Kinetic Target-Guided Synthesis of Small-Molecule G-Quadruplex Stabilizers

Article abstract of DOI:10.1002/open.202000261

The formation of a G-quadruplex motif in the promoter region of the c-MYC protooncogene prevents its expression. Accordingly, G-quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4-(4-m

Full text of DOI:10.1002/open.202000261

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Kinetic Target-Guided Synthesis of Small-Molecule
G-Quadruplex Stabilizers
+ [a, b]
+ [a, c]
[a]
[a]
[a]
Alice Pomeislová ,
Lukáš Vrzal , Jaroslav Kozák, Juraj Dobiaš, Martin Hubálek,
[c]
[a]
[a]
[a, d]
Hana Dvořáková, Paul E. Reyes-Gutiérrez, Filip Teplý, and Václav Veverka*
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The formation of a G-quadruplex motif in the promoter region
of the c-MYC protooncogene prevents its expression. Accord-
ingly, G-quadruplex stabilization by a suitable ligand may be a
viable approach for anticancer therapy. In our study, we used
the 4-(4-methylpiperazin-1-yl)aniline molecule, previously iden-
tified as a fragment library screen hit, as a template for the SAR-
guided design of a new small library of clickable fragments and
subjected them to click reactions, including kinetic target-
guided synthesis in the presence of a G-quadruplex forming
oligonucleotide Pu24. We tested the clickable fragments and
products of click reactions for their G-quadruplex stabilizing
activity and determined their mode of binding to the c-MYC G-
quadruplex by NMR spectroscopy. The enhanced stabilizing
potency of click products in biology assays (FRET, Polymerase
extension assay) matched the increased yields of in situ click
reactions. In conclusion, we identified the newly synthesized
click products of bis-amino derivatives of 4-(4-methylpiperazin-
1-yl)aniline as potent stabilizers of c-MYC G-quadruplex, and
their further evolution may lead to the development of an
efficient tool for cancer treatment.
[
6–9]
1
. Introduction
mortality rates).
expression is one of the potential anticancer strategies.
The G4 motif, a key regulatory element of the human c-MYC
Therefore, G4 stabilization to reduce c-MYC
[
4,9]
Poly-guanine tracts in nucleic acids form non-canonical struc-
tures known as G-quadruplexes (G4s), which were recognized
as an attractive class of targets for therapeutic intervention in
[10]
gene, is located downstream of the promoter region of the c-
MYC gene in the nuclease hypersensitive element III (NHE III )
that controls over 80% of the c-MYC transcriptional activity.
1
1
[1–3]
[5,11]
cancer,
particularly for their role in regulating the expression
[4]
of key oncogenes. It was found that stabilization of G4 near
The NHE III comprises the core 27-nucleotide sequence of the
1
the c-MYC gene promoter using a small molecule can reduce
wild-type c-MYC (Pu27) that contains six guanine tracts and can
[5]
[5,12–15]
the levels of the c-MYC oncoprotein in the nucleus. Increased
c-MYC oncoprotein expression is typical of many cancers and
often associated with poor prognosis (aggressive tumors, poor
clinical outcome and increased metastasis, recurrence and
thus form multiple G4s.
Apart from that, other biologically
relevant sequences have been described, e.g., the ’propeller
type’ parallel-stranded G4 sequence containing the four central
guanine tracts of Pu27, or the 24-nucleotide five-guanine-tract
[
13]
sequence (Pu24) forming a parallel-stranded foldback G4.
Generally, the c-MYC ligands are planar aromatic molecules
that interact with the G4 scaffold via external π-π stacking. In
addition, electrostatic interactions between positively charged
ligands and negatively charged G4-DNA scaffolds contribute to
+
+
[
a] A. Pomeislová, Dr. L. Vrzal, Dr. J. Kozák, Dr. J. Dobiaš, Dr. M. Hubálek,
Dr. P. E. Reyes-Gutiérrez, Dr. F. Teplý, Dr. V. Veverka
Institute of Organic Chemistry and Biochemistry
The Czech Academy of Sciences
[16]
Flemingovo nam. 2
Prague (Czech Republic)
G4 stabilization. The known c-MYC binders include e.g., the
[5]
cationic porphyrin TMPyP4, the synthetic fluorescent dye
E-mail: vaclav.veverka@uochb.cas.cz
[
17–18]
[19–20]
[21]
+
Hoechst 33258,
quindoline
and berberine
deriva-
[
[
[
b] A. Pomeislová,
[22]
[23]
Department of Organic Chemistry
Charles University
Prague (Czech Republic)
c] Dr. L. Vrzal Dr. H. Dvořáková
NMR laboratory
University of Chemistry and Technology
Prague (Czech Republic)
d] Dr. V. Veverka
tives, carbazoles, perylene derivatives, the fluoroquinolone
derivative quarfloxin,
9,10-dione derivative MYRA-A.
anilinoquinazoline derivatives as well as the crescent-shaped
[24–25]
and the 1,4-dihydroxyanthracene-
[
26]
+
More recently, various 4-
[27]
[28]
thiazole peptide TH3
were found to down-regulate tran-
scription and expression of the c-MYC gene in HeLa cells. Other
Department of Cell Biology
Charles University
Prague (Czech Republic)
newly reported c-MYC ligands comprised functionalized imida-
[29]
zo[2,1-i]purine derivatives
amines.
and fluorescent binaphthyl
[30]
+
[ ] These authors contributed equally to this work
The rational design of novel potent and selective G4 ligands
is inherently difficult due to the dynamic nature of typical G4
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.202000261
[13]
©
2020 The Authors. Published by Wiley-VCH GmbH. This is an open access
structures.
