Divalent Naphthalene Diimide Ligands Display High Selectivity for the Human Telomeric G-quadruplex in K+ Buffer

Article abstract of DOI:10.1002/chem.201700140

Selective G-quadruplex ligands offer great promise for the development of anti-cancer therapies. A novel series of divalent cationic naphthalene diimide ligands that selectively bind to the hybrid form of the human telomeric G-quadruplex in K⁺ buffer are described herein. We demonstrate that an imidazolium-bearing mannoside-conjugate is the most selective ligand to date for this quadruplex against several other quadruplex and duplex structures. We also show that a similarly selective methylpiperazine-bearing ligand was more toxic to HeLa cancer cells than doxorubicin, whilst exhibiting three times less toxicity towards fetal lung fibroblasts WI-38.

Full text of DOI:10.1002/chem.201700140

A Journal of
Accepted Article
Title: Divalent Naphthalene Diimide Ligands Display High Selectivity for
the Human Telomeric G-quadruplex in K+ Buffer
Authors: Steven Street, Donovan Chin, Gregory Hollingworth, Monica
Berry, Juan Carlos Morales, and M. Carmen Galan
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700140
Link to VoR: http://dx.doi.org/10.1002/chem.201700140
Supported by

Chemistry - A European Journal
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COMMUNICATION
Divalent Naphthalene Diimide Ligands Display High Selectivity for
+
the Human Telomeric G-quadruplex in K Buffer
[
a]
[b]
[c]
[a]
Steven T. G. Street, Donovan N. Chin, Gregory J. Hollingworth, Monica Berry, Juan C.
[
d]
[a]
Morales* and M. Carmen Galan*
Abstract: Selective G-quadruplex ligands offer great promise for the
development of anti-cancer therapies. A novel series of divalent
cationic naphthalene diimide ligands that selectively bind to the
+
hybrid form of the human telomeric G-quadruplex in K buffer are
described here. We demonstrate that an imidazolium-bearing
mannoside-conjugate is the most selective ligand to date for this
quadruplex against several other quadruplex and duplex structures.
We also show that a similarly selective methylpiperazine-bearing
ligand was more toxic to HeLa cancer cells than doxorubicin, whilst
exhibiting three times less toxicity towards fetal lung fibroblasts WI-
3
8.
G-quadruplexes are four-stranded secondary structures that
occur in guanine rich regions of DNA and are over-represented
in telomeres and gene promoter regions including oncogenes
1
and tumor suppressors. It has been proposed that stabilization
of these polynucleotide sequences by small molecules could
2
Figure 1. Structure of new NDI ligands 1-7.
lead to novel anticancer treatments. However, the development
of anti-cancer, drug-like, bioavailable ligands to selectively
stabilize a specific G-quadruplex structure is still a major
challenge, given the approximately 716,310 putative G-
stability of the G-quadruplex structure as well as hydrogen bond
7
3
to purines. Indeed, a few examples of carbohydrate-based G-
quadruplex forming sequences in the human genome . Another
quadruplex ligands point towards sugars able to bind to grooves
hurdle to G-quadruplex ligands reaching clinical development is
8
4
and/or loops of the quadruplex. We thus proposed that
their insufficient drug-like character. Whilst some progress has
glycosides should be both tolerated and useful binding motifs in
quadruplex ligand design.
been made, none of the G-quadruplex ligands entering clinical
5
trials have succeeded to date. Several molecules have been
To test our hypothesis, we decided to replace classical
charged amine-derived side chains with charged sugars on the
quadruplex ligands. We chose the naphthalene diimide (NDI)
scaffold since NDI-based compounds are some of the most
active and highly studied G-quadruplex ligands to date, such as
reported that can selectively stabilize one quadruplex structure
6
or topology over another. For instance, telomestatin analogue
6
c
TOxaPy is one of the most selective ligands discovered to date
+
for the human telomeric G-quadruplex in Na buffer. Another
ligand, N-methyl mesoporphyrin (NMM), can stabilize the human
9
+
tetra-substituted NDI MM41 developed by the Neidle group.
telomeric G-quadruplex in
K
buffer with no observed
+
Despite tri- and tetra-substituted NDI ligands having been
stabilization in Na buffer, although it binds to other parallel
1
0,11
6
a
extensively researched,
di-substituted NDI scaffolds have
quadruplexes.
