The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions with G-Quadruplex DNA

Article abstract of DOI:10.1002/chem.202000579

G-quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis-indoles (3,3-diindolyl-methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which for example, show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure–activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

Full text of DOI:10.1002/chem.202000579

A Journal of
Accepted Article
Title: The Relation Between Position and Chemical Composition of
Bis-Indole Substituents Determines their Interactions with G-
Quadruplex DNA
Authors: Bagineni Prasad, Rabindra Nath Das, Jan Jamroskovic,
Rajendra Kumar, Mattias Hedenström, Nasim Sabouri, and
Erik Chorell
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10.1002/chem.202000579
Chemistry - A European Journal
The Relation Between Position and Chemical Composition
of Bis-Indole Substituents Determines their Interactions
with G-Quadruplex DNA
Bagineni Prasada, Rabindra Nath Dasa, Jan Jamroskovicb, Rajendra Kumara, Mattias
Hedenströma, Nasim Sabourib*, Erik Chorella*
[a] Dr. B. Prasad, Dr. R. N. Das, Dr. R. Kumar, Dr. M. Hedenström, and Dr. E. Chorell.
Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
[b] Dr. J. Jamroskovic and Dr. N. Sabouri. Department of Medical Biochemistry and Biophysics,
Umeå University, 901 87 Umeå, Sweden.
*corresponding authors: nasim.sabouri@umu.se and erik.chorell@umu.se
Supporting information is available for this article.
Abstract
G-quadruplex (G4) DNA structures are linked to fundamental biological processes and
human diseases, which has triggered the development of compounds that affect these
DNA structures. However, more knowledge is needed about how small molecules interact
with G4 DNA structures. This study describes the development of a new class of bis-
indoles (3,3-diindolyl-methyl derivatives) and detailed studies of how they interact with
G4 DNA using orthogonal assays, biophysical techniques, and computational studies.
This revealed compounds that strongly bind and stabilize G4 DNA structures, and
detailed binding interactions which e.g. show that charge variance can play a key role in
G4 DNA binding. Furthermore, the structure-activity relationships generated opened the
possibilities to replace or introduce new substituents on the core structure, which is of key
importance to optimize compound properties or introduce probes to further expand the
possibilities of these compounds as tailored research tools to study G4 biology.
Introduction
Single-stranded DNA sequences containing four repeats of guanines (normally two-
three), bridged by random nucleotides, can fold into G4 DNA structures. In the G4 DNA
structure, the guanines bind to each other by Hoogsten hydrogen bonds and form a
quartet of four guanines, one from each guanine stretch, called G-tetrads. The G-tetrads
stack on top of each other to form strong pi-pi interactions and are further stabilized by
central monovalent cations such as potassium or sodium.[1, 2] G4 DNA structures can be
more stable than the classical double-stranded helical DNA.[3] However, the number of
G-tetrads and the length and base composition of the nucleotides that bridge the guanine
stretches, called loops, are highly important for the stability of the G4 DNA structure.[4-9]
G4 DNA structures can form when DNA is single-stranded, for instance during cellular
processes, such as, DNA replication, transcription, and DNA repair. Furthermore,
superhelical stress,[10] molecular crowding,[11] and G4 DNA binding proteins can also favor
their formation.[12]
There are about 700,000 predicted G4 DNA structures in the human genome and many
of these are evolutionary conserved,[13, 14] suggesting that they have important functions.
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One of the first reported biological implications of G4 DNA structures was their presence
at the telomeres, the chromosomal ends.[15] Since then, it has also been shown that G4
DNA structures can inhibit or enhance gene expression and G4 DNA motifs are present
in every second human promoter, and most frequently at promoters of oncogenes and
regulatory genes.[16, 17] Additionally, G4 DNA motifs are also present in the promoters of
other organisms.[18-20] G4 DNA structures have thus been linked to fundamental cellular
events such as maintenance of the telomeres and transcription, but they also affect DNA
replication progression and many proteins and enzymes are reported to bind and/or
resolve G4 DNA structures.[15, 21-23]
G4 DNA structures are also connected to human diseases such as neurodegenerative
diseases and different types of cancers.[24-27] One of the most extensively studied G4 DNA
structures in this regard is located in the c-MYC promoter, which is a transcriptional factor
affecting cell proliferation that is overexpressed in many human cancers.[28] Mutations that
prevent formation of the G4 DNA structure in the c-MYC promoter downregulates
transcription of this gene,[29] and selective stabilization of this G4 DNA structure is
therefore a potential chemotherapeutic strategy. The G4-forming region of the c-MYC
promoter is a 27 nucleotide sequence, Pu27, that is important for regulating transcription
of the c-MYC gene.[30, 31] The Pu27 sequence form parallel polymorphic G4 DNA
structures but the predominant G4 DNA structure involved in c-MYC transcriptional
regulation is captured in the modified and truncated versions of the Pu27 sequence,
Pu24T (G13T) and Pu22 (G14T/G23T) c-MYC G4 DNA structures.[32, 33]
The prevalence and biological relevance of G4 DNA structures as well as their links to
human diseases has resulted in a significant interest in the development of compounds
that stabilize G4 DNA structures. However, although there are many examples of G4 DNA
stabilizing compounds, the reports that fully explore the structure-activity relationships
and correlates this to the compound’s interactions with G4 DNA structures are scarce.
This type of information is of key importance to develop this field of research both towards
tailored research tools and to efficiently explore G4 DNA structures as therapeutic targets.
Indoles are frequently occurring in drugs and biologically active compounds and are
therefore considered to be privileged structures. Bis-indoles have been shown to
efficiently bind and stabilize G4 structures with effects similar or even improved to the
most well-known and efficient G4 stabilizing compound Phen-DC3.[34-37] However, these
reported bis-indoles are either linked at the indole nitrogen or in position 2, leaving bis-
indoles linked at position 3 unexplored. Furthermore, these studies also show that
flexibility is important in the design of G4 ligands and that diindolyl-methyl derivatives can
be efficient G4 binders even though their most energetically favorable conformation is
non-planar.[35-36] In this work, we have thus designed and synthesized 3,3-diindolyl-methyl
analogues with different side chains to achieve structure-activity relationships (SARs).
Additionally, we have developed synthetic methods that allowed us to vary the side chains
on the 3,3-bisindolyl analogues in all open positions for all side chains and for a
combination of side chains to enhance the SAR study (Figure 1). To analyze the
compounds´ abilities to bind and stabilize G4 DNA structures, we used a wide range of
methods including fluorescence resonance energy transfer (FRET) melting assay,
circular dichroism (CD) spectroscopy, microscale thermophoresis (MST), Fluorescence
Intercalator Displacement (FID) assay, Nuclear Magnetic Resonance (NMR), and
computational techniques such as molecular dynamics (MD) simulations to understand
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10.1002/chem.202000579
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the molecular details of the results. Overall, this revealed compounds that strongly bind
and stabilize G4 DNA structures and key components in the design of these types of
compounds and their interactions with G4 DNA structures.
Figure 1. Summary of the 3,3-diindolyl-methyl central fragment and the variations of the
substituents and their position.
Results and discussion
Synthesis
To determine how the position and composition of different side chains of the 3,3-
diindolyl-methyl affect G4 binding and stabilization, we synthesized different analogues.