Recently, kinetic target-guided synthesis (KTGS)
article under the terms of the Creative Commons Attribution Non-Com-
mercial NoDerivs License, which permits use and distribution in any med-
ium, provided the original work is properly cited, the use is non-commercial
and no modifications or adaptations are made.
proved successful in the development of novel inhibitors of
several enzymes with improved selectivity.
target molecule is exploited to irreversibly assemble its own
[31–36]
In KTGS, the
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inhibitor (ligand) in situ by joining two suitable fragments with 2. Results and Discussion
complementary reactive groups via a ’click reaction’. This term
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typically refers to a 1,3-dipolar cycloaddition between azides
and terminal alkynes that leads to triazole formation, although
2.1. Choice of Fragment Template
[37]
there are several alternative reactions (as reviewed in ref. ).
Specific interactions between fragments and the biological
target bring the two molecules into close proximity with a
Initially, we tested the 11 fragments (Figure 1), which were
[42]
reported by Nasiri et al as c-MYC ligands, in in vitro assays to
evaluate their c-MYC G4-stabilizing potential. In particular, we
employed a standard polymerase extension assay (PEA) using a
template strand containing the Pu27 sequence and tested
thermal stabilization of G4-forming Pu25 oligonucleotide
detected by Förster resonance energy transfer (FRET). All
compounds from this set produced a stop band in PEA at
500 μM, and compound 7 was the strongest G4 stabilizer (ESI,
Section 3.1, Figure S3.1 and S3.2). In addition, compound 7
induced a significant thermal stabilization effect on the labeled
Pu25 oligonucleotide in FRET, with a melting temperature
increase of 10.4�0.6°C at 100 μM, indicating that this com-
pound has a significantly higher binding affinity to c-MYC G4
than other compounds in the respective set (data not shown).
Additionally, a significant fraction of compound 7 is
incorporated into the fluorescent dye Hoechst 333258 that was
¼6
suitable orientation, which decreases the negative ~S values
of 1,3-dipolar cycloaddition, thereby overcoming the otherwise
low reaction rate under non-catalyzed conditions. During in situ
click experiments, the target molecule controls the assembly of
the best alkyne and azide within a pool of available fragments
present in the reaction mixture. By directing the choice of the
best binding partner, the active site of a biological target
promotes the synthesis of the most efficient ligand/
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[31–36,38–40]
inhibitor.
KTGS employing in situ click chemistry allows
to build novel and unique ligands by combining suitable
fragments and to improve previously established ligands
[
41]
towards fine-tuning their selectivity and bioavailability.
date, KTGS has been only used to stabilize telomeric G4.
To
[
38–39]
Here, we present the in situ development of c-MYC G4
stabilizers by KTGS using a 4-(4-methylpiperazin-1-yl)aniline
molecule that was active in the previous library screen targeting
[
43–44]
previously reported as a c-MYC ligand.
decided to use the structure of 7 as a template to build a
focused fragment library through synthetic elaboration.
Therefore, we
[42]
c-MYC G4.
We successfully modified this scaffold for click
chemistry and prepared a series of compounds through both
in situ and conventional chemical synthesis. The clickable frag-
ments and the synthesized click products were tested for their
G4 stabilizing activity, and their mode of binding to c-MYC G4
was characterized using NMR spectroscopy. The combination of
biochemical assays, in situ click experiments, and NMR data
provided a basis for the characterization of structure-activity
relationships that led to the successful development of c-MYC
G4 stabilizers.
2.2. Focused Fragment Library
In order to design new molecules with enhanced c-MYC affinity,
we aimed to construct a small focused fragment library derived
from compound 7 that would be amenable for KTGS. Before
functionalizing compound 7 for click chemistry, it was necessary
to ascertain the role of various chemical moieties of this parent
compound 7 in c-MYC stabilization. For this purpose, a set of
commercially available derivatives of compound 7 that con-
tained modifications at the piperazine and/or at the aniline
moiety was provided (Figure 2, 12–23). All the compounds 12–
2
3 were tested in FRET melting assay and in PEA at 100 μM for
their c-MYC binding ability and compared to the parent
compound 7 (for PEA gel images, see ESI, Section 3.2, Fig-
[
42]
Figure 1. The 11 published compounds (1-11) which were found as c-MYC ligands in a previously performed fragment library screen. The FRET and PEA
assays suggested fragment 7 as the best ligand and it was therefore selected for further elaboration.
