Macrocyclization of bis-indole quinolines for selective stabilization of G-quadruplex DNA structures

Article abstract of DOI:10.1039/d0sc03519j

The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the connection between G4s and the evolvement of different diseases, has sparked interest in developing small organic molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds. The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure-activity relationship of importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4 biology and potential therapeutics targeting G4-related diseases.

Full text of DOI:10.1039/d0sc03519j

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DOI: 10.1039/D0SC03519J
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Macrocyclization of Bis-Indole Quinolines for Selective
Stabilization of G-quadruplex DNA Structures
a†
a†
a
a
Rabindra Nath Das, Måns Andréasson, Rajendra Kumar, and Erik Chorell *
Received 00th January 20xx,
Accepted 00th January 20xx
The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the
connection between G4s and the evolvement of different diseases, has sparked the interest to develop small organic
molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the
design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds.
The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical
assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and
stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could
be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure-activity relationship of
importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic
compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4
biology and potential therapeutics targeting G4-related diseases.
DOI: 10.1039/x0xx00000x
across the human genome, with increased abundance in
sequences such as promoter regions, transcriptional regulators,
Introduction
The Secondary nucleic acid structures known as G-
quadruplexes, discovered in the mid 20 century, have over the
last decades received a lot of attention for their established
7
, 8, 9
and telomeric ends.
G4 structures are also more common
in oncogenes and have found to play a role in telomeric
maintenance and the development of diseases, such as
th
1
0
neurodegenerative disorders and different types of cancers.
1
relevance as important biological regulators. G-quadruplex
In addition, there is a 90% occurrence rate of potential G4-
forming sequences at the DNA replication origins, which
together with their stability and rapid folding kinetics suggest
(G4) structures can form in guanine-rich nucleotide sequences,
including both DNA and RNA. G4 structures are formed by π–π
stacking of G-quartets which are stabilized by Hoogsteen
7
,
that G4s can play a central role in the regulation of replication.
+
+
hydrogen bonds and cation coordination, usually to Na or K .
These secondary structures also display different topologies,
such as parallel, anti-parallel, and hybrid arrangements. The
type of topology is dependent on the nature of the base
sequence, G4-loop (nucleotides connecting the guanines)
length, strand orientation, and the type of cations, which all
directly correlate to the energetic properties of the G4.
fact, the G4s can be more thermodynamically stable than the
DNA-helix and can have rapid folding kinetics. The formation of
DNA G4-structures requires disruption of the helical structure
and they are thus believed to be formed during e.g. replication,
1
1, 12, 13
As an example, the c-MYC gene is associated with cell
cycle regulation and is upregulated in 70% of all identified
1
4
cancers. Expression of the c-MYC gene is mostly (~90%)
regulated by the gene promoter region (NHEIII) containing the
guanine-rich Pu27 which can fold into multiple G4-structures.
The predominant G4 structure in Pu27 is an intramolecular-
parallel structure that dominates the mutated sequences Pu22
2
, 3
In
(
deletion of G1-3/G19-20 and G14/G23 to T exchange) and
4
1
5, 16, 17
Pu24T (deletion of G1-3 and G10 to T exchange).
Ligand-
induced stabilization of the G4 in this sequence has shown to
1
8
prevent the expression of c-MYC. This can offer a novel cancer
therapeutic strategy that avoids direct interactions with the
MYC protein which has a short half-life and is difficult to target
5
transcription, or negative supercoiling.
Approximately 700 000 nucleotide sequences with potential to
form a G4-structure have been identified. Many of these are
evolutionary conserved and display a non-random distribution
1
9
with common ligand-protein interactions. Another example of
G4s associated with prevalent oncogenes is the c-KIT and its
dysregulation has been reported in the development of several
types of cancers and is the main cause of gastrointestinal
6
2
0
cancer. There are two sequences in the c-KIT promoter region
capable of folding into G4s, the c-KIT G4 and, c-KIT1 G4. The G4s
are located upstream of the transcription initiation site, and
there is a correlation between the stabilization of these G4-
aDepartment of Chemistry, Umeå University, 90187 Umeå, Sweden.