The nitro-indoles (1a-d) were the key to investigate the effect of the position and
composition of the side chains of the 3,3-diindolyl-methyl derivatives. The 4- and 5-nitro-
indoles are commercially available whereas the 6- and 7-nitro-indoles were synthesized
according to published procedures in two and five steps, respectively (supporting
information). The 7-nitro-indole synthesis also resulted in a 5-bromo substituent for this
set of derivatives (which depending on the data generated from these derivatives
potentially can be used for late stage functionalization or be dehalogenated). From these
four nitro-indoles, attempts to synthesize the di-nitro-3,3-diindolyl-methyl intermediates
were performed by reacting the corresponding nitro-indoles with formaldehyde in water
and 3 equivalents of acetic acid under refluxing conditions for 12 h. This gave the desired
di-nitro-3,3-diindolyl-methyl intermediates (2a-2c) in 65-75% yields. Unfortunately, the 5-
Bromo-7-nitro indole (1d) did not react under these conditions. However, when the nitro
indoles were reacted with formaldehyde using 10% sodium dodecyl sulfate (SDS) in water
under microwave irradiation at 100 °C for 2 h, all the desired di-nitro-3,3-diindolyl-methyl
intermediates (2a-2d) were obtained in 88-92% yields. Hydrogenation of the di-nitro-3,3-
diindolyl-methyl analogues under Pd/C conditions in methanol at room temperature
afforded di-amino-3,3-diindolyl-methyl analogues (3a-d) in 86-96% yields. Next, the four
di-amino-3,3-diindolyl-methyl analogues were each coupled with 4 different carboxylic
acid substituted side chains using N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) as
coupling reagent and diisopropylethylamine (DIPEA) as base to provide the 16 desired
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10.1002/chem.202000579
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analogues (5aa-ad, 5ba-bd, 5ca-cd, and 5da-dd) in high yields. The choice of side
chains was based on both aromatic and aliphatic systems with possibilities to form
stacking interactions and/or positive charges under physiological pH or through
permanent methylations. Hence, the analogues having quinolines as side chains were
also quaternized using methyliodide (MeI) in dimethylformamide (DMF) at 40 °C for 24 h
to give the four methylated analogues (7aa-da) in 88-96% yields. Unfortunately,
methylations of the side chain containing benzo[d]thiazoles (5ab, 5bb, 5cb, and 5db)
were not successful even though various methods were applied (Summarized in Scheme
S1). To further expand the scope of the study, an analogue with the reversed amide (5ea
and 7ea) as well as an example with an ethyl substituent on the central carbon (5fa and
7fa) was also synthesized (scheme 2). In total 24 derivatives (5aa-5fa and 7aa-7fa) with
different side chain composition and position on the 3,3-diindolyl-methyl central fragment
was prepared.
Scheme 1. Synthesis of 3,3-diindolyl-methyl analogues. Reaction conditions: a) HCHO, 10% SDS in H2O,
MW, 100 °C, 2 h; b) EtCHO, AcOH, H2O, reflux, 12 h; c) Pd/C, H2, Methanol, rt, 3 h; d) LiOH, THF, 2 h; e)
R-COOH (quinoline-3-carboxylic acid (4a)/benzo[d]thiazole-2-carboxylic acid (4b)/4-(4-methylpiperazin-1-
yl)butanoic acid) (4c)/4-morpholinobutanoic acid (4d), HATU, DIPEA, DMF, rt or 50 °C, 12-24 h; f) MeI (6a),
DMF, 40 °C, 24 h; g) MeOTf (6b), DCE, 85 °C, 3 h.
FRET Melting assay
We next analyzed how the variations of the side chains, both composition and position,
affect the compounds ability to stabilize G4 DNA structures by the FRET melting assay.
This assay measures the ligand induced change in melting temperature (∆Tm) of the
labeled parallel Pu24T c-MYC G4 DNA structure (see Experimental Section for details,
the oligonucleotides used in this study are summarized in Table S1). This showed that
both the position and the composition of the side chains on the 3,3-diindolyl-methyl is
crucial for an efficient G4 stabilization (Figure 2A and Figure S1). The derivatives with
methylated quaternized quinoline side chains in position 5 and 6 (7ba and 7ca) clearly
gave the most efficient stabilization with a dramatic difference compared to analogues
with substituents in position 4 and 7 (7aa and 7da). Furthermore, the methylated quinoline
side chain in the 5- and 6-substituted derivatives (7ba and 7ca) proved to be of utmost
importance for an efficient G4 stabilization when compared to their homologs with non-
methylated quinoline side chains, suggesting that the charge is important for the quinoline
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side chains. This was supported by the derivatives with neutral benzo[d]thiazoles side
chains (5ab-5db) that showed very week stabilization. However, the charge alone could
not be responsible for the observed effects as the piperazine and morpholine derivatives
(5ac-5dc and 5ad-5dd, respectively), which are positively charged in the physiological
pH used in the experiments, only showed very week stabilization. The piperazine and
morpholine side chains lack the ability to form pi-stacking interactions which suggest that
this is important and cannot in full be compensated for by the potential electrostatic
interactions of these derivatives. Reversing the amides (7ea compared to 7da) did not
have any major impact on the compounds ability to stabilize G4 DNA in this assay.
Importantly, all compounds showed good selectivity for G4 DNA structures over double-
stranded DNA (Figure S2). A potential problem with the FRET assay is the risk that
compounds can interfere with the FRET process and thereby give false positive/negative
results. However, these effects should appear immediately and not be temperature
dependent. None of the tested compounds displayed a temperature independent effect
on the FRET process thus suggesting that they do not affect the fluorophores or disrupt
the G4 DNA structure (except 7fa with a central ethyl group which affected the FRET
process likely by interacting with the Carboxy tetramethyl-rhodamine (TAMRA)
fluorophore, which can be the reason for the decreased stabilization ability observed for
this compound compared to 7ca).
Taq polymerase stop assay
The synthesized analogues were next evaluated for their ability to affect the progression
of the Taq DNA polymerase on a DNA template with the Pu24T c-MYC G4 DNA structure
and compared to a non-G4 control template. Due to their intrinsic stability, G4 DNA
structures can form obstacles for DNA replication resulting in DNA synthesis pausing just
before the G4 DNA structure on the template strand. Compounds that stabilize G4 DNA
structures can thus be detected by an increased pausing before the G4 DNA structure
and hence a reduced amount of full-length products. Importantly, compounds that bind
DNA non-selectively and therefore cause DNA replication arrest, can also be detected
when running the experiments in parallel with a non-G4 control template. We first
performed a primary screen that showed a clear G4 stabilization by 7ba-7fa as indicated
by a strong replication pausing one or two nucleotides before the first G-tract in the G4
structure (Figure 2B, denoted with asterisks; Figure S3B). 7ba-7fa also displayed a clear
selectivity for G4 DNA compared to the non-G4 control template (Figure 2 versus S4)
although they also slightly suppressed primer utilization for both the G4 and non-G4 DNA
(Figure 2B, Figure S3A, S4A, S4C). In line with the FRET melting studies, none of the
tested neutral 3,3-diindolyl-methyl derivatives (5) showed G4 stabilization in this assay
(Figure 2). Both the FRET melting studies and this assay clearly showed that 7ba induced
the strongest stabilization (Figure 2). The 4- and 7-substituted derivatives 7aa and 7da
both have low effects, which likely can be attributed to sterical hindrance to adopt a
conformation that match the G-tetrad and also suggest that the 5-bromo substituent likely
is not of key importance for the effect of 7da. In the Taq polymerase stop assay, we also
detected strong pausing and reduced amount of full-length products by 7fa, however this
was not found with the FRET melting assay (Figure 2). Furthermore, both the central ethyl
substituent and reversing the amide had only mild impact on the stabilization ability (7fa
versus 7ca and 7ea versus 7da, respectively) in this assay.
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Next, we performed dose-response Taq polymerase stop assays to determine the
efficiency and selectivity of 7aa, 7ba, and 7ca by calculating the IC50 values (Table 1,
Figure S5, S6). All three compounds showed a clear dose response, and the IC50 for the
G4 DNA template was 1.5, 0.17, and 0.24 µM for 7aa, 7ba, and 7ca, respectively (Table
1, Figure S5). For the non-G4 DNA template, the IC50 values were >5, 2, and 1.7 µM,
respectively (Table 1, Figure S6). These data demonstrate the lowest IC50 values for
7ba and 7ca, and a strong selectivity by both compounds for the G4 DNA template
compared to the non-G4 DNA.