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Figure 2. A set of 4-(4-methylpiperazin-1-yl)aniline derivatives containing various chemical moieties. Compounds 12–23 were obtained from commercial
sources.
ure S3.3 and S3.4). The only molecule that exhibited signifi-
cantly increased FRET activity and retention of PEA activity
compared to compound 7 was the bis-amino derivative 17
containing an additional amino moiety in position 2 of the
aromatic ring. With ΔTm increase exceeding 35°C, compound
obtained from the nitro-alkyne 39 and served as a negative
control to assess whether the amino group is essential for
electrostatic interactions between the fragments and the c-MYC
G4. Similarly, compound 12 served as a negative control for
comparison with 7.
1
7 exhibited more than 3-fold ΔTm increase in FRET relative to
The bis-amino series (Figure 3B) was prepared by nucleo-
philic aromatic substitution of 5-chloro-2-nitroaniline with
the parent molecule 7. Furthermore, the reduced activity of the
nitro analogue 12 and inactivity of the compounds 13 and 14
isomeric to 7 suggested necessity of the amino group at the
para-position of the aromatic ring for interaction of the ligand
with c-MYC. Therefore, this amino moiety cannot be used for
further derivatization. Finally, inactivity of compound 15 with
[46]
piperazine, resulting in compound 41
with a nitroaniline
moiety, which was then reduced by Pd/C under hydrogen
[46]
atmosphere to 42.
Compound 42, which contains an o-
phenylendiamine moiety, was N-alkylated with three alkynyl
halides of different linker length to form the homologous series
of compounds 43–45 (Figure 3B) carrying 1–3 methylene units
in the alkynyl chain. Within this bis-amino series, we decided to
prepare only click products derived from alkyne 45 with 3
methylene units because the previously performed SAR study
regarding the mono-amino series (Table 2 for PEA results, and
Table 3 for in situ click results) showed that the Pu27-stabilizing
activity of click products 35 and 38 positively correlated with
the length of the alkynyl linker: the N-alkynyl chain with three
methylene units showed the best Pu27 stabilizing activity.
Furthermore, azides 54 and 55 provided the most active mono-
4
an unsubstituted N -piperazine position and partial restoration
of PEA activity for compound 16 underlines the need of an
aliphatic substituent at this piperazine nitrogen.
Based on these findings, we constructed a fragment library
(
Figure 3A and 3B) that consisted of two different families of N-
alkyne compounds: a mono-amino series directly derived from
(Figure 3A) and a bis-amino series containing an additional
amino functional group at position 2 of the aromatic ring
7
(
Figure 3B). Both series included ’clickable’ compounds alkyny-
4
lated at the N -piperazine position and the resulting click
products. The design of both series was guided by a SAR study
performed simultaneously with compound synthesis.
Table 1. FRET and PEA results at 100 μM for commercial compound 7
derivatives 12–23 in comparison to the parent molecule 7.
The mono-amino series was synthesized by N-alkylation of
the piperazine moiety of 4-(piperazin-1-yl)aniline (compound
Cpd. No
PEA at
100 μM
ΔTm/°C FRET
1
5) with three different alkynyl halides of various chain lengths
at 100 μM
to investigate influence of molecule flexibility on interaction
with c-MYC. Thus, we prepared a homologous series of three
alkynes with 1, 2 or 3 methylene units in the alkynyl chain, that
7
12
1.0
0.2
0.2
–
0.2
0.6
1.1
0.2
0.3
0.1
0.2
0.2
0.2
10.4�0.6
3.7�0.5
0.0
13
[45]
is, 24, 25, and 26 (Figure 3A), respectively. Compounds 24–26
were directly coupled to four selected azides 52–55 (Figure 3C)
14
À 0.3
15
1.6
16
1.7
>35
1.1
1.8
0.9
1.2
1.1
1.1
I
in a Cu-catalyzed 1,3-dipolar cycloaddition (CuAAC), referred to
17
as ’click reaction’, upon 1,4-triazole formation (products 27–38,
18
[38]
19
Figure 3A). The choice of azides 52–53 and 55 was directly
20
[38]
inspired by the study performed by Di Antonio et al and
azide 54 was included as a tertiary-amine analogue to
compound 53. The click product 40, a nitro analog of 38, was
21
22
23
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Figure 3. 4-(piperazin-1-yl)aniline-derived fragment library constructed in the present study. (A) Mono-amino series. (B) Bis-amino series. (C) Azides used for
CuAAC. Compounds from both series (A and B) are sorted by type of reaction whereby they were prepared.
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Table 2. SAR data summary showing both the FRET and PEA results at
00 μM. The PEA values (ESI, Section 3.3, Supplementary Figure S3.5–S3.9)
are determined as a quotient of stop band density of the respective
Table 2. continued
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1
Cpd. Structure
No
PEA at
100 μM FRET at
ΔTm/°C
compound divided by the stop band density of the parent compound 7.