1
1a,12
not yet been fully explored.
. Herein we report a highly
Work within the Morales group has shown that
carbohydrates can stack onto the G-C base pair, and affect the
modular synthesis of a small library of di-substituted NDI ligands
bearing charged carbohydrate moieties alongside classical and
non-classical charged groups, and their evaluation as selective
G-quadruplex ligands (Fig. 1) and their in vitro cell cytotoxic
profile in model systems.
[a]
Mr. S. T. G. Street, Dr. M. C. Galan
School of Chemistry, University of Bristol
Cantock’s Close, Bristol BS8 1TS (UK)
E-mail: m.c.galan@bristol.ac.uk
Dr. M. Berry
Initial efforts focused on targeting glucose- and mannose-
based NDIs. We hypothesized that the different stereochemical
presentation of the OH at C-2 and anomeric linkage at C-1
should yield useful SAR information on the role the carbohydrate
scaffold plays in binding and its ability to pick up additional polar
interactions. In addition, a methylimidazolium cationic group was
School of Physics, University of Bristol
HH Wills Physics Laboratory, Bristol, BS8 1TL (UK)
Dr. D. N. Chin
[b]
[c]
[d]
Novartis Institutes for Biomedical Research
250 Massachusetts Ave., Cambridge, Massachusetts 02139 (USA)
1
3
Dr. G. J. Hollingworth
introduced at C-6 of the glycosides to aid binding. As a control,
Novartis Institutes for Biomedical Research
Novartis Campus, CH-4002, Basel (Switzerland)
Dr. J. C. Morales
uncharged glucoside 1 was also targeted, alongside novel NDIs
4
and 5 bearing only the methylimidazolium group in the
Instituto de Parasitología y Biomedicina
Avenida del Conocimiento, s/n 18016, Armilla, Granada (Spain)
E-mail: jcmorales@iiq.csic.es
absence of the sugar but differing in chain length. Finally, di-
substituted methylpiperazine- and dimethylamine-containing 6
14
and 7 were also prepared, as these motifs had been previously
1
1a, b
Supporting information for this article is given via a link at the end of
the document.
reported as components of other NDI ligands.
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COMMUNICATION
With all the compounds in hand, G-quadruplex and duplex
DNA stabilization was evaluated in a FRET melting assay at a
range of concentrations (1-10µM), (Table 1, Fig. 2 and Tables
+
S1-3 in ESI). Sequences tested were F21T K (human telomeric
+
+
G-quadruplex in K buffer), F21T Na (human telomeric G-
+
quadruplex in Na buffer), F-Myc-T (c-Myc Pu-27 G-quadruplex)
and F10T (duplex DNA) (See ESI). Uncharged glucoside 1 did
not stabilize any of the DNA sequences, which is in accordance
with earlier observations that cationic charge is needed for
quadruplex DNA stabilization. Excitingly, all of the charged
ligands 2-6 showed different degrees of stabilization of a single
Scheme 1. Synthetic route to monosaccharide-NDIs.
+
G-quadruplex (F21T K ), with no stabilization of the antiparallel
+
F21T Na quadruplex observed, whilst exhibiting significantly
[
a]
Table 1. DNA stabilization in FRET melting assay.
lower stabilization of duplex DNA (F10T). This is somewhat
interesting, as it might be expected for smaller, linear
compounds such as 6 to be able to intercalate into duplex
+
+
Compound
F21T K
F21T Na
F-Myc-T
F10T
1
7
DNA. At high concentrations of ligand (e.g. 10 µM), some
binding to both the C-Myc quadruplex and to a lesser extent
duplex DNA is observed, however as the concentration of ligand
1
2
3
4
5
6
7
0.4 ± 0.1
2.9 ± 0.3
10.1 ± 0.3
1.3 ± 0.4
3.7 ± 0.3
9.8 ± 0.6
2.3 ± 0.5
-0.1 ± 0.5
-1.5 ± 0.4
-1.4 ± 0.3
-0.6 ± 0.5
-0.5 ± 0.4
-2.3 ± 0.3
-1.5 ± 0.2
-0.3 ± 0.4
0.9 ± 0.7
-0.3 ± 0.7
1 ± 0.4
0.5 ± 0.2
0.1 ± 0.2
-0.1 ± 0.1
0.4 ± 0.2
0.1 ± 0.1
0.3 ± 0.2
1.1 ± 0.1
+
decreases the selectivity for F21T K increases (see Fig. 2).