Figure 2. (A) Evaluation of the compounds ability to stabilize the Pu24T c-MYC G4 DNA structure using
the FRET assay (at 2 µM ligand concentration). Tm in absence of ligands of FPu24TT is 62.7 ± 0.3 °C. (B)
Primary screening of the compounds (at 1 µM ligand concentration) by Taq-polymerase stop assay using
Pu24T c-MYC G4 DNA template. *indicates pausing sites. Grey arrow represents full-length product, black
arrow represent primer – start of the reaction. (C) Relative quantification of full-length product in each
reaction. Error bars correspond to SD of at least three independent experiments.
Synthesis of asymmetrical compounds
Based on the data above, the positioning of the side chains on the 3,3-diindolyl-methyl
central fragment proved to be highly important with position 5 giving the most efficient G4
DNA stabilizing compounds in both assays. Derivative 7ba with methylated quinoline side
chains in position 5 was the most promising derivative and other side chains in position 5
did not result in compounds with any G4 stabilizing capacity at the tested concentrations.
However, we hypothesized that not both side chains are required for an efficient binding
and stabilization, which thus would open for the possibility to use one of the side chain
positions for e.g. improving compound properties or reaching additional electrostatic
interactions with the phosphate backbone. Hence, a combined derivative with one
quinoline and one piperazine side chain in position 5 of the 3,3-diindolyl-methyl central
fragment was synthesized (Scheme 2).
Many different strategies were tested to achieve these derivatives (Scheme S2).
However, the successful synthetic method (Scheme 2) started with a coupling reaction
between one amino group of 3,3'-methylenebis(1H-indol-5-amine) 3b with 4-(4-
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methylpiperazin-1-yl)butanoic acid directly followed by the coupling of the other amino
group with quinoline-3-carboxylic acid using HATU and DIPEA conditions to afford the
unsymmetrical 5bac in 70% yield. Subsequent methylation gave the tri methylated
compound 7bac in 88% yield.
Scheme 2. Synthesis of 1,1,4-trimethyl-4-(4-((3-((5-(1-methylquinolin-1-ium-3-carboxamido)-1H-indol-3-
yl)methyl)-1H-indol-5-yl)amino)-4-oxobutyl)piperazine-1,4-diium iodide. Reaction conditions: a) 4-(4-
methylpiperazin-1-yl)butanoic acid (4c), quinoline-3-carboxylic acid (4a), HATU, DIPEA, DMF, rt, 12 h, 70%
yield; b) MeI (6a), DMF, 40 °C, 24 h, 88% yield.
G4 stabilization by the asymmetrical analogues
Next, the 3,3-diindolyl-methyl derivatives with one quinoline and one piperazine side
chain (5bac and 7bac) were tested for their ability to affect the progression of the Taq
DNA polymerase with Pu24T c-MYC G4 DNA and non-G4 DNA (Figure S7). Similar to
the other 5 and 7 analogues, the charged 7bac stabilized the G4 structure whereas the
neutral 5bac did not (Figure S7). To test the efficiency of 7bac to selectively stabilize G4
structures, we also performed the dose-response Taq polymerase stop assay (Figure S5-
S6). 7bac displayed a clear dose response and efficiently blocked the Taq polymerase
1-2 nucleotides before the G4 DNA structure. In fact, the 3,3-diindolyl-methyl derivative
with one quinoline and one piperazine side chain, 7bac, displayed an IC50 of 0.2 µM,
showing that it is equally active as the derivative with two methylated quinolines (7ba)
(Table 1). Furthermore, this asymmetric analogue demonstrates an impressive selectivity
for the G4 template over the non-G4 template (20-folds).
To further confirm that these derivatives stabilize the Pu24T c-MYC G4 DNA structure,
we used thermal CD spectroscopy. Thermal CD melting experiments again showed that
the compounds with methylated side chains are able to stabilize the G4 DNA structure
from thermal melting, whereas the derivative with only piperazine side chains (5ac) did
not have any effect (Figure S8). Importantly, the CD studies also showed that the
compounds did not affect the structural folding of the Pu24T G4 DNA structure (Figure
S9). However, this assay did not give the same internal order of efficiency as the Taq
DNA polymerase stop assay and showed that 7bac (with one methylated quinoline and
one piperazine side chain) stabilized the G4 DNA structure much more efficient than 7ba
(with two methylated quinolines). This can likely be explained by the difference in the
compound’s interactions with the G4 DNA (stacking versus electrostatic interactions)
which may affect these assays differently. Furthermore, the FRET melting assay showed
similar results as the Taq polymerase stop assay with high melting temperatures for both
7ba and 7bac (Table 1, Figure S10). The FRET assay further showed intermediate
stabilization for 7ca whereas both 5bac (the non-methylated version of 7bac) and 7aa
were unable to stabilize the Pu24T c-MYC G4 DNA structure from thermal denaturation.
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Binding affinity studies
To investigate the compounds (7aa, 7ba, 5bac, and 7bac) binding affinity to the Pu24T
c-MYC G4 DNA structure, we used MST (Table 1, Figure S11). The 3,3-diindolyl-methyl
derivative 7ba with two methylated quinoline side chains in position 5 was the strongest
binder in this assay with a Kd of 0.49 µM, whereas 7aa with two methylated quinoline side
chains in position 4 did not reach saturation at the highest concentration (10 µM). This
again emphasize the importance of the correct positioning of the side chains on the
central fragment for an efficient binding to the G4 DNA structure. As expected, the
methylated 7bac (with one quinoline and one piperazine side chain) displayed a much
stronger binding affinity (3.05 µM) compared to the non-methylated homolog 5bac which
did not bind to the Pu24T c-MYC G4 DNA in this assay. However, the difference in binding
affinity between 7ba and 7bac was surprisingly high. Therefore, the FID assay with
thiazole orange (TO) was used to confirm these binding affinities (Table 1, Figure S12-
13). This assay ranked the compounds in the same internal order, but showed a more
similar binding affinity (1.4 µM and 1.7 µM for 7ba and 7bac, respectively).
Table 1. Summary of binding and stabilization of the most promising 3,3-diindolyl-methyl derivatives.
∆Tm
∆Tm °Ca
IC50 (µM)c
e
Apparent Kd
(µM)
°Cb
Ligands
7aa
Kdd (µM)
1 µM
2 µM
5 µM
G4 DNA
dsDNA
>5
1.55 ±
0.13
no
no
0.7 ± 0.2 0.9 ± 0.1
1.8 ± 0.3
5.3
saturation displacement
0.17 ±
0.06
0.24 ±
0.06
7ba
7ca
4.2 ± 0.8 10.9 ± 0.6 17.5 ± 1.5 4.0
2.01 ± 0.3 0.49 ± 0.04
1.42 ± 0.06
n.d.
1.72 ±
n.d.
0.8 ± 0.1 5.2 ± 0.6
0.2 ± 0.1
1.5 ± 0.8 8.8 ± 1.3
10.4 ± 1.0
0.6 ± 0.3
3
0.07
5bac
7bac
no
0
n.d.
n.d.
n.d.
no binding
displacement
0.19±
0.015
12.4 ± 0.9 > 12
4.02± 0.45 3.05 ± 0.3
1.67 ± 0.05
aUsing the FRET melting assay at the tabulated compound concentrations (see also figure S1 and S10).
c
bUsing CD melting (see also figure S8). Using the Taq polymerase stop assay (see also figure S5-6).
e
dUsing MST (see also figure S11). Using FID assay (see also figure S12-13). n.d. – not determined.