100 μM
Cpd. Structure
No
PEA at
100 μM FRET at
ΔTm/°C
1
00 μM
3
5
0.8
4.2�0.8
7
1.0
0.5
10.4�0.6
3.7�0.5
1
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
24
0.1
3.3�0.4
3
3
6
7
0.5
0.6
3.0�0.8
3.9�0.4
2
2
5
6
0.1
0.5
2.4�0.9
2.4�0.4
2
7
8
0.4
0.5
À 2.3�0.7
À 1.9�0.3
3
8
1.2
7.6�0.4
2
4
4
0
1
0.1
0.3
3.9�0.1
4.1�1.1
29
0.5
À 2.7�0.2
3
0
0.6
0.5
À 1.4�0.3
À 2.5�0.4
42
1.6
0.5
0.5
1.6
0.1
>35
4
3
4
13.0�0.9
15.9�1.0
29.5�0.3
5.6�0.4
31
4
45
3
2
0.7
À 2.3�0.3
4
6
33
0.7
0.9
3.6�0.5
3.6�0.8
47
0.5
3.5�0.4
3
4
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amino series with an additional NH - group. Both series
included the clickable fragments alkynylated on the piperazine
nitrogen as well as the respective click products resulting from
CuAAC with selected cationic azides.
Table 2. continued
2
1
2
3
4
5
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7
8
9
0
1
2
3
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0
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8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
Cpd. Structure
No
PEA at
100 μM FRET at
ΔTm/°C
100 μM
2
.3. Structure-Activity Relationships
4
8
0.7
4.5
5.1�0.7
Here, we investigated how different chemical moieties, present
in compound 7 derivatives, and their respective positions
contribute to c-MYC G4 stabilization. For this purpose, a starting
ligand concentration of 100 μM was used in both the FRET and
PEA biochemical assays; the results are compared to the parent
compound 7 and summarized in Table 2 (for PEA gel images,
see ESI, Section 3.3, Figure S3.5 to S3.9.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
49
33.9�0.2
Amino groups are a common feature of well-established G4
ligands because they bind to phosphate groups of the G4
backbone via electrostatic interactions mediated by bridging
[47]
water molecules.
As expected, the fragments containing
amino groups 7 and 17 exhibited higher stabilization activity at
100 μM in both FRET and PEA than their nitro analogs 12 and
5
0
4.5
>35
5
1, respectively (Table 2), suggesting that a free amino group in
para-position of the aromatic ring is required for the interaction
between the ligand and the c-MYC G4. Additionally, a higher
number of amino groups also showed enhanced c-MYC affinity
5
1
1
7
0.1
1.6
1.0�1.0
>35
(Table 2, e.g., 17 versus 7). These results confirm the original
findings obtained during FRET and PEA evaluation of commer-
cial derivatives of compound 7 (Table 1).
Apart from that, we found that the piperazine moiety of 4-
4
(
piperazin-1-yl)aniline can be N-alkylated at the N position by
different-length alkynyl groups (with one to three methylene
units) without affecting its ability to bind to G4. This was
observed in scaffolds derived from the compound 17 carrying
two amino groups at the aromatic ring (43–45), whereas the
activity was abolished in mono-amino derivatives (24–26).
Interestingly, we observed considerable restoration of the G4
stabilizing activity for the click products 34–35 (in PEA) and 38
(in both PEA and FRET), all of which were prepared from the N-
alkynylated mono-amino derivatives (Table 2). In general, click
products 33–38, derived from compounds 54 and 55, exhibited
partially restored FRET and PEA activity when compared to the
parent compound 7, although the extent of activity restoration
varied widely. Nevertheless, all these click products showed
overall better activity than click products 27–32, derived from
52 and 53, which exhibited a small decrease in Pu25 melting
temperature in FRET at 100 μM, indicating their inactivity.
Moreover, the click product 38 derived from compound 26 with
three methylene units in the alkynyl linker was more active in
both FRET and PEA than its shorter-linker analogs, compounds
36 and 37. A similar trend was also observed in FRET for the
click product 35 and its analogs 33 and 34, although the
difference in activity was rather small. It is obvious that
increased flexibility of the central part of the molecule facilitates
interaction of certain ligand moieties with c-MYC. Accordingly,
amino click products. However, the analogous direct click
reaction of the bis-amino alkyne derivative 45 with azides 54
and 55 was not performed due to the low stability of the o-
phenylendiamine moiety. Instead, o-nitroaniline compound 41
was alkylated with 5-iodopent-1-yne to form 46. In a CuAAC
with azides 54 and 55, alkyne 46 formed triazoles 47 and 48,
respectively. The resulting o-nitroaniline click products 47 and
[38]
4
8 were subsequently reduced by Pd/C under hydrogen
atmosphere to their diamino counterparts, 49 and 50, and
immediately converted into hydrochloride salts to stabilize
them. Lastly, compounds 51 and 17 (Figure 3B), bis-amino
analogs of 7, were prepared as described for compounds 41
and 42, respectively: in a nucleophilic aromatic substitution
with 1-methylpiperazine, 5-chloro-2-nitroaniline formed com-
pound 51; compound 17 was then prepared from 51 by
reduction of the nitro group by Pd/C under hydrogen
atmosphere.