*
†
Corresponding author, E-mail: erik.chorell@umu.se.
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: Experimental procedures, G4-
binding studies, and characterization of the compounds. See
DOI: 10.1039/x0xx00000x
2
1
structures and the downregulation of c-KIT gene-expression.
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number of possible conformations it could occupy. This issue
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DOI: 10.1039/D0SC03519J
has previously been attempted to be counteracted by locking
the structure in a more rigid conformation, as in compound 1,
illustrated in figure 1. Unfortunately, this ultimately resulted in
a too static compound not optimally accommodating the G4
binding surface which caused a reduction in stabilization as
3
1
compared to 2. This thus constitutes an ideal model system to
evaluate if macrocyclization of known G4 ligands can generate
improved compounds as this will lock the compounds closer to
the bioactive conformation which would reduce the energetic
penalty upon binding, but still retain some degree of flexibility
(Figure 1).
In this project, we have therefore developed synthetic methods
to construct a novel set of macrocyclic bis-indole quinolines
with variations in both the ring-size and conformation to
investigate the ideal design for optimal stacking on the G4-
surface. Additionally, examples with a nitrogen atom in the
linker were made with the aim to improve solubility and reach
additional interactions with the DNA (Figure 2). The ability of
these novel macrocyclic ligands to bind selectively and stabilize
G4-structures was evaluated by using several assays;
fluorescence intercalator displacement (FID) assay,
fluorescence resonance energy transfer (FRET) melting assay,
binding induced fluorescence quenching assay, circular
dichroism (CD) spectroscopy, and NMR experiments. The
evaluation of the results obtained from the assays was further
corroborated with molecular dynamics (MD) simulations. We
also expanded the scope of the general concept of
macrocyclization using a simple bis-quinoline to both show on
the general applicability of the synthetic method and to
corroborate the positive effects of macrocyclization on G4 DNA
stabilization. From our results, we have identified novel
macrocycles that display strong selective binding and
2, stabilization of G4-structures together with important
structure-activity relationships which, taken together, show
that macrocyclization can indeed be an efficient strategy to
optimize G4 ligands.
Figure 1. Reported bis-indole compounds (1-3) that efficiently bind and stabilize G4 DNA
structures. The transition from a compound with linear conformations (3) to a semi-rigid
macrocyclic compound will lock the compound closer to the bio-active conformation and
prevent potential off-target binding to dsDNA and thus improve both binding and
selectivity.
Despite extensive studies over the past years, the biological
roles of G4s are far from fully explored. Small organic molecules
with the ability to selectively bind and stabilize G4s thus
represent a powerful tool to gain further insight into G4 biology.
Furthermore, such compounds can serve as starting points for
further developments towards therapeutics targeting G4-
structures. A large pool of ligands has indeed been reported to
bind and stabilize G4-structures but reports that describe the
further developments of these towards potent and selective
compounds with drug-like properties are still scarce. A deeper
knowledge of how to advance G4 ligands in terms of what
properties make them potent and selective stabilizers of G4s
2
would, therefore, be a valuable addition to the scientific field.
2
3, 24
Along this line, macrocyclic structures are known to have
positive effects on the pharmacokinetics and allow properties
beyond Lipinski’s rule of 5. It is also attributed that macrocycles
can interact selectively with flat and featureless binding sites
which is a very important property, considering the non-specific
Results & Discussion
G4 binding surface.2
5, 26
Furthermore, a macrocyclic structure
should also increase the selectivity for G4 DNA over dsDNA
because of its conformational constraints. There are indeed
macrocycles reported with good abilities to bind and stabilize
Synthesis of Macrocycles
The novel macrocyclic compounds were planned to be constructed
via the convergent pathway depicted in Figure 2. The
macrocyclization was proposed to be achieved via the combination
of the bis-indole amine and the bis-quinoline acids via an initial
amide-coupling followed by a theoretically more favourable
2
7, 28
G4 DNA, e.g. isolated from natural sources.