NMR studies
To further probe the compounds’ abilities to bind the Pu24T c-MYC G4 DNA structure,
we performed NMR titration studies with 7aa, 7ba, 5bac, and 7bac, ranging from 0.1-5
equivalents and using the peaks in the imino-region of the spectra as probes for G4
interactions (Figure 3). This showed some clear differences between the compounds;
7bac induced the biggest changes on the G4 structure with a strong effect even at a 0.1:1
compound to DNA ratio, and an almost complete disappearance of NMR signals at the
higher concentrations was found. Based on the other assays with 7bac, it is unlikely that
this compound induced unfolding of the G4 DNA. Instead, the most likely explanation for
the observed effect is exchange between free and bound form of the G4 DNA on an
intermediate time-scale, resulting in line-broadening. Compound 7aa also induced strong
effects on the G4 DNA structure at substochiometric concentrations whereas 7ba
required almost equimolar concentrations of compound to DNA before a clear effect could
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be observed. However, at these concentrations of 7ba, a new set of peaks starts to
appear that was not observed with the other compounds, suggesting that 7ba have a
stronger binding, resulting in a shift from intermediate to slow exchange on the NMR time-
scale where peaks from both free and bound form of the DNA are observed. As expected,
5bac did not induce any big changes. However, at equimolar and higher amounts of 5bac
compared to DNA, a clear effect on the guanines at the 5´-G-tetrad was clearly visible
indicating that this compound indeed binds G4 DNA structures although it does not
stabilize them to the same extent as the other compounds. All compounds primarily
affected the guanines on the 5´-G-tetrad although almost all guanines were affected at
the higher concentrations of 7aa, 7ba, and 7bac.
Figure 3. NMR spectra of Pu24T c-MYC G4 DNA in presence of different amount of 7aa, 7ba 5bac, and
7bac (0.0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 5:1 compound:G4 DNA). Numbers below peaks in the lower
panel refers to the nucleotide position in the DNA sequence.
Molecular dynamics simulations
We next used molecular dynamics (MD) simulations to understand the molecular details
of the observed G4 DNA binding and stabilization. This will also give valuable information
about the binding mode of the 3,3-diindolyl-methyl derivatives with G4 DNA. Based on
the NMR results, we modeled compounds 7aa, 7ba, 5bac, and 7bac at the 5´-G-tetrad
of the Pu24T c-MYC G4 DNA structure and performed 1 µs MD simulations of these
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complexes. The sampled binding modes for these compounds were subsequently
clustered using principal component analysis (PCA).
Five clusters were obtained for 7aa and contrary to the expected binding mode, all these
clusters showed a partially self-stacked conformation of 7aa with one indole stacking on
a quinoline (Figure 4, Figure S14, and Table S2). This conformation reduced the stacking
interaction of 7aa with the top G-tetrad due to the loss of the planar compound
conformation. The self-stacked conformation of 7aa was also present in its unbound form
thus highlighting the rigidity of this conformation, which also can explain the high
selectivity for 7aa to G4 DNA over other types of DNA. The indole in 7aa that is not
involved in self-stacking and one of the quinolines were involved in stacking interactions
with the top G-tetrad while the other free quinoline either made strong van der Waals
interactions with the phosphate and sugar moiety or stacks with A-12 (first and second vs
third and fourth clusters, respectively).
The MD simulations with 7ba could also be clustered into five main binding modes on the
top G-tetrad (Figure 4, Figure S15, and Table S3). During 50% of the simulations (first
and fourth clusters by occurrence), all four aromatic rings of 7ba were stacked on top of
the G-tetrad. 7ba was also sandwiched by A-12 and G-11 from above in the first binding
mode, which made this the most stable binding mode. When these additional stacking
interactions were temporarily lost, 7ba was able to rotate by 90 degrees to attain the
fourth binding mode (fourth by occurrence, weakest binding affinity, Figure S15). Although
7ba was stacked by A-3 from above, this fourth binding mode was flexible and could shift
towards either the third or the fifth binding modes. This shift pushed both the quinoline
rings outside of the G-tetrad and these rings either stacked with A-12 or interacted with
phosphate and sugar moieties. The second binding mode was stabilized by strong
stacking interactions with one quinoline and one indole group while the other quinoline
ring remained outside of the G-tetrad. Overall, 7ba could stack very efficiently with all the
four top guanines and was also further stabilized by stacking interactions with A-3, A-12,
and G-11, however it remained flexible and was able to rotate on the top of the G-tetrad.
Because of the asymmetry in the 3-3´bisindolylmethanes with two different side chains,
5bac and 7bac, we started with two different binding modes in these simulations, which
gave seven and six binding modes from the MD simulations, respectively (Figure 4 and
Figure S16-17). One of the starting binding modes of 7bac, where the piprazine is close
to G-13, was surprisingly stable and this binding mode was populated at 42% of the
simulation time which was the highest occurrence of all compounds (Figure S17 and table
S5). This binding mode also included interactions between the methylated piperazine and
the DNA backbone and gave the highest binding affinity of all binding modes for all
compounds. A similar starting binding mode for 5bac did not give the same stable binding
mode and yielded three different clusters (second, third and fourth clusters, Figure S16
and Table S4). The other starting binding mode in both 5bac and 7bac yielded four
clusters. The obtained single largest cluster in 7bac suggest that both the piperazine and
the quinoline groups were interacting much stronger with the G4 DNA than that of 5bac.
Overall, the obtained binding energy suggests that 7bac is the strongest binder while
5bac is a very weak binder (Table S6). However, the computed binding energy does not
include entropic contribution and therefore, these cannot be directly compared with
experimental values.
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Additionally, when the quinoline side chain was methylated, the partial charge variance
significantly decreased (Figure S18). This reduction in partial charge variance strengthen
the stacking interaction with the guanines in the G-tetrad because guanine also possess
large charge variance and two groups with large charge variance will thus repulse each
other which will destabilize the stacking interaction. Therefore, a low partial charge
variance is preferable for a strong stacking interaction with the guanines in the G-tetrad,
as observed for the methylated quinoline in 7bac compared to the non-methylated
quinoline in 5bac.
Figure 4. Representative binding poses of compounds during MD simulations. The 5´-G-tetrad (light blue),
nucleotides flanking the G4 DNA structure (orange), and compounds (ball-stick model) from the largest
cluster’s central structure 7aa, 7ba, 5bac, and 7bac. The interactions of 7bac with the DNA backbone is
highlighted with a space filling model. The potassium ion (central purple sphere) is also shown. The binding
energies are tabulated in Table S6.
Selectivity studies
Finally, we investigated if 7ba and 7bac were able to bind and stabilize other types of G4
DNA structures in addition to the Pu24T c-MYC G4 DNA structure. A selection of 11
different G4 DNA structures were screened using the FRET assay and the results showed
that 7ba and 7bac efficiently bind to many types of G4 structures (Figure 5A and Figure
S19). The c-MYC Pu22 and Pu24T G4 DNA structures are very similar and it is thus not
surprising that both 7ba and 7bac effectively stabilize both these structures. Furthermore,
the compounds were also able to stabilize several other G4 DNA structures correlated to
oncogenes, such as the c-Kit and BCL-2 G4 DNA structures.[38, 39] This can be a valuable
attribute as it has been proposed that it in fact can be more effective to target several G4
DNA structures simultaneously in cancer therapy.[40]
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To further study the selectivity of 7ba and 7bac to G4 DNA, we investigated if double-
stranded DNA can compete out the binding of 7ba and 7bac to the G4 DNA structure.
We therefore performed the FRET melting assay with G4 DNA, compound, and
increasing concentrations of double-stranded DNA. This again showed the compound’s
selectivity for G4 DNA, as even 50-fold excess of double-stranded DNA hardly affected
ΔTm in the FRET melting studies (Figure 5B).
Figure 5. A) Selectivity study of 7ba and 7bac (2 μM) for their ability to stabilize different G4 DNA (0.2
μM) and dsDNA (0.2 μM) (see also Figure S19). Pu24T, Pu22 (C-MYC promoter); Ckit1, Ckit2 (C-KIT
promoter); K-RAS (K-RAS gene) and 25ceb (human minisatellite) are parallel G4 forming sequences.