In conclusion, FRET and PEA evaluation of various 4-
(piperazin-1-yl)aniline derivatives showed that the amino group
at the para-position of the aromatic ring was found necessary
for interaction of the ligand with c-MYC. Also, an additional
amino moiety in position 2 of the aromatic ring further
increased the c-MYC stabilizing potency. Therefore, we con-
structed a fragment library that contained the mono-amino
series directly inspired by the parent 7 molecule and the bis-
[48–51]
previously performed studies
also reported the impact of
the linker length or side-chain length and thus molecule
flexibility on interaction with various G4 s. Taken together, these
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Table 3. The relative formation yields of compounds 27–38 given by reaction ratios. X-1, Y-1, and Z-1-the combination reactions of three alkynes with the
corresponding azides; A, B and C-in situ click reactions with and without Pu24 and a control reaction, respectively. The ratio was calculated by comparing
the area of specific m/z value from the extracted ion chromatography in LC-MS experiment.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Compound
CH
units
2
Experiment X-1
Experiment Y-1
Experiment Z-1
A/B
A/C
A/B
A/C
A/B
A/C
27
1
1.8
1.3
1.3
–
–
–
–
–
–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
2
2
8
9
2
3
1.7
4.4
–
–
–
–
3.0
–
–
3
0
1
2
–
–
–
–
1.4
1.5
1.2
1.2
–
–
–
–
31
3
3
2
3
3
–
–
2.5
1.9
–
–
1
2
7.1
5.2
4.7
3.1
–
–
–
–
6.6
7.4
5.1
4.4
34
3
5
3
22.7
10.8
–
–
55.0
42.7
3
3
6
7
1
2
–
–
–
–
4.8
3.4
3.8
2.5
5.4
3.7
4.5
2.6
38
3
–
–
9.8
5.5
16.9
10.5
4
results show that a simple N -alkynylation of the piperazine
moiety at the 4-(piperazin-1-yl)aniline template can destroy the
binding activity of the resulting mono-amino fragment, but an
additional click reaction of the inactive molecule with a suitable
azide can lead to a click product with partly restored activity.
Generally, the bis-amino series exhibited higher affinity
towards c-MYC than the mono-amino series. The FRET activity
of piperazine-alkylated bis-amino compounds 43–45 was lower
than that of the bis-amino standard 17, albeit similar to or
better than the activity of the mono-amino template 7. Both
bis-amino click products 49 and 50 exhibited a significant
increase in activity in FRET experiments at 100 μM, which was
comparable to that of 45 and 17 and even 3-fold higher than
the activity of 7 (Table 1). Moreover, based on PEA measure-
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ments, 49 and 50 appeared to be strongest ligands prepared in
this study with 4.5-fold activity increase compared to com-
pound 7 and significantly higher activity than 45 and 17. The
inactivity of the parent nitroaniline compounds 47 and 48
underlines the importance of the aromatic 1,2-diamino moiety
in 49 and 50 for Pu27 stabilization potency.
Interestingly, the alkynylated derivatives of both series as
well as most mono-amino click products exhibited decreased
activity when compared to the respective parent molecule 7 or
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1
7. We hypothesize that alkynylated derivatives and click
products interact with the G4 structure via both the 4-
piperazin-1-yl)-aniline motif and the alkynyl chain or triazole
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
(
residue, respectively. If the linker connecting the piperazinyl
moiety with the triple bond or triazole ring is short, the
molecule is rigid and interacts with G4 via both moieties
inefficiently. This leads to activity decrease of such fragment.
On the other hand, three methylene units in the linker provide
the molecule with increased flexibility that facilitates effective
binding of the molecule to the G4 structure via both moieties.
This way we could explain activity restoration in the case of the
mono-amino click product 38 and the bis-amino alkyne 45, and
PEA activity increase in the case of the bis-amino click products
Figure 4. Comparative FRET analysis at 50 μM using F-myc25T and FdsT
control), performed with the bis-amino series in compaison to compounds
7 and 12 of the mono-amino series.
(
4
9 and 50.