Despite this,
the development of novel macrocyclic-based ligands or
macrocyclization of known ligands targeting G4s has barely
2
9
been attempted to date.
intramolecular amide-cyclization as compared to
a second
Bis-indoles 2 & 3 have displayed excellent abilities to bind and intermolecular amide-coupling. Synthesis of the bis-indole has
3
0, 31, 32
stabilize G4 DNA structures.
However, energy previously been reported by the condensation of two indoles with an
minimizations show that this compound class prefers mixed aldehyde.33 Generation of the bis-quinoline acids were proposed to
non-planar low energy conformations in contrast to the more be achieved with a tandem amide coupling of two quinoline amines
planar crescent-shaped bioactive conformation illustrated in with a di-acid linker. This assembly was considered attractive since it
Figure 1. This is believed to result in a higher energetic penalty
upon binding to a G4 because it will force the compound out of
its most energetically favourable conformation and restrict the
would easily allow variations, both in the amine position on the
quinoline and in the length and composition of the linker prior to the
2
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crucial macrocyclization step. Finally, our quinolines were proposed intramolecular cyclization reaction.
to be made from the azide precursors via a light-mediated radical
View Article Online
DOI: 10.1039/D0SC03519J
Figure 2. Retrosynthetic outline on how we envisioned the assembly of our macrocyclic compounds.
The bis-indole amine 5 was synthesized as planned from 5- However, propanephosphonic acid anhydride (T3P) has been
nitroindole by condensation with formaldehyde at elevated used successfully in the coupling of acids with poorly
temperature followed by subsequent nitro reduction to the di- nucleophilic amines without undesired side reactions between
3
4
amine 5 with hydrogen in the presence Pd/C-10% (Figure 3). The the amine and the coupling reagent. Fortunately, the amide-
synthesis of the azide precursors 8a/b for the quinoline coupling between the different di-acid linkers and the quinoline
formation was initiated from nitro benzaldehyde, with the amines proceeded smoothly with T3P as a coupling reagent and
2 2
nitro-group either in the meta- or para-position, through a N-methylmorpholine as the base in CH Cl at ambient
Bailys-Hillman reaction to give 6a/b followed by acetylation of temperature to afford the different bis-quinoline linkers 11 in
the newly formed alcohol to generate 7a/b. The acetylated satisfactory yields (79-95%) without the need for
compounds were treated with NaN followed by a light- chromatographic purification. Subsequent saponification of the
3
mediated radical promoted cyclization of 8a/b in the presence ethyl ester afforded bis-quinoline acids 12 in high yields (Figure
of NBS (N-bromosuccinimide) to give the quinoline products 3).
9
a/b. The concentration for this reaction proved to be critical
and when performed at 0.2 M, mostly intermolecular radical Our hypothesis for the key macrocyclization reaction between
side reactions took place resulting in a quinoline yield of only di-amine 5 and di-acids 12 was that a first intermolecular amide-
1
5-20%. However, when the reaction was performed at a lower coupling would occur and that an intramolecular cyclization
concentration (~20 mM) a significant improvement of the yield then would be faster and more favourable compared to another
was observed (75-87%). Several radical initiators were also intermolecular amide-coupling that would result in unwanted
investigated;
iodosuccinimide), AIBN (azobisisobutyronitrile), TBHP (t-butyl with the same conditions as for the quinoline linker-coupling
hydroperoxide) and DBPO (benzoyl peroxide), but none of them from 10 to 11, with DMF as the solvent instead of CH Cl . Upon
NCS
(N-chlorosuccinimide),
NIS
(N- polymer/dimer side-products. The reaction was first attempted
2
2
gave any improvement in the yield. In the case of 3-nitro monitoring the reaction, the desired product was formed in
substituted (8a), the cyclization can occur either in ortho- or very small amounts and after extensive condition-screening we
para-position to the nitro-group and unfortunately the only obtained the macrocycles in very low yields (<5%), giving a