Bcl2 (Bcl2 promoter) and 21g (human telomere) are hybrid G4 forming sequences. Bom17 (Bombyx
telomere) and TBA (thrombin binding aptamer) are antiparallel G4 forming sequences. ∆Tm could not be
calculated for dsDNA. B) Stabilization of Pu24T c-MYC G4 DNA (0.2 µM) by the ligands 7ba, 7bac, and
5bac (2 µM) in the presence of various concentrations of a double-stranded competitor dsDNA (ds26).
Comparison of ∆Tm in the absence of competitor (blue) vs. ∆Tm in the presence of ds26 (brown and black
bars).
Conclusions
Bis-indoles are known to have the potential to efficiently bind and stabilize G4 DNA. In
here, we have developed a new bis-indole core structure and evaluated the effect of both
the position and chemical composition of the side chains attached to this scaffold.
Multistep synthetic method developments generated convenient methods to synthesize
the target compounds, starting from readily available starting materials. Using these
methods, a set of 26 target compounds were synthesized with varying substituents both
in terms of their position on the core structure and their chemical composition. The
compounds were evaluated using an array of different biochemical and biophysical
techniques (FRET melting assay, CD, NMR, Taq polymerase stop assay, MST, FID) as
well as computational techniques (MD simulations and charge variance calculations).
This revealed compounds that bind and stabilize G4 DNA structures with well-defined
dose-response curves and similar or even improved selectivity and activity compared to
previously reported bis-indoles. Furthermore, this work also resulted in important
structure-activity relationships that were correlated to the compound’s interactions with
the G4 DNA to understand why certain structural motifs are so important for the
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interactions with G4 DNA structures. For example, the methylated quinoline side chains
generated the most efficient bis-indoles in terms of G4 binding and stabilization, and the
methylation of the quinoline proved crucial for this activity. The MD simulations and
charge variance calculations show that the reason for this does not lie in the possibility
for electrostatic interactions but rather the formation of a substituent with low charge
variance that can stack efficiently on the top 5’ G-tetrad. However, even though this
charged quinoline is required for binding, it did not prove to be crucial to have two of these
substituents as one could be replaced, while still retaining most of the binding and
stabilization abilities. Moreover, the position of the substituents proved crucial for both the
selectivity and stabilization ability with substitutions in position 5 being preferred as this
has the ability to adopt a conformation that place the substituents in the right position to
bind the G-tetrad. This type of detailed structure-activity relationships connected to a deep
understanding of the compounds G4 DNA interactions open the possibilities to replace or
introduce new substituents. This is of key importance to improve pharmacokinetic
properties or to broaden the use of these G4 binding compounds through e.g. the
introduction of chemical handles for pull-down experiments or fluorophores and,
ultimately, to better understand G4 biology and its therapeutic potential.
Experimental Section
Selected synthesis
Procedure for the preparation of N,N'-(3,3'-methylene-bis(1H-indole-4,3-
diyl))bis(quino-line-3-carboxamide) (ECH-108) (5aa):
HATU (322 mg, 0.85 mmol) was added to the solution of quinoline-3-carboxylic acid (4a)
(140 mg, 0.80 mmol) in a dry DMF (3 mL) and the reaction mixture was stirred at room
temperature until the solids were dissolved completely. DIPEA (155 mg, 1.2 mmol) was
thereafter slowly added to the reaction mixture that was stirred for 10 min followed by
slow addition of di-amine 3a (110 mg, 0.40 mmol) in DMF (3 mL). The mixture was stirred
at room temperature under nitrogen atmosphere for 12 h. The progress of the reaction
was monitored by TLC. Upon completion, the reaction mixture was poured into ice and
filtered through sintered funnel. The solid was dried under vacuum. The pure compound
5aa was isolated in 92% yield as a light-yellow solid.
1H NMR (600 MHz, DMSO-d6): δ = 10.93 (d, J = 2.5 Hz, 1H), 10.06 (s, 1H), 9.31 (s, 1H),
8.44 (d, J = 2.3 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.85 (t, J = 8.4 Hz, 1H), 7.69 (d, J = 8.1
Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.25 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.03
(t, J = 7.8 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 4.62 (s, 1H); 13C NMR (150 MHz, DMSO): δ
= 164.84, 149.22, 138.60, 136.11, 131.85, 130.28, 129.95, 128.47, 127.67, 127.57,
126.80, 124.54, 122.41, 121.47, 121.21, 116.37, 114.09, 110.23, 23.70; MS (ES mass):
m/z 587.3 (M+1); HRMS: m/z calcd for C37H26N6O2[M+H]+ 587.2190, found 587.2180;
Purity: 96.8% (according to HPLC).
Procedure for the preparation of N,N'-(3,3'-methylenebis(1H-indole-4,3-diyl))bis-
(benzo[d]-thiazole-2-carboxamide) (ECH-109) (5ab):
HATU (322 mg, 0.85 mmol) was added to the solution of benzo[d]thiazole-2-carboxylic
acid (4b) (143 mg, 0.80 mmol) in a dry DMF (3 mL) and the reaction mixture was stirred
at room temperature until the solids were dissolved completely. DIPEA (155 mg, 1.2
mmol) was thereafter slowly added to the reaction mixture that was stirred for 10 min
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followed by slow addition of di-amine 3a (110 mg, 0.40 mmol) in DMF (3 mL). The mixture
was stirred at room temperature under nitrogen atmosphere for 12 h. The progress of the
reaction was monitored by TLC. Upon completion, the reaction mixture was poured into
ice and filtered through sintered funnel. The solid was dried under vacuum. The pure
compound 5ab was isolated in 89% yield as a light-yellow solid.
1H NMR (600 MHz, DMSO-d6): δ = 11.02 (s, 1H), 10.23 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H),
7.55 (dd, J = 8.2, 4.5 Hz, 2H), 7.52 – 7.47 (m, 2H), 7.28 (d, J = 8.1 Hz, 1H), 7.12 (t, J =
7.9 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 4.74 (s, 1H); 13C NMR (150 MHz, DMSO): δ =
164.52, 158.14, 152.70, 138.72, 136.66, 129.74, 127.33, 127.29, 124.67, 123.21, 121.61,
120.61, 113.61, 113.50, 110.11, 24.52; MS (ES mass): m/z 599.2 (M+1); HRMS: m/z
calcd for C33H22N6O2S2[M+H]+ 599.1318, found 599.1298; Purity: 99.1% (according to
HPLC).
Procedure for the preparation of N,N'-(3,3'-methylenebis(1H-indole-4,3-diyl))bis(4-
(4-methylpiperazin-1-yl)butanamide) (ECH-110) (5ac):
HATU (456 mg, 1.2 mmol) was added to the solution of 4-(4-methylpiperazin-1-
yl)butanoic acid (4c) (149 mg, 0.80 mmol) in a dry DMF (3 mL) and the reaction mixture
was stirred at room temperature until the solids were dissolved completely. DIPEA (155
mg, 1.2 mmol) was thereafter slowly added to the reaction mixture that was stirred for 10
min followed by slow addition of di-amine 3a (110 mg, 0.40 mmol) in DMF (3 mL). The
mixture was stirred at room temperature under nitrogen atmosphere for 12 h. The
progress of the reaction was monitored by TLC. Upon completion, the reaction mixture
was diluted with (15 mL) cold water and extracted with isopropanol and dichloromethane
(3:1) (3 × 20 mL). The combined organic phases were washed with brine (50 mL), dried
over anhydrous Na2SO4, filtered, and concentrated under a reduced pressure. The
residue was purified by column chromatography over basic alumina using MeOH–DCM
(0.1:9.9) to give N,N'-(3,3'-methylenebis(1H-indole-4,3-diyl))bis(4-(4-methylpiperazin-1-
yl)butanamide) 5ac in 73% yield as white solid.