Remarkably,
at
1µM
imidazolium-mannoside
3
and
methylpiperazine 6 displayed a 10.1°C and 9.8°C stabilization
+
for the F21T sequence in K buffer respectively, with no
1.8 ± 0.8
0.5 ± 0.3
3.9 ± 0.2
observable binding to any other sequence.
mannoside 3 showed a much higher stabilization than glucoside
. This could be due to either differences in hydrogen bonding
Furthermore,
2
interactions (OH at C-2 in mannose is axial vs equatorial in
glucose), or the relative orientation of the glycoside due to their
anomeric linkage (axial vs equatorial). These compounds, as far
as we are aware, are the most selective compounds for the
[
a] Displayed as ΔTmax in °C.
1 µM ligand and 200 nM DNA
concentration. For further information, see ESI.
+
hybrid/parallel type K human telomeric G-quadruplex over the
3
3
3
3
F21T K+
F-myc-T
F10T
+
2
1
1
0
5
0
5
0
5
antiparallel Na equivalent, and in addition exhibit the highest
+
selectivity for the hybrid/parallel type K human telomeric G-
F21T Na+
quadruplex over the parallel type c-Myc promoter quadruplex.
Next, we sought to further investigate the quadruplex/duplex
DNA selectivity of the most interesting compounds 2, 3, 5 and 6,
by using a FRET competition assay with quadruplex forming
6
F21T K+
6
6
F-myc-T
F10T
6 F21T Na+
+
oligonucleotide F21T K and unlabeled ds26 competitor duplex
DNA (Tables S4-6 and Fig. S2-4 in ESI). In general, the
compounds displayed good quadruplex/duplex selectivity,
2
4
6
8
10
-
retaining
64-39 %
stabilization
even
at
a
1:217
[
Ligand] (µM)
quadruplex:duplex DNA ratio (See Fig. S2 in ESI). It is
noteworthy that the quadruplex stabilization effect for glycosides
Figure 2. Stabilization of various DNA quadruplex and duplex sequences as a
function of concentration by compound 3 and 6.
2
and 3 was less perturbed than for their non-carbohydrate
counterparts (5 and 6, Fig. S4 in ESI) suggesting that the former
might be more selective towards quadruplex sequences.
The synthesis of glyco-conjugates 2 and 3 was achieved in
1
5
14
four steps from azidoalkyl glycosides 8 and 9 (Scheme 1).
To further understand the binding and selectivity observed for
compounds 3, 5 & 6 with the human telomeric G-quadruplex,
Circular Dichroism (CD) titrations were performed (Fig. 3 and
Figs. S5-S10 in ESI) with unlabeled human telomeric
Selective mesylation of the C6-hydroxyl using MsCl and pyridine,
followed by S
imidazolium-bearing glycosides 12 and 13 in two steps and 23-
3% overall yield. Subsequent azido reduction by
N
2 displacement with methyl imidazole afforded
3
+
+
oligonucleotide telo23 in pH 7.2 and either 100 mM Na or K
hydrogenolysis followed by condensation with 1,4,5,8-
naphthalenetetra-carboxylic dianhydride (NTCDA) under basic
phosphate buffer. All three ligands appeared to cause significant
perturbation to the observed CD spectrum, indicating that these
compounds are able to induce changes to the conformation of
conditions afforded the desired NDI products
2 and 3.
Analogously, ligands 1 and 4-7 were obtained by reaction of
+
1
4
the oligonucleotide. As shown in Fig. 3, in K buffer and at low
NTCDA
with
either
aminoethyl
glucoside
or
1-
ligand concentrations (1-3 equivs. of 3) stabilization of the
hybrid-type topology was observed, as evidenced by the
negative band at 235 nm, a reduction in the shoulder at 270 nm
methylimidazolium amines S5 and S6 in ethanol (See ESI) and
1
6
3
-dimethylaminopropylamine
or 1-(3-aminopropyl)-4-methyl-
piperazine in toluene, respectively.