unwanted ortho-cyclized derivative was always obtained as the dimeric product in excess. We instead performed the reaction
major product (59% yield) and the para-cyclized derivative as at high dilutions (2-6 mM) with several known amide-coupling
the minor product (28% yield) likely because of steric effects reagents, hoping to avoid di-amine side-reactions and reduce
and electronic directions by the nitro-group. The nitro-group in dimer-formation. The change in conditions, run at both
9
a/b was next reduced to the amines 10a/b (87-92% yield) elevated temperature and ambient temperature, gave slight
through palladium catalysed hydrogenation. The amide- improvements in yield. We then utilized TCFH (N,N,N',N'-
coupling between the different di-acid linkers and the quinoline tetramethylchloroformamidinium hexafluorophosphate) as
3
5
amines 10a/b was attempted with several different coupling activating reagent and N-methylimidazole as the base in DMF
agents, including PyBOP (benzotriazol-1-yl- and ran the reaction with a dilution of 2 mM. This afforded the
oxytripyrrolidinophosphonium hexafluorophosphate), PyAOP macrocycles in yields between 20-28%, which we deemed to be
(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium acceptable. Furthermore, this reaction setup also offered us
(
hexafluorophosphate), EDC•HCl (N-(3-Dimethylaminopropyl)- easier purifications by column chromatography. Subsequent
N′-ethylcarbodiimide hydrochloride) and TFFH (Fluoro- methylations of the quinolines were performed at 40 °C using
N,N,N′,N′-tetramethylformamidinium hexafluorophosphate), methyl iodide (CH I) and afforded the different methylated
3
but none of these conditions gave satisfactory results.
macrocycles (14) in near quantitative yields. In the case of the
This journal is © The Royal Society of Chemistry 20xx
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Figure 3. Total synthetic scheme for the macrocyclic compounds and a summary of the 11 different macrocycles.
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NBoc analogues (14a1 and 14b1) the nitrogen was deprotected internal hydrogen bonding. However, since the purity and
DOI: 10.1039/D0SC03519J
2 2
in the presence of 10% TFA in CH Cl at ambient temperature. characterization of 15a was not confirmed, we cannot fully
LC-MS and HRMS analysis suggested the formation of the validate these results. More importantly, FRET melting
desired macrocycles (15a/15b) and although we were not able experiments to investigate possible interactions with double-
to obtain clear NMR spectra to fully characterize these stranded DNA (ds26) were also performed, and while
compounds, we thus still decided to include them in the compound 3 showed a ∆T
m
of around 8 °C at 5 µM the
evaluation of the compounds’ abilities to stabilize G4 DNA macrocyclic compounds (except for 14a1) had a very small or
(
Figure 3).
unmeasurable effect on the stabilization of dsDNA (Figure 4d
and Figure S3). This supports our hypothesis that the
macrocyclic design increases the selectivity by preventing
conformations that can interact with dsDNA.
FRET Melting Assay
All compounds displayed good solubility properties and were
initially screened in a FRET melting assay. In this assay, we
measure the differences in the melting temperature (∆T ) of
m
fluorescently labelled G4 DNA (c-MYC Pu22, c-MYC Pu24T, and
c-KIT2, for sequences see Table S1) in the presence and absence
of macrocycles 14a1-4 and 14b1-5 (Figure 4) to investigate their
abilities to stabilize G4 structures at different concentrations (1,
To confirm that the compounds do not influence the topological
change of G4-DNA, we measured CD spectra in the absence and
presence of the compounds. No major changes were observed
which confirms that the conformation of the G4 structures
remained intact in the presence of the macrocycles (Figure S4).
CD melting studies were also performed for G4 DNA with 14a1
and 14a2 and 3-18 C stabilization was observed (Table S2,
Figure S5).
2
, and 5 µM) using the most efficient bis-indole quinoline
reported (3) as a reference (Figure 4a-c, Figure S1-S3).
FID and Fluorescence Quenching Assays
To investigate the binding affinities of the macrocyclic ligands we
performed a Fluorescence Intercalator Displacement (FID) assay. The
FID assay was performed with a selection of the most interesting
macrocycles from the first FRET screen (14a2, 14a3, 14b3, and 14b5).