1H NMR (600 MHz, DMSO-d6): δ = 10.90 (s, 1H), 8.85 (s, 1H), 7.17 (t, J = 8.0 Hz, 2H),
7.02 (t, J = 7.9 Hz, 1H), 6.81 (s, 1H), 4.39 (s, 1H), 2.40 – 2.21 (m, 8H), 2.11 (s, 3H), 2.03
(d, J = 7.5 Hz, 2H), 1.90 (d, J = 8.1 Hz, 2H), 1.45 (q, J = 7.5 Hz, 2H);13C NMR (150 MHz,
DMSO): δ = 171.73, 138.64, 130.99, 124.30, 121.55, 120.92, 114.61, 113.78, 109.19,
57.60, 55.15, 52.99, 46.18, 34.35, 23.73, 22.61; MS (ES mass): m/z 613.2 (M+1); HRMS:
m/z calcd for C35H48N8O2[M+H]+ 613.3973, found 613.3998; Purity: 99.1% (according to
HPLC).
Procedure for the preparation of N,N'-(3,3'-methylenebis(1H-indole-4,3-diyl))bis(4-
mor-pholinobutanamide) (ECH-111) (5ad):
HATU (456 mg, 1.2 mmol) was added to the solution of 4-morpholinobutanoic acid (4d)
(138 mg, 0.80 mmol) in a dry DMF (3 mL) and the reaction mixture was stirred at room
temperature until the solids were dissolved completely. DIPEA (155 mg, 1.2 mmol) was
thereafter slowly added to the reaction mixture that was stirred for 10 min followed by
slow addition of di-amine 3a (110 mg, 0.40 mmol) in DMF (3 mL). The mixture was stirred
at room temperature under nitrogen atmosphere for 12 h. The progress of the reaction
was monitored by TLC. Upon completion, the reaction mixture was diluted with (15 mL)
cold water and extracted with isopropanol and dichloromethane (3:1) (3 × 20 mL). The
combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4,
filtered, and concentrated under a reduced pressure. The residue was purified by column
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chromatography over basic alumina using MeOH–DCM (0.1:9.9) to give N,N'-(3,3'-
methylenebis(1H-indole-4,3-diyl))bis(4-(4-methylpiperazin-1-yl)butanamide) 5ad in 71%
yield as white solid.
1H NMR (600 MHz, DMSO-d6): δ = 10.90 (s, 1H), 8.87 (s, 1H), 7.19 – 7.16 (m, 2H), 7.02
(t, J = 7.8 Hz, 1H), 6.82 (s, 1H), 4.39 (s, 1H), 3.50 (t, J = 4.7 Hz, 4H), 2.33 – 2.21 (m, 4H),
1.92 (t, J = 7.4 Hz, 2H), 1.47 (t, J = 7.4 Hz, 2H); 13C NMR (150 MHz, DMSO): δ = 171.66,
138.64, 130.96, 124.35, 121.57, 120.95, 114.64, 113.78, 109.24, 66.46, 57.92, 53.55,
34.18, 23.72, 22.05; MS (ES mass): m/z 587.2 (M+1); HRMS: m/z calcd for C33H42N6O4
[M+H]+ 587.3340, found 587.3329; Purity: 98.2% (according to HPLC).
Procedure for the preparation of 3,3'-(((3,3'-methylenebis(1H-indole-4,3-
diyl))bis(azanedi-yl))bis(carbonyl))bis(1-methylquinolin-1-ium) iodide (ECH-112)
(7aa):
To the compound 5aa (30 mg, 0.05 mmol) dissolved in DMF (2 mL) was added
methyliodide (6a) (212 mg, 1.5 mmol) drop wise at room temperature under nitrogen
atmosphere. The reaction mixture was stirred at room temperature for 24 h. The progress
of the reaction was monitored by LC-MS. Upon completion, the reaction mixture was
precipitated by slow addition of acetone. The precipitate was filtered through a sintered
funnel and washed with diethyl ether and acetone. The solid was dried under vacuum to
obtain pure compound 7aa as a light brown solid in 93% yield.
1H NMR (600 MHz, DMSO-d6): δ = 10.97 (s, 1H), 10.37 (s, 1H), 9.72 (s, 2H), 9.26 (s, 1H),
8.53 (d, J = 8.9 Hz, 1H), 8.37 (m, 1H), 8.29 (d, J = 8.9 Hz, 1H), 8.08 (t, J = 7.6 Hz, 1H),
7.13 (d, J = 8.0 Hz, 1H), 6.85 – 6.82 (m, 2H), 6.78 (d, J = 7.4 Hz, 1H), 4.67 (s, 6H), 4.40
(s, 1H); 13C NMR (150 MHz, DMSO): δ = 161.37, 149.43, 144.46, 138.65, 138.02, 136.86,
131.61, 130.34, 128.39, 127.82, 127.08, 124.67, 122.53, 120.80, 119.11, 116.64, 113.53,
110.29, 45.73, 34.39; MS (ES mass): m/z 615.2 (M-1); HRMS: m/z calcd for C39H32N6O2
[M-H]+ 616.2581, found 616.2517; Purity: 98.7% (according to HPLC).
Procedure for the preparation of 3,3'-(((3,3'-methylenebis(1H-indole-5,3-
diyl))bis(azanedi-yl))bis(carbonyl))bis(1-methylquinolin-1-ium) iodide (ECH-113)
(7ba):
To the compound 5ba (30 mg, 0.05 mmol) dissolved in DMF (2 mL) was added
methyliodide (212 mg, 1.5 mmol) drop wise at room temperature under nitrogen
atmosphere. The reaction mixture was stirred at room temperature for 24 h. The progress
of the reaction was monitored by LC-MS. Upon completion, the reaction mixture was
precipitated by slow addition of acetone. The precipitate was filtered through a sintered
funnel and washed with diethyl ether and acetone. The solid was dried under vacuum to
obtain pure compound 7ba as a light brown solid in 95% yield.
1H NMR (600 MHz, DMSO-d6): δ = 10.89 (s, 1H), 10.79 (s, 1H), 10.01 (s, 1H), 9.80 (s,
1H), 8.59 – 8.56 (m, 2H), 8.41 – 8.38 (m, 1H), 8.15 (t, J = 7.6 Hz, 1H), 8.01 (d, J = 1.9
Hz, 1H), 7.49 (dd, J = 8.7, 2.0 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 2.2 Hz, 1H),
4.73 (s, 3H), 4.17 (s, 1H);13C NMR (150 MHz, DMSO): δ = 160.60, 150.53, 145.66,
139.09, 137.23, 134.53, 131.80, 131.12, 130.01, 129.04, 128.56, 127.40, 124.51, 119.84,
116.08, 114.41, 111.96, 111.37, 46.07, 21.61; MS (ES mass): m/z 615.2 (M-1); HRMS:
m/z calcd for C39H32N6O2 [M - H]+ 615,2503, found 615.2517; Purity: 99.3% (according to
HPLC).
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Procedure for the preparation of N-(3-((4-(4-(4-methylpiperazin-1-yl)butanamido)-
1H-indol-3-yl)methyl)-1H-indol-4-yl)quinoline-3-carboxamide (ECH-114) (5bac):
DIPEA (155 mg, 1.2 mmol) was added to a solution of di-amine 3b (110 mg, 0.40 mmol)
in a dry DMF (2 mL) and then 4-(4-methylpiperazin-1-yl)butanoic acid (4c) (59 mg, 0.32
mmol) and HATU (125 mg, 0.33 mmol) in a dry DMF (2 mL) was slowly added drop wise
over 6 h under nitrogen atmosphere at room temperature. Next, quinoline-3-carboxylic
acid (4a) (69 mg, 0.4 mmol) and HATU (152 mg, 0.4 mmol) in a dry DMF (2 mL) was
added drop wise to the reaction mixture. The mixture was stirred at room temperature
under nitrogen atmosphere for 2 h. The progress of the reaction was monitored by LCMS.
