy and M. E. Weber, Chem. Rev., 2004,
104, 2723–2750.
W.-W. Tso and W.-P. Fung, Inorg. Chim. Acta, 1980, 46, L33–
L340.
W.-W. Tso and W.-P. Fung, Inorg. Chim. Acta, 1981, 55, 129–
1
34.
W.-W. Tso, W.-P. Fung and M.-Y. Tso, J. Inorg. Biochem., 1981,
4, 237–244.
1
D. L. Bennett, A. J. Clark, J. Huang, S. G. Waxman and
1
S. D. Dib-Hajj, Physiol. Rev., 2019, 99, 1079–1151.
10 E. Bourinet, C. Altier, M. E. Hildebrand, T. Trang,
1
M. W. Salter and G. W. Zamponi, Physiol. Rev., 2014, 94,
81–140.
2
11 M. Endo, Physiol. Rev., 2009, 89, 1153–1176.
12 A. Horowitz, C. B. Menice, R. Laporte and K. G. Morgan,
Physiol. Rev., 1996, 76, 967–1003.
1
1
1
1
1
1
1
2
3 E. Balse, D. F. Steele, H. Abriel, A. Coulombe, D. Fedida and
S. N. Hatem, Physiol. Rev., 2012, 92, 1317–1358.
4 M. N. Foster and W. A. Coetzee, Physiol. Rev., 2016, 96, 177–
2
017, 10, 1–9.
3
6 G. Holtmann, E. P. Bakker, N. Uozumi and E. Bremer, J.
Bacteriol., 2003, 185, 1289–1298.
7 J. Humphries, L. Xiong, J. Liu, A. Prindle, F. Yuan,
H. A. Arjes, L. Tsimring and G. M. S u¨ el, Cell, 2017, 168,
2
52.
5 N. Schmitt, M. Grunnet and S. P. Olesen, Physiol. Rev., 2014,
4, 609–653.
3
9
200–209.
6 L. Aguilar-Bryan, J. Bryan and M. Nakazaki, Recent Prog.
Horm. Res., 2001, 56, 47–68.
7 P. Rorsman and F. M. Ashcro, Physiol. Rev., 2018, 98, 117–
3
3
8 A. Prindle, J. Liu, M. Asally, S. Ly, J. Garcia-Ojalvo and
G. M. S u¨ el, Nature, 2015, 527, 59–63.
9 G. Eisenman and O. Alvarez, in Biomembrane Structure &
Function - The State of the Art, ed. B. P. Gaber and K. R. K.
Easwaran, Adenine Press, Schenectady, NY, 1992, pp. 321–
2
14.
8 J. R. M. Harvey, A. E. Plante and A. L. Meredith, Physiol. Rev.,
020, 100, 1415–1454.
2
3
51.
0 S. Varma, D. Sabo and S. B. Rempe, J. Mol. Biol., 2008, 376,
3–22.
1 N. Richter, A. Zienert and W. Hummel, Eng. Life Sci., 2011,
1, 26–36.
9 Q. Xie, Y. Li, G. Gokel, J. Hern ´a ndez and L. Echegoyen, J. Am.
Chem. Soc., 1994, 116, 690–696.
0 W. M. Leevy, M. E. Weber, M. R. Gokel, G. B. Hughes-
Strange, D. D. Daranciang, R. Ferdani and G. W. Gokel,
Org. Biomol. Chem., 2005, 3, 1647–1652.
4
4
4
1
1
2 Y. Zhang and X. Gao, Acta Crystallogr., Sect. F: Struct. Biol.
Cryst. Commun., 2012, 68, 63–65.
21 S. Negin, M. B. Patel, M. R. Gokel, J. W. Meisel and
G. W. Gokel, Chembiochem, 2016, 17, 2153–2161.
40394 | RSC Adv., 2020, 10, 40391–40394
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