As compounds 42–45, 49–50, and 17 from the bis-amino
series exhibited sufficiently increased c-MYC stabilization poten-
tial at 100 μM, they were also evaluated by comparative FRET
assay at 50 μM together with their o-nitroanaline analogues 41,
4
6–48, and 51 for their G4-specificity over duplex DNA; the
parent compound 7 and its nitro-analogue 12 were included
for comparison. Therefore, apart from using F-myc25T, all these
compounds were tested in control FRET experiments with a
dual-labelled FdsT oligonucleotide containing a 26-mer ’Q
[52]
sequence’ (ds26) that forms a double-stranded hairpin. The
FRET results for F-myc25T and FdsT at 50 μM are displayed in
Figure 4 (FRET data available in ESI, Section 3.5, Table S3.1). The
data regarding c-MYC stabilization showed significantly in-
creased ΔT values of the amino-, resp. bis-amino derivatives in
Figure 5. Competitive FRET analysis at 25 μM and 10 μM using F-myc25T
and ds26 in the 1:10 ratio. The assay was performed only for selected
compounds that exhibited significant FRET activity at 50 μM.
m
comparison with their nitro-, resp. o-nitroaniline analogs. In
addition, the FRET activity of the homologous series of bis-
amino alkynes 43–45 at 50 μM increased with the number of
methylene units in the alkynyl linker, which is in line with the
FRET measurements at 100 μM (Table 2). The control FRET
experiments with FdsT showed in most cases zero stabilization
potential at 50 μM. Although a slight ΔTm increase was
observed for compound 42, the value was negligible when
compared to ΔTm increase induced for c-MYC (F-myc25T)
stabilization. Therefore, we can conclude that our ligands,
especially the bis-amino compounds 17, 42, 45, 49, and 50,
selectively interact with the c-MYC G4 motif.
The compounds 17, 42, 45, 49, and 50 were tested in a
competitive FRET assay at 25 μM and 10 μM concentration. For
each ligand concentration, two parallel FRET experiments were
performed: one with F-myc25T (0.2 μM), and the second with
the mixture of F-myc25T (0.2 μM) and 10-fold excess of non-
labelled oligonucleotide ds26 (2.0 μM). The results are summar-
ized in Figure 5 (FRET data available in ESI, section 3.5,
Figure 6. Average intensity of click products 27–29 and 33–35 during in situ
click experiment X-1.
Table S3.2). In spite of an apparent ΔT decrease in competitive
m
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Section 3.4, Figure S3.10) clearly showed formation of a stop
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
product in the presence of the Pu27 template strand for all
compound-containing samples (7, 17, TMPyP4, and 5-AMI) at
given concentrations. Moreover, the 100 μM KCl solution
+
produced the stop band as well, as the K ions efficiently
stabilized the G4 s formed within the Pu27 sequence. In
contrast, no stop band was observed in the presence of the
Pu27 all-mut template for any of the samples including the
standard TMPyP4, 5AMI, and the KCl solution. Therefore, we can
confirm that in PEA, the mechanism of stopping the chain
reaction is due to G4 formation induced by the compounds
evaluated in this study. Accordingly, the c-MYC stabilization
potential of the compounds tested corresponds with density of
their stop band.
Taken together, both the FRET and PEA assays showed that
all compounds of the bis-amino series, especially the com-
pounds 17, 42, 45, 49, and 45, are substantially stronger c-MYC
ligands than compounds of the mono-amino series. The
requirement for the free amino group in para-position of the
aniline moiety was confirmed as the fragments containing
amino groups from both series exhibited better c-MYC stabiliza-
tion efficiency in FRET and PEA than their respective nitro- or o-
nitroaniline- analogues. Furthermore, partial restoration of FRET
and PEA activity was observed for mono-amino click products
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Figure 7. Average intensity of click products 30–32 and 36–38 during in situ
click experiment Y-1.
3
3–38, which originated from azides 54 and 55 containing a
tertiary amino moiety. Moreover, FRET activity of these mono-
amino click products 33–38 as well as of the bis-amino alkynes
4
3–45 positively correlated with the number of methylene units
in the alkynyl linker. Finally, FRET and PEA control experiments
performed with G4 non-forming templates confirmed validity
and relevance of our measurements.
2
.4. KTGS Using In Situ Click Experiments
Figure 8. Average intensity of click products 33–38 during in situ click
experiment Z-1.
To evaluate the co-catalytic activity of G4 on formation of the
respective click products, a series of in situ click experiments
employing the 1,3-dipolar azide-alkyne cycloaddition was
carried out. However, KTGS was performed only for the mono-
amino series, because the bis-amino alkynes 43–45 suffered
from poor stability as reported above regarding their synthesis
and isolation. It was assumed that performing a direct click
reaction of the bis-amino alkynes 43–45 with azides 52–55 in
aqueous solution, in the presence of air, and under prolonged
(2-hour) incubation at RT would lead to a quick oxidation of the
bis-amino moiety. Additionally, we decided to carry out all
assays in the presence of the 10-fold excess of ds26, all
compounds retained their c-MYC stabilization ability. However,
a significant ΔTm decrease was observed at lower ligand
concentration, especially for click products 49 and 50 that
showed almost zero activity at 10 μM. Moreover, activity of
compounds 17 and 42 at 10 μM was below 10°C. Therefore,
competitive FRET measurements at lower concentrations were
not performed as no compound activity would be expected.