1
8
and an increase in the maximum at 290 nm.
As the
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Chemistry - A European Journal
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concentration of ligand increases (up to 30 equivs.), the
maximum at 290 nm and the shoulder at 270 nm reverses
direction. This effect could imply that these ligands appear to
a hydrogen bonding interaction between the protonated tertiary
amine and the phosphate in the groove.
The additional
interaction of the positively charged imidazolium with the
+
stabilize a hybrid-type topology of telomeric DNA in K buffer at
negatively charged phosphate of 3 in the groove is consistent
low concentrations, yet shift to stabilize a parallel-type topology
a
with its enhanced K over 6. Further work is still needed to fully
at higher concentrations, with mannoside 3 exerting the biggest
understand the precise mechanism of cell cytotoxicity for these
compounds and whether the observed activity is directly
+
shift in conformation. CD titrations in Na buffer, where the DNA
2
a,21
sequence displays an antiparallel type topology showed a shift
towards a hybrid-type topology (decrease at 240 nm, increase at
attributed to G-quadruplex based processes.
2
60 nm and 295 nm, Fig. S5). This would indicate that ligands in
ratio DNA : 3
1:0
:1
1:2
:3
1:4
:5
1:6
:7
1:10
:15
1:30 annealed
question still have some form of interaction with the antiparallel
G-quadruplex, though this does not appear to result in
stabilization of the structure in the FRET melting assay. Even in
the presence of telo23 without any buffer, 3 was able to induce
the formation of a hybrid type G-quadruplex (decrease in band
at 258 nm, increase in band at 288 nm, Fig. S10). These results
suggest that these NDI ligands can induce significant topological
changes to the quadruplex upon binding, and are possibly
interacting with a hybrid/parallel-type conformation of the human
telomeric G-quadruplex.
1
5
4
1
1
3
1
2
1
1
0
-1
-2
-3
2
20
240
260
λ (nm)
Figure 3. Circular Dichroism titration of 3 with telo23 K
280
300
Isothermal Titration Calorimetry (ITC) was then used to
obtain quantitative thermodynamic information on the binding of
+
+
3
, 5 & 6 to the telo23 K human telomeric G-quadruplex (Table 2
+
Table 2. Thermodynamics of binding of 3,5 & 6 to telo23 K measured by
ITC. K = association constant, N = stoichiometry, ΔG = Gibbs free energy
and Fig. S11-S13 in ESI. Thermodynamic analysis revealed that
the ligands differ in their binding affinity with a K of the order of
M , with imidazolium 5 having the largest K , followed by
a
a
change, ΔH = enthalpy change, ΔS = entropy change and T = temperature.
Error (in brackets) represents a confidence interval of 68.3%. For more
information, see ESI.
6
-1
1
0
a
mannoside 3 and then 6. Further analysis of the data revealed
that enthalpy was the main driving force for binding for all three
compounds tested. Entropic contributions were negative in each
case, although they were much less for mannoside 3 than for
imidazolium 5 or methylpiperazine 6. Cationic mannoside 3
Compound
3
5
6
6
-1
K
a
(× 10 M )
1.30
2.16
1.03
(
1.02,1.64)
(1.85,2.52)
(0.865,1.23)
+
showed the highest stabilization effect for F21T K by FRET
analysis and also exhibited the second highest ΔG values, with
the lowest entropic penalty of all three ligands investigated. This
suggests that the sugar side chain in the ligand can better
displace water molecules from the complex surface than the
other motifs. The ITC-derived binding stoichiometry for 5 and 6
showed approximately two ligands interacting with the
quadruplex, whilst the stoichiometry for 3 was 2.7. These
differences in binding thermodynamics between the different
ligands indicate that besides the NDI scaffold, the side chains
play an important role in binding to the oligonucleotide.