Upon completion, the reaction mixture was diluted with (15 mL) cold water and extracted
with isopropanol and dichloromethane (3:1) (3 × 20 mL). The combined organic phases
were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by column chromatography over basic
alumina using MeOH–DCM (0.1:9.9) to give N,N'-(3,3'-methylenebis(1H-indole-4,3-
diyl))bis(4-(4-methylpiperazin-1-yl)butanamide) 5bac in 70% yield as a white solid.
1H NMR (600 MHz, DMSO-d6): δ = 10.78 (s, 1H), 10.67 (s, 1H), 10.45 (s, 1H), 9.62 (s,
1H), 9.39 (s, 1H), 8.97 (s, 1H), 8.15 (d, J = 8.25 Hz, 1H), 8.12 (d, J = 8.32 Hz, 1H), 8.01
(s, 1H), 7.90 (t, J = 7.8 Hz, 1H), 7.85 (s, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 8.8 Hz,
1H), 7.35 (d, J = 8.6 Hz, 1H), 7.23 (s, 2H), 7.06 (d, J = 18.1 Hz, 2H), 4.08 (s, 2H), 2.34 –
13
2.22 (m, 8H), 2.14 (s, 3H), 1.71 (t, J = 7.3 Hz, 2H), 1.03 (s, 4H); C NMR (150 MHz,
DMSO): δ = 170.85, 163.96, 149.67, 148.87, 136.12, 134.17, 133.60, 131.60, 131.32,
130.67, 129.60, 129.25, 128.54, 127.86, 127.38, 127.33, 127.00, 124.13, 123.89, 118.79,
116.31, 115.25, 114.58, 114.36, 111.57, 111.45, 111.37, 109.75, 57.78, 55.14, 53.01,
46.13, 46.09, 34.76, 22.94, 21.44; MS (ES mass): m/z 600.4 (M+1); HRMS: m/z calcd for
C36H37N7O2 [M+H]+ 600.3081, found 600.3088; Purity: 97.1% (according to HPLC).
Procedure for the preparation of 1,1,4-trimethyl-4-(4-((3-((4-(1-methylquinolin-1-
ium-3-carboxamido)-1H-indol-3-yl)methyl)-1H-indol-4-yl)amino)-4-
oxobutyl)piperazine-1,4-diium iodide (ECH-115) (7bac):
To the compound 5bac (30 mg, 0.05 mmol) dissolved in DMF (2 mL) was added
methyliodide (212 mg, 1.5 mmol) drop wise at room temperature under nitrogen
atmosphere. The reaction mixture was stirred at room temperature for 24 h. The progress
of the reaction was monitored by LC-MS. Upon completion, the reaction mixture was
precipitated by slow addition of acetone. The precipitate was filtered through a sintered
funnel and washed with diethyl ether and acetone. The solid was dried under vacuum to
obtain pure compound 7bac as a light brown solid in 88% yield.
1H NMR (600 MHz, DMSO-d6): δ = 10.87 (s, 1H), 10.78 (s, 1H), 10.72 (s, 1H), 10.01 (s,
1H), 9.81 (d, J = 4.3 Hz, 2H), 8.61 – 8.57 (m, 2H), 8.41 (t, J = 8.0 Hz, 1H), 8.16 (t, J = 7.7
Hz, 1H), 7.98 (s, 1H), 7.84 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.29–
7.25 (m, 2H), 7.10 (s, 1H), 7.04 (s, 1H), 4.74 (s, 3H), 4.10 (s, 2H), 3.85 (s, 6H), 3.64 (dd,
J= 10.8, 5.8 Hz, 2H), 3.38 (s, 8H), 3.30 (s, 2H), 3.26 (s, 3H), 2.04 (s, 2H); 13C NMR (150
MHz, DMSO): δ = 169.17, 160.58, 150.51, 145.66, 139.12, 137.25, 134.50, 133.78,
131.82, 131.14, 131.00, 130.01, 129.03, 128.57, 127.40, 127.34, 124.45, 124.06, 119.87,
116.05, 115.29, 114.44, 114.06, 111.96, 111.63, 111.32, 109.78, 54.87, 53.75, 46.09,
34.87, 32.52, 26.67, 21.58, 17.65; MS (ES mass): m/z 642.2 (M-2); HRMS: m/z calcd for
C39H44N7O2 [M-2H]+ 642.3551, found 642.3536; Purity: 96.9% (according to HPLC).
Folding of G4 structures for FRET study
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Synthetic oligonucleotides for FRET study were purchased from Eurofins Genomics.
Stock solutions were prepared in water at 100 µM concentration. The sequences used
are listed in supplementary Table S1. All the oligonucleotides except Pu22 were prefolded
in 10 mM lithium cacodylate buffer (pH 7.4), with 10 mM KCl and 90 mM LiCl by heating
for 5 min at 95 °C and then quick cooling on ice. Pu22 was folded in 10 mM lithium
cacodylate buffer (pH 7.4), with 2 mM KCl and 98 mM LiCl.
FRET melting assay
The fluorescence resonance energy transfer (FRET) occurs between two dyes (5'-FAM
as donor and 3'- TAMRA as acceptor) linked at both extremities of a DNA oligonucleotide.
When the oligonucleotides are folded into G4 structures, the donor and acceptor are in
close proximity, which results in an energy transfer from the donor to the acceptor. This
process can be detected by a reduction in the fluorescence emission of the donor.
Fluorescence emission of the donor is recovered when the temperature increment
triggers the thermal denaturation of the G4 structure. The experiments were performed in
a Bio-rad CFX96 real-time PCR device at temperatures from 10 to 95 °C with a heating
rate of 1.5 °C/min using a 492-nm excitation wavelength and a 516-nm detection
wavelength in 96-well plates. Each condition was tested in duplicate and analysis of the
data was carried out by using Excel and Origin 8 software. In each well, 200 nM of labelled
oligonucleotide was heated in the presence or absence of the ligand (and with or without
the competitor dsDNA) at the specified concentrations. Emission of 5'-FAM was
normalized between 0 and 1, and the melting temperature (Tm) is defined as the
temperature at which 50% of the G4 structures are denatured (the temperature when the
normalized emission was 0.5). The stabilization (∆Tm) is calculated from comparison of
Tm of the fluorescently labeled oligonucleotide in the presence or absence of the ligand.
Taq-polymerase stop assay
1 µM TET-labeled primer was annealed to 1.25 µM template DNA in 50 mM KCl by
heating the reaction at 95 °C for 5 min and slowly cool down overnight to room
temperature. Folding of the G4 structure and primer annealing proceeded simultaneously.
For each reaction in the screening procedure, 40 nM annealed DNA was incubated with
1 µM compounds or 1% (v/v) DMSO (control reaction) for at least 30 min in 50 mM KCl,
10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, and 200 mM dNTPs at room temperature. DNA
synthesis was started by the addition of 0.625 U of recombinant Taq DNA-polymerase
(Thermo Scientific) into the reaction mixture (10 µL), and incubated 30 min at 50 °C. The
reaction was stopped by addition of one volume of stop solution (95% formamide, 20 mM
ethylenediaminetetraacetic acid (EDTA), and 0.1% bromophenol blue) into the reaction.
A total of 8 µL of the mixture was loaded into a 10% polyacrylamide gel containing 8 M
urea, 25% formamide, and 1x tris/borate/ethylenediaminetetraacetic acid (TBE). The gel
was visualized with a Typhoon Scanner 9400 (GE Healthcare) at λ = 532 nm and
quantified with the Image Quant TL 8.1 software (GE Healthcare). Quantifications are
displayed as an average of two experiments ± absolute error.