In the FID assay, we investigate the ability of the macrocycles to
displace the well-known G4 binder thiazole orange (TO) upon binding
to the G4 DNAs (c-MYC Pu22, c-MYC Pu24T, and c-KIT2), which can
be measured by a decrease in fluorescence (Figure 5a, Figure S6-
S10). The results (see Table 1) follow the same trend as for the FRET
assay, 14a2 and 14a3 have the lowest K
with 14a2 as the best compound with binding constants of 0.99 µM,
.23 µM, and 0.44 µM for Pu24T, Pu22, and c-KIT2 G4 DNA
respectively. The results underscore the importance of the
macrocyclic ring-size and the position of the macrocyclic linker
position on the quinoline for an optimal binding to the G4 surface.
d
of the tested macrocycles
Figure 4. Stabilizing ability of the ligands by FRET melting assay at 2 M of concentration
for (a) Pu24T G4 DNA, (b) Pu22 G4 DNA, (c) Ckit2 G4 DNA, and (d) double standard DNA.
0
m
DNA concentration is 0.2 M. T in absence of ligands of Pu24T G4 DNA is 62.5 ± 0.3 °C,
Pu22 G4 DNA is 64.3 ± 0.2 °C, Ckit2 G4 DNA is 63.9 ± 0.1 °C and ds DNA is 68.0 ± 0.2 °C.
All the compounds were able to stabilize both of the two FID assay was further employed for dsDNA with the best macrocycle
parallel c-MYC and the parallel c-KIT2 G4 structures. 14a2 and reference compound 3 to compare their ability to displace
Macrocycles 14a1, 14a2, and 14a3, with the linker in position 6 TO from dsDNA. This shows that both 14a2 and 3 can displace TO at
on the quinoline, displayed highly efficient stabilization effects higher concentrations which is not surprising considering that the
with a ∆T of almost 20 C (at 5 µM of 14a2, see Figure 4a-c and compounds are positively charged and are therefore likely to interact
m
Figure S1-2), thus being comparable to the strongly stabilizing
reference compound 3. The macrocycles 14b1-14b5, with the
linker in position 7 on the quinoline, show a large drop in their
activity compared to macrocycles 14a1, 14a2 and 14a3 with the
linker in position 6 on the quinoline. A clear trend in the size of
the macrocycles with the linker in position 6 (14a1-4) was
observed where the smaller macrocycles more effectively
stabilized the G4 structures. The same trend could not be seen
for the macrocycles with the linker in position 7 (14b1-5). The
macrocycles with an aliphatic amine in the linker (15a) had an
apparent reduced stabilization potential compared to the boc-
protected version (14a1). This might be due to solvent penalties
for the amine upon binding to the G4 surface or by breaking up
in an unspecific manner with the negatively charged DNA in this in
vitro setting. Importantly, macrocyclization of 3 to form 14a2 reduce
this unspecific interaction ( DC50 = 1.34 µM; dsDC50
ds
3
14a2
= 2.40 µM)
(Figure S11a, S12). We have also investigated the compounds ability
to stabilize dsDNA using CD (Figure S11b). This shows that 14a2 does
not affect dsDNA whereas compound 3 does, in fact, affect dsDNA
stability which again underscores the value of macrocyclization to
achieve selectivity.
To confirm the binding affinities obtained from the FID assay, we also
employed a ligand induced fluorescence-quenching assay with a 5'-
Cy5-labelled G4 DNA (c-MYC Pu22, c-MYC Pu24T, and c-KIT2) by using
Microscale Thermophoresis (MST). The results from the binding
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induced fluorescence quenching assays corroborate the FID data proved to be the better overall binder comparedVtieow AtrhtiecleoOtnhliener
with a very similar internal trend in the binding affinities for the macrocycles. The binding constants (K
different macrocycles (Figure 5b, Figure S13). Once again, 14a2 MST assays are summarized in Table 1.