Furthermore, binding of imidazolium-glycoside 3, imidazolium 5
N
2.7 - 2.6
-8.34
2.0 - 1.9
-8.64
1.9 - 2.0
-8.20
ΔG (kcal/mol)
ΔH (kcal/mol)
-10.0
-13.9
-14.5
(-8.80,-11.9)
(-13.4,-14.4)
(-13.7,-15.4)
ΔS (cal/mol K)
-5.55
-17.6
298
-21.0
298
T (K)
298
+
and methylpiperazine 6 to telo23 K is greater than that of
cationic porphyrin TmPyP4 as measured by Bončina et al., and
of a similar magnitude as that of the quadruplex ligand Phen-
1
9
DC3.
Molecular docking simulations were used to try to understand
the binding mode of our ligands 2-6 with several of G-
quadruplex (g4) structures (parallel, antiparallel and 3+1 hybrid
human telomeric g4, and the Pu27T c-Myc g4) and with a NDI
2
0
derivative bound (PDB ID: 4DA3) using Molsoft ICM-Pro (Fig. 4,
Table S7-9 and Fig. S14-41). All of the compounds evaluated
exhibited the NDI end-stacking onto the quadruplex, with one
side chain pointing into a groove. Models of binding for both 3
and 2 with the parallel human telomeric G-quadruplex revealed
several putative hydrogen bonds and imidazolium stacking
interactions within this groove of the quadruplex, with one of the
carbohydrate side chains inserted. Compound 6 also displayed
+
Figure 4. Model of 3 bound to the parallel K human telomeric G-quadruplex
(based on PDB ID: 4DA3). See ESI for further details.
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COMMUNICATION
(
Fig. 5-B & 5-F), the uptake for 3 is much lower (Fig. 5-C & 5-G),
In order to evaluate the cell cytotoxicity profile of compounds
and a further reduction in fluorescence is registered after 16h
Fig. 5-D & 5-H). This suggests that the lack of cytotoxicity for
glyco-conjugate 3, despite showing selective G-quadruplex
1
-7 and doxorubicin as the benchmark, we compared 72 h
(
incubations with WI-38 (embryonic human lung fibroblasts),
HeLa (human cervical cancer cells), MDA-MB231 and MCF7
2
2
binding, could be attributed to poor cellular uptake. Further
work is still needed to fully understand the precise mechanism of
cell cytotoxicity for these compounds, and whether the observed
activity is directly attributed to G-quadruplex based
(
human breast cancer cells) over the range of 10 fM-100 µM
ligand, quantifying the number of live cells (Calcein fluorescence,
Table 3) and metabolic competence (Alamarblue, measure of
reductive metabolism, Table S10). While glyco-conjugates 1-3
did not appear to be toxic at concentrations up to 100 µM for all
the cell cultures tested, all other compounds in the class
appeared to exhibit varying degrees of cytotoxicity, with
increased toxicity towards cancer cells. HeLa cells were the
most susceptible of all the cancer cells screened e.g. 6 and 7
displayed 410 and 380 nM IC50 values respectively, a slight
improvement in potency when compared to doxorubicin (530
nM). Methylpiperizine 6 displayed the most potent in vitro
cancer-cell killing activity of all the compounds screened (e.g.
IC50 of 410 nM for HeLa and 810 nM for MDA-MB32 vs 2.28 µM
for the healthy WI-38 cells) (Table 3). It is important to highlight
that while doxorubicin displayed a two-fold selectivity for HeLa
over WI-38, 6 displayed a six-fold selectivity for HeLa over WI-
2
a,21
processes.
3
8, making it three times as selective. Compounds 4 and 5,
which did not show significant G-quadruplex stabilization, were
about one order of magnitude less toxic to HeLa, MCF-7 and
WI-38 than 6. A non-monotonic dose response was observed
when 4 and 5 were tested against MDA-MB231 cells and IC50
values could not be obtained, which suggest a different mode of
action.
[
a]
Table 3. Cytotoxicity of 1-7 and doxorubicin (Dox) after 72 h incubation.