Additionally, control PEA experiments were performed with
selected compounds to exclude a different inhibition mecha-
nism of the polymerase chain reaction, e.g. by forming a non-
canonical PEA bulge. In regard of the control assay, we used a
G4-non-forming oligonucleotide template ’Pu27 all-mut’ along
with Pu27 on the same gel. In this control assay, we tested the
parent compound 7 (100 μM) and its bis-amino analogue 17
I
in situ click experiments with a catalytic amount of Cu species,
as our preliminary experiments serving for layout optimization
did not show any product formation in the absence of copper
catalysis (data not shown). The same finding regarding the lack
I
of product formation in the absence of the Cu species was also
[38]
reported earlier in the study targeting H-Telo.
For the mono-amino series, three sets of copper catalyzed
[5]
[38]
(
100 μM), the well-established c-MYC ligand TMPyP4 (1 μM), 1-
in situ click experiments were set up.
Each experiment
methyl-1H-indol-5-amine (5AMI, 500 μM) reported as a c-MYC
combined three homologous alkynes 24–26 with two different
azides to form the 1,4-click products (the combination reactions
of three alkynes with the corresponding azides are denoted
[42]
binder by Nasiri et al, and a KCl solution (100 μM) to observe
+
the effect of G4 stabilization by the K ion. The results (ESI,
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here as X, Y and Z). For the products of X, Y, and Z reactions,
three different in situ click experiments were performed simul-
taneously: with Pu24 (A), without Pu24 (B), and a control
reaction (C), wherein Pu24 was added just before quenching
the reaction with TFA. All experiments were performed in
triplicates.
Furthermore, control in situ click experiments were per-
formed to confirm that the co-catalytic activity of the Pu24
oligonucleotide on ligand assembly is due to the G4 secondary
structure formed.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
The control in situ click experiments were carried out with a
26-mer ’Q sequence’ (ds26) that forms a double-stranded
[52]
Experiment X consisted of in situ click reactions of alkynes
hairpin, and in the same manner as the standard in situ click
experiments using the G4-forming Pu24 oligonucleotide. The
results (ESI, Section 7, Table S7.7, S7.9, S7.11 and Chart S7.7,
S7.9, S7.11 for click products; Table S7.8, S7.10, S7.12 and Chart
S7.8, S7.10, S7.12 for alkyne precursors) showed no remarkable
product formation increase in experiments A with ds26 over
experiments B without ds26 and the control experiment C,
because the A/B and A/C ratios were in most cases close to 1.0
(ESI, Section 7, Table S7.13). A substantial A/B and A/C ratio
decrease for some compounds – especially for click products
27, 28, and 30 derived from azides 52 and 53 with a primary
amino moiety-suggests their binding to the double-stranded
ds26 sequence. Nevertheless, based on the results obtained, we
can assume that the increase of click product formation in
presence of Pu24 is the outcome of the c-MYC G4 co-catalytic
activity.
24–26 with azides 52 and 54 resulting in the click products 27–
29 and 33–35, respectively. Experiment Y combined alkynes
24–26 with azides 53 and 55 resulting in click products 30–32
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
and 36–38, respectively. Based on previously performed PEA
measurements that identified the click products of azides 54
and 55 more active than click products derived from azides 52
and 53, we performed an additional in situ click reactions
(experiment Z) combining alkynes 24–26 with azides 54 and 55
to obtain click products 33–38. The average yields of the in situ
click experiments AÀ C for all 6 products of reactions XÀ Z are
shown in Figure 6 and 5 and in ESI, Section 7, Table S7.1, S7.3,
S7.5 and Chart S7.1, S7.3, S7.5. The LC residues (alkyne
precursors) are shown in ESI, Section 7, Table S7.2, S7.4, S7.6
and Chart S7.2, S7.4, S7.6.
In a standard in situ click experiment, detecting a click
product in a templated reaction directly suggests the templat-
ing effect. However, the amounts of the triazole formed in
complex reaction mixtures are usually small and difficult to
detect and quantify. Therefore, all in situ click experiments
Overall, KTGS performed for the mono-amino series in the
presence of a copper catalyst showed the same SAR trends
regarding c-MYC affinity as FRET and PEA biology assays. The
formation yield in the presence of Pu24 was substantially higher
for click products containing three methylene units at the
piperazine-triazole linker than for compounds with one and two
methylene units. It is apparent that increased flexibility of the
aliphatic linker attached to the central scaffold facilitates
suitable orientation of key structural motifs and thus efficient
interaction of the ligand with c-MYC. Moreover, the yield of click
product formation derived from azides 54 and 55 containing a
tertiary amino moiety was higher than for click products
derived from azides 52 and 53 bearing a primary amino moiety.
Our results of control in situ click experiment with G4 non-
forming oligonucleotide showed no substantial increase of click
product formation. Therefore, we can suppose that the click
product formation yield increase in presence of Pu24 is due to
the c-MYC G4 co-catalytic activity.