) obtDaiOnIe:d10f.r1o0m39/tDh0eSCFI0D35a1n9dJ
d
d d
Table 1. Summary of the apparent K -values obtained from the FID assay and K -values obtained from the MST assay with the different G4 DNA (c-MYC Pu24T, c-MYC Pu22, and
c-KIT2) for the macrocycles (14a2, 14a3, 14b3, and 14b5).
d
K (µM) for
Pu24Ta
K
d
(µM) for
Pu22a
K
d
(µM) for
c-KIT2a
d
K (µM) for
Pu24Tb
K
d
(µM) for
Pu22b
K
d
(µM) for
c-KIT2b
Macrocycle
1
1
4a2
4a3
0.99 ± 0.02
1.25 ± 0.04
0.23 ± 0.01
0.33 ± 0.02
0.44 ± 0.02
0.54 ± 0.06
0.77 ± 0.09
1.32 ± 0.24
0.90 ± 0.11
1.09 ± 0.10
0.31 ± 0.03
0.35 ± 0.04
1
1
4b3
4b5
2.31 ± 0.06
1.80 ± 0.03
0.60 ± 0.04
0.68 ± 0.05
0.93 ± 0.04
1.22 ± 0.08
1.54 ± 0.21
1.39 ± 0.20
0.74 ± 0.09
3.56 ± 0.45
0.62 ± 0.08
nd
a
Using the FID assay (see also Figure 5a and Figure S6-10). b Using fluorescence quenching assay (see also Figure 5b and Figure S13).
‘nd’ indicates not determined.
Figure 5. (a) FID based binding affinity plot induced by the ligand 14a2 for Pu24T, Pu22
and c-KIT2 G4-DNA. (b) Fluorescence quenching based binding affinity plot of 14a2 for
Pu24T, Pu22, and c-KIT2 G4-DNA.
1H NMR Titrations
1
Figure 6.
H NMR (850 MHz) titrations for c-MYC Pu24T (a), c-MYC Pu22 (b), and c-KIT2
(
c) with 14a2. The initial DNA concentration was 90 µM and macrocycle was then added
NMR experiments were next utilized to probe both the
macrocycles’ abilities to bind the G4 structures (c-MYC Pu22, c-
MYC Pu24T, and c-KIT2) and to learn more about these
interactions. In these experiments, the G4-DNA was titrated
with macrocycle spanning a ratio from 1:0.1 to 1:2
so the last addition corresponded to a total molar ratio of G4 DNA:macrocycle 1:2.
The broadening of the imino proton signals is most likely due to
an on-off rate in the interaction between the DNA and
macrocycle on a timescale that yields line-broadening due to
chemical exchange. A clear broadening of the imino protons
was also observed for 14a3 and 14b3 in all three G4 structures,
the effect was stronger with 14b3 indicating a difference in their
binding interactions (Figure S15-S16). The appearance of new
peaks was noticed (e.g. when increased amounts of 14a2 were
added to the c-KIT2 G4 DNA) which indicates a slower exchange
(DNA:macrocycle) and the changes in the imino protons for the
hydrogen bonding guanines in the G-tetrads in the NMR
spectrum were compared at the different concentration ratios.
No significant changes in the chemical shift for the imino
protons were observed upon binding of the macrocycles.
However, an evident broadening was instead seen, and upon
the addition of 2 equivalents of macrocycle 14a2, the intensities
of the peaks from the imino protons in the c-MYC G4s are
strongly reduced (Figure 6 and Figure S14-S16).
(
lower on-off rate) where both the free and bound form of DNA
are observed which indicate a stronger binding.
Competition Assay and Selectivity
In the FRET screening assay, we concluded that the macrocycles
had very little or no effect on dsDNA and to further confirm
these results a FRET melting competition assay was conducted.
The thermal stability was first measured only for the respective
G4-DNA with 14a2 and compound 3 (0.2 µM DNA:2 µM 14a2/3)
and dsDNA were sequentially added to investigate if any major
6
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changes in the thermal stability were observed. In the presence the experimentally determined structure-activity VrieewlaAtritoiclnesOhnilpines
of a large excess of dsDNA (20 µM, 100 equivalents compared for the macrocycles. Furthermore, this mDoOdI:e1l0c.1a0n39a/lDs0oSCe0x3p5la19inJ
to G4 DNA), no major changes in the thermal stabilization for the reduced activity observed by the macrocycles linked in
the G4 DNA were observed in case of 14a2, but for compound position 7 on the quinolines as the planar conformation
3
m
the stabilization (T ) decreases by 2-4 °C. This show that required to efficiently stack on the G-tetrad would be
macrocycle 14a2 has a strong preference for binding G4 DNA energetically disfavoured in this design. These macrocycles
over dsDNA (Figure 7a, Figure S17) and confirm that would also be less dependent on the linker length, which also is
macrocyclization also increases the selectivity. We next in good agreement with the observed results for the 14b
investigated if the macrocycles display selectivity over different macrocycles linked in position 7 of the quinolines.