WI-38
HeLa
MCF7
MDA-MB32
Dox
1.19
0.53
0.43
0.80
(
1.05,1.33)
(0.42,0.62)
(0.37,0.50)
(0.66,0.97)
1
2
3
4
>100
>100
>100
21.46
>100
>100
>100
>100
>100
>100
>100
>100
>100
n/m
6.23
52.76
(
(
16.39,28.10)
(4.35,8.92)
(38.41,72.48)
5
6
7
40.22
3.63
11.77
n/m
28.83,56.11)
(2.59,5.08)
(9.67,14.33)
2.28
0.41
3.09
0.81
(
(
2.10,2.47)
(0.35,0.47)
(2.83,3.38)
(0.66,1.01)
0.72
0.38
0.53
8.10
0.67,0.76)
(0.32,0.46)
(0.48,0.58)
(6.73,9.74)
[
a] Absolute IC50 values measured in µM using calcein fluorescence to
assess live cells, with 95 % confidence interval in brackets. For metabolic
effects (Alamar Blue Assay) see ESI. n/m = Non monotonic dose response
observed.
Taking advantage of the inherent fluorescence of our ligands,
confocal microscopy was used to assess the intracellular uptake
in HeLa cells for compounds 3 and 6 (100 µM) after 30 min. and
Figure 5. HeLa Cell uptake of 3 and 6 monitored by fluorescence
confocal microscopy. Fluorescence images and the merged bright-
field/fluorescence images of: A and E) untreated control; B and F) after
3
0 min exposure to 100 µM of 6; C and G) after 30 min exposure to 100
1
6
h
incubations. As shown in Fig. 5, while significant
µM of 3; D and H) after 16 h exposure to 100 µM of 3. For more
intracellular uptake is seen for methylpiperazine 6 after 30 min.
information, see ESI.
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COMMUNICATION
R. Lucas, E. Vengut-Climent, I. Gómez-Pinto, A. Aviñó, R. Eritja, C.
González, J. C. Morales, Chem. Commun. 2012, 48, 2991-2993; c) R.
Lucas, P. Peñalver, I. Gómez-Pinto, E. Vengut-Climent, L. Mtashobya,
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In conclusion, the modular synthesis and evaluation of a
series of di-substituted naphthalene diimide G-quadruplex
ligands that display high selectivity towards the hybrid-type
+
topology of the K human telomeric G-quadruplex is described.
We showed that imidazolium mannoside 3 is the most selective
+
ligand towards the F21T K quadruplex sequence to date.
Excitingly, compound 6, which is easily accessible in one step, is
more toxic than doxorubicin towards cancer cells whilst
exhibiting three times more selectivity. Our findings suggest that
lead divalent compounds 3, 5 and 6 provide an interesting
platform for further development and that charged carbohydrates
can be exploited as binding motifs that can be tuned to interact
with G-quadruplex grooves. These results represent an exciting
step towards developing more selective and bioactive G-
quadruplex ligands with potentially improved anti-cancer activity.
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Acknowledgements
STGS thanks Dr. A. Izaac (Novartis) for support with ITC, Dr. A.
Bailey (Bristol) for use of qPCR, NanoDrop UV-Vis
spectrophotometer and Plate Reader, Dr. A. Leard and the
Wolfson Bioimaging Facility (BB/L014181/1) for support with
Confocal Microscopy and EPSRC EP/G036764/1 for a PhD
studentship. MCG thanks the EPSRC EP/J002542/1 and ERC-
COG 648239 for funding. JCM thanks Ministerio de Economía y
Competitividad (CTQ2012-35360 and CTQ2015-64275-P).
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Chemistry - A European Journal
10.1002/chem.201700140
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Get into the groove… A series of
divalent naphthalene diimide ligands
selectively bind to the human telomeric
Steven T. G. Street, Donovan N. Chin,
Gregory J. Hollingworth, Monica Berry,
Juan C. Morales* and M. Carmen
Galan*
+
G-quadruplex in K buffer. An
imidazolium-bearing mannoside
conjugate is the most selective ligand
discovered to date for this quadruplex
structure, and a similarly selective
methylpiperazine-bearing ligand was
more toxic to HeLa cancer cells than
doxorubicin, but with 3 times less
toxicity towards fetal lung fibroblasts
WI-38.
Page No. – Page No.
Divalent Naphthalene Diimide Ligands
Display High Selectivity for the
+
Human Telomeric G-quadruplex in K
Buffer
This article is protected by copyright. All rights reserved.