For the determination of IC50 values, the DNA was incubated with different concentrations
(5, 2, 0.8, 0.32, 0.13 0.05, 0.025 and 0.008 µM) of the tested compounds or 5% (v/v)
DMSO. IC50 values were calculated by fitting the full-length products into the dose
response function in the OriginPro 2016 software. All reactions were independently
repeated three times and standard deviation was calculated.
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10.1002/chem.202000579
Chemistry - A European Journal
CD melting experiment
50 µM folded oligonucleotides diluted to 5 µM concentration into 10 mM Tris (pH 7.5),
10 mM KCl and 10 µM compounds or 1.25% DMSO were used for CD melting studies.
A quartz cuvette with a path length of 1 mm was used for the measurements by
JASCO-720 spectropolarimeter (Jasco Internatiol Co. Ltd.). First, CD spectra were
recorded at 25 °C over λ = 230-350 nm with an interval of 0.2 nm and a scan rate of 100
nm/min. The corresponding buffer was used alone for baseline correction of each
measurement. Melting curves were recorded at 264 nm between 25-95 °C at a speed of
1 °C/min. Melting temperature (Tm) is defined as the temperature at which 50% of the
G4 structures are unfolded. Tm values were estimated by fitting the melting curves into
a dose response function using the OriginPro 2016 software.
Fluorescence Intercalator Displacement Experiments
The experiments were performed at 25 °C on a Jasco FP- 6500 spectrofluorometer
equipped with a temperature controller. 0.25 μM of prefolded G4-DNA in 10 mM
potassium phosphate (pH 7.4), 100 mM KCl buffer was mixed with 0.50 μM Thiazole
Orange (TO) and incubated for 2 minutes before the fluorescence spectrum was recorded
(λex = 501 nm; λem = 510 − 650 nm). Then ligands were added to the mixture stepwise
with a 2 min equilibration period, and the fluorescence spectra were recorded. The
percentage of TO displacement was calculated from the fluorescence intensity (ꢀ) at the
emission maxima, using the following equation:
ꢀ
ꢁꢂꢃꢄꢂꢅꢆꢇꢈꢂ ꢉꢊ ꢋꢌ ꢍꢎꢏꢐꢑꢇꢄꢂꢒꢂꢅꢆ = 100 − ( ꢔ100)
ꢀꢓ
where, ꢀ0 is the initial fluorescence intensity of TO bound to G4-DNA.
The percentage of TO displacement was plotted as a function of the concentration of
added ligands and DC50 is determined. The association binding constant (Ka) of the
ligands were calculated from the following equation using ꢕꢖꢗꢘas 5.01X106 M-1:
ꢕꢖꢗꢞꢔ [ꢋꢌ]
[ꢑꢎꢈꢇꢅꢍ]ꢟꢓ
ꢕꢖꢙꢚꢛꢖꢜꢝ
=
Micro Scale Thermophoresis (MST)
The Pu24T G4 DNA sequence with a 5' CY5 label was folded in KCl buffer (10 mM
phosphate, 100 mM KCl, pH 7.4) by heating at 95 ºC for 5 min and then cooling to room
temperature. All the experiments were performed in 10 mM phosphate pH 7.4, 100 mM
KCl, 0.05% Tween20 and 4% BSA. The labelled DNA concentration is held constant at
25 nM and ligand concentration is varied from 0.15 nM to 10 µM (fourteen 1:1 dilutions).
The samples were loaded into standard MST graded glass capillaries and MST
experiment is performed using Monolith NT.115 (Nano Temper, Germany) with 40% LED
power. Data was analyzed using the Nano Temper analysis software and plotted in
OriginPro 8.
Nuclear magnetic resonance
The G4 DNA stock solutions was prepared by folding 100 µM c-MYC Pu24T in 10 mM
potassium phosphate buffer (pH = 7.4) and 35 mM KCl by heating to 95 °C and slowly
cooling to room temperature overnight. 10% D2O and 10% DMSO-d6 was added to the
DNA stock solutions, yielding a final DNA concentration of 82 μM. NMR samples were
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10.1002/chem.202000579
Chemistry - A European Journal
prepared by sequential addition of 7aa, 7ba 5bac, and 7bac from 20 mM DMSO stock
solutions to 220 μL of the DNA solution which was then transferred to 3 mm NMR tubes.
Control samples with Pu24T c-MYC G4 DNA with and without 10% DMSO was also
performed to verify that DMSO did not have a significant effect on the DNA structure. All
spectra were recorded at 298 K on a Bruker 850 MHz Avance III HD spectrometer
equipped with a 5 mm TCI cryoprobe. Excitation sculpting was used in the 1D 1H
experiments, and 256 scans were recorded. Processing was performed in Topspin 3.5
(Bruker Biospin, Germany).
G4 complex modeling
For Pu24T c-MYC G4 DNA, structure coordinates were downloaded from PDB with PDB-
ID 2MGN[41]. From the Pu24T structure, the bound ligand was removed. In the next step,
three-dimensional structural coordinates of 7aa, 7ba 5bac, and 7bac were generated
using Avogadro package[42]. To model the Pu24T c-MYC G4 DNA complexes, docking
was performed at the 5'-terminal G-tetrad using Autodock Vina[43]. Two potassium ions
were preserved inside the G4 channel to maintain its stability during the simulations.
Molecular dynamics simulations
All G4-compound complexes were prepared for molecular dynamics simulations using
GROMACS[44] by placing these at the center of a periodic dodecahedron box and
solvating with water molecules. Subsequently, the system was neutralized by adding an
excess of 0.100 M KCl using the GROMACS tools. For the DNA, Amber99SB[45] with
PARMBSC1[46] improvements were used as force-field parameters. For water, the TIP3P
model[47] was considered while ion parameters were taken from the following reference[48].
For 7aa, 7ba 5bac, and 7bac, at first, their geometry was optimized and ESP was
calculated with HF/6-31G* basis set using Gaussian-16[49] and subsequently the partial
atomic charges were calculated with the RESP method using the AmberTools
package[50]. All four compounds’ force-field parameters were generated from GAFF using
the AmberTools package and converted to GROMACS format using acpype script[51].
Subsequently, MD simulations were performed using GROMACS package.[44] Parameter
settings for all these stages were previously described in the following reference[36].
Length of the MD simulations were 5*200 ns for each compound. All trajectories were
merged for respective complexes, processed, and further used for the analysis.
Conformational clustering for each compound bound to their corresponding G4 DNA
structure was performed with the gmx_clusterByFeatures tool using PCA based
conformational
clustering
(https://github.com/rjdkmr/gmx_clusterByFeatures).
Subsequently, the first 50 frames of each cluster were considered for binding energy
calculation using the g_mmpbsa tool [52, 53]. The obtained MD trajectories were visualized
and images were rendered using VMD[54].
Acknowledgements
Work in the Chorell lab was supported by the Kempe foundations (SMK-1632) and the
Swedish Research Council (2017-05235). Work in the Sabouri lab was supported by Knut
and Alice Wallenberg Foundation (KAW2015-0189), the Swedish Research Council (VR-
MH 2018-2651), the Swedish Cancer Society (CAN2019/126), and the Medical Faculty
of Umeå University. The MD simulations were performed on resources provided by the
Swedish National Infrastructure for Computing (SNIC) at HPC2N Umeå, Sweden. We
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10.1002/chem.202000579
Chemistry - A European Journal
also thank the Knut and Alice Wallenberg foundation program NMR for Life
(www.nmrforlife.se) for NMR spectroscopy support.
Keywords
Bis-indole, DNA structures, Drug design, G-Quadruplexes, Nitrogen heterocycles
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Table of contents graphical abstract
In this study, synthetic methods to a new bis-indole core
structure with various substituents in different positions are
developed. The compounds’ interactions with G4 DNA are
evaluated using an array of experimental and computational
techniques revealing highly efficient G4 stabilizing
compounds and information about key components for
efficient G4 interactions.
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