G4 topologies by measuring their ability to stabilize different G4 The MD simulations also show that the size of the macrocycle
structures using FRET. This shows that 14a2 strongly stabilizes may not be optimal for occupying the G4-surface. With the
parallel and hybrid G4 structures whilst completely present design, only 3 of the 4 aromatic systems seem to be able
discriminating against the anti-parallel structures (Figure 7b, to partake in stacking interactions with the G-tetrad. This
Figure S18-S19). This discrimination could not be seen with the suggests that further improvements of the macrocyclic design
open-chain analogue 3 which also stabilizes the anti-parallel should be possible by adjusting the ring-size and the number of
structures.28 This difference in selectivity could thus be aromatic systems to achieve a more optimal size and an even
attributed to the macrocyclic design of the compounds. The further improved stacking interaction with the G4-surface.
other macrocyclic compounds also showed the same strong Next, we calculated and compared the drug-like descriptors
selectivity for G4 DNA over dsDNA and also the same selectivity (molecular weight, logP, hydrogen bond-donor, hydrogen
trend between different types of G4 DNA structures, which bond-acceptor, and the total polar surface area (TPSA))
support the value of the macrocyclic design in the observed between the macrocycles and compound 3 (Table S3). This
selectivity.
show that the smaller macrocycles have a lower logP and a
dramatically increased TPSA in comparison to 3 despite their
increased molecular weight. Furthermore, we notice that the
lowest energy conformations of the unbound form of the
macrocycles can shield functionalities through intramolecular
hydrogen bonds, non-classical hydrogen bonds, and π -π
interactions (Figure S20-23), which can have a positive impact
2
5
on drug-like properties such as cellular uptake. This again
underscores the potential of macrocyclization to improve drug-
Figure 7. (a) Stabilization ability of 14a2 (2 µM) for Pu24T, Pu22 and c-KIT2 G4-DNA (0.2 like properties.
µM) in presence of excess amount (0-100 equivalent) of a double-stranded competitor
dsDNA (ds26). (b) Selectivity study of 14a2 (2 µM) for their ability to stabilize of Ds-DNA
(0.2 µM) and different G4 DNA (0.2 µM) (see also Figure S14, S15). Pu24T, Pu22 (c-MYC
promoter); c-KIt1, c-Kit2 (c-KIT promoter); Kras (K-RAS gene) and 25ceb (human
minisatellite) are parallel G4 forming sequences. Bcl-2 (BCL-2 promoter) and 21g (human
telomere) are hybrid G4 forming sequences. Bom17 (Bombyx telomere) and TBA
(thrombin binding aptamer) are antiparallel G4 forming sequences.
Computational studies
Molecular dynamics simulations were next performed to study
the details of the interaction between macrocycle 14a2 and G4
DNA. Based on previous studies of the reference compound 3,
Figure 8. Representative binding pose of 14a2 with Pu24T c-MYC G4 DNA during MD
simulations. The 5´-G-tetrad (green), nucleotides flanking the G4 DNA structure (light
we expect the macrocycles to bind by end-stacking to the blue), and compounds (ball-stick model) from the largest cluster’s central structure.
terminal G-tetrads. The Pu22 c-MYC G4 has both G-tetrads
accessible for binding and the macrocycles thus likely bind at
both the 3´- and the 5´-G-tetrads. However, the Pu24T c-MYC
G4 has an accessible 5´-G-tetrad and a partly blocked 3´-G-
tetrad. We thus modelled 14a2 on the top of the 5´-G-tetrad of
the Pu24T c-MYC G4 structure and performed molecular
dynamics simulations for a total of 1 µs. This showed that 14a2
efficiently stack on the top G-tetrad and is further sandwiched
by strong interactions with the nucleotides flanking the G-
tetrad (Figure 8, Table S4 and Figure S20). From the simulations,
it becomes evident that increasing the linker length in
macrocycle 14a2 will be detrimental to the ability to efficiently
stack on the top G-tetrad as it would partly push one of the
quinolines out of the G-tetrad. This is in well agreement with
General applicability of the strategy
To expand the scope of this study, both in terms of the general
applicability of the synthetic strategy and the macrocyclization
strategy to improve G4 binding and stabilization, we employed
our strategy on the simple bis-quinoline 16 to afford a
macrocyclic counterpart 17 (Figure 9a). The two compounds’
abilities to stabilize G4 DNA (c-MYC Pu24T and c-MYC Pu22)
were evaluated by the FRET melting assay. This show that
macrocyclization of 16 clearly increased the ability to stabilize
G4 DNA (at 8 µM 16: ∆T
Pu22), 17: ∆T = 7.5 °C (Pu24T) and ∆T
b-c). Furthermore, none of 16 and 17 were able to stabilize
m
= 2.6 °C (Pu24T) and ∆T
m
= 3.2 °C
= 9.9 °C (Pu22)) (Figure
(
m
m
9
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J. Name., 2013, 00, 1-3 | 7
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PleaseC dh oe mn oi ct a al dS cj ui es nt cme argins
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ARTICLE
Journal Name
dsDNA, and macrocycle 17 also displayed great selectivity for relationships, MD simulations based on the NMVRiewdAarttiaclewOnelirnee
G4 DNA over dsDNA (Figure S21-S22). This thus emphasizes the performed. This clarified the reason behDinOdI:t1h0e.10o3b9/sDe0rSvCe0d35S1A9RJ
value of macrocyclization as a design element in the with the ring size and linker position being of key importance to
optimization of G4 interacting ligands in general and for the keep the macrocycle aligned for efficient end-stacking on the
frequently appearing bis-quinoline derivatives in particular.
top G-tetrad. In addition, the MD simulations also suggest that
it should be possible to construct macrocyclic compounds with
even further improved affinity, selectivity, and stabilization for
G4 structures. Finally, we applied our strategy on a simpler bis-
quinoline derivative which resulted in increased G4 stabilization
with great G4 selectivity which confirmed the potential of
macrocyclization.
Taken together, we have shown that macrocyclization is a
valuable design-element for the advancement of ligands that
target G4 DNA structures, with the potential for improved drug-
like properties, G4 stabilization, selectivity, and affinity. We
show that this strategy can be adopted on various G4 ligands
with the aim to advance them towards research tools,
diagnostic tools, and as therapeutics for the treatment of G4-
related diseases.
Figure 9. Bis-quinoline 16 and a macrocyclic analogue 17. Stabilizing ability of the ligands
by FRET melting assay at 1, 2, 5, and 8 M concentrations for (a) Pu24T G4 DNA, (b) Pu22
G4 DNA. DNA concentration is 0.2 M. T
m
in absence of ligands of Pu24T G4 DNA is 62.5
Conflicts of interest
±
0.3 °C and Pu22 G4 DNA is 64.3 ± 0.2 °C.
There are no conflicts to declare
Conclusions
Acknowledgements
We have used bis-indole quinoline G4 ligands as a model system
to evaluate the potential of macrocyclization in the optimization Work in the Chorell lab was supported by the Kempe
of G4 ligands in terms of G4 binding and stabilization properties. foundations (SMK-1632) and the Swedish Research Council (VR-
Key to succeed with this was substantial synthetic NT 2017-05235). RND was supported by a fellowship from the
developments for the construction of the novel macrocyclic Wenner-Gren foundation. We also thank Dr. Mattias Hedenström
compounds with variations in both the amide-linker length and at the Knut and Alice Wallenberg foundation program, NMR for
the quinoline-amine position. All the synthesized target Life (www.nmrforlife.se), for NMR spectroscopy support.
macrocycles were first evaluated by a FRET melting assay and
three different G4 structures which identified highly efficient G4
stabilizing compounds. Their stabilizing abilities were confirmed
using CD melting and four highly promising derivatives 14a2,
1
4a3, 14b3, and 14b5 were selected for further studies. NMR,
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