[38]
contained catalytic copper to increase triazole adduct for-
mation and to facilitate subsequent analysis. Furthermore, the
co-catalytic effect of Pu24 G4 is expressed as an A/B ratio of
area integral of specific m/z in experiment A (with Pu24) and B
(without Pu24). Experiment C and the related A/C ratio were
introduced to assess the Pu24 effect on the LC/MS analysis. All
compounds 27–38 were prepared with increased yields in the
presence of Pu24, thus suggesting a possible co-catalytic
activity for G4 during the formation of the click products
(Table 2, A/B and A/C ratios).
Our data revealed that the presence of three methylene
4
units at the alkynyl chain attached to the N position of the
piperazine moiety increased the stability and yield of the
reaction products 29, 32, 35, and 38, with no significant
differences in compounds with one and two methylene units
(Table 3). Moreover, the A/B and A/C ratios of in situ click
products derived from azides 54 and 55 were higher than those
of click products derived from azides 52 and 53. In this regard,
click products 33–35 derived from azide 54 had the highest A/B
and A/C ratios. Compound 35 had the highest yield of the
in situ click experiments, followed by 38, both of which were
derived from compound 26 with the longest alkynyl linker (3
methylene units). These results were in agreement with the
previously performed SAR study (Table 2) except for compound
2.5. Structural Basis of Compound Binding
The G4 core of Pu24 is formed by the three guanine tetrads
[4–5]
(Figure 4A; ESI, Section 8, Figure S8.1).
To assess the binding
modes of the studied compounds, we followed chemical shift
1
changes in the guanine imino-region of H NMR spectra of the
Pu24-formed G4 upon ligand addition. In particular, the
compounds were sorted based on the perturbation or dis-
appearance of individual assigned imino signals in Pu24 into
three distinct classes (Figure 4B). The class I ligands induced 5’
G-tetrad destabilization because the imino signals of G4, G13,
G8 and G17 disappeared. The residues from the central (G18,
G5, G14) and from the 3’ (G15, G6, G19) G-tetrad were shifted
significantly, indicating stacking or intercalation of these
compounds. The class II compounds induced perturbations
3
8, which showed higher activity in PEA than 35. Interestingly,
the control reaction C showed small but significant increases in
reaction yields when compared with B, suggesting that adding
Pu24 oligonucleotide just before quenching leads to rapid click
product formation. Therefore, the A/B ratio should be consid-
ered the corresponding relative formation yield of a compound.
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within the 5’ G-tetrad (G4, G13, G8 and G17) and central G-
tetrad (G5, G14), indicating stacking of the ligand on the top.
The class III compounds induced only perturbations of two
guanine NH signals from the 5’ G-tetrad (G4, G17), suggesting a
specific interaction with only a minimal effect on the overall
conformation of the Pu24 oligonucleotide. The compounds
inducing no significant change in the imino region were
classified as non-binders.
The classification is summarized in Figure 9B (schematic
changes within NMR spectra are shown in ESI, Sections 8.1–8.4,
Figures S8.2–S8.6). The class I binders 42, 49, 50, and 17 (ESI,
Section 8.1, Figure S8.2) contained a bis-amino moiety and
exhibited high Pu24-stabilizing activity in both PEA and FRET. In
addition, 49 and 50 were the most active click products
prepared in this study. All the class I binders induced significant
shifts in selected peaks and extreme broadening of other peaks,
indicating 5’ G-tetrad disruption and possible binding on the
top of the central tetrad or a ligand intercalation between the
central tetrad and the 3’ tetrad. The class II binders comprised
the homology series of bis-amino N-alkynylated derivatives 43–
weakest because only two guanine NH signals from the 5’ G-
tetrad were affected. Moreover, chemical shift changes were
only moderate compared with class I and II compounds.
Correspondingly, the activity of 40, 46 and 51 was similar to
that of the negative control in PEA and lower than that of 7 in
FRET.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
No significant effects were observed in the imino regions of
NMR spectra of all mono-amino click products 27–38, their N-
alkynylated clickable precursors 24–26, non-alkylated o-nitro-
aniline derivative 41, compound 12 as a nitro-analog of 7 and,
surprisingly, compound 7 itself (ESI, Section 8.4, Figure S8.5 and
S8.6). For compounds 12, 24–26, and 41 and for most mono-
amino click products, this result is consistent with FRET (zero or
only partially restored activity compared to 7) and PEA (activity
similar to negative control) assays, which suggest low binding
affinity to c-MYC G4. However, this category also includes the
template molecule 7 and several mono-amino click products,
34, 35, 37, and 38, which exhibited some stabilizing activity
both in FRET or PEA, and their formation was promoted in the
presence of Pu24 during in situ click experiments. Hence, these
compounds likely have an alternative binding mechanism,
which does not perturb imino groups in the core G4 tetrads.
Notably, mono-amino click products 27–32 that exhibited small
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
4
5 and the o-nitroaniline click products 47 and 48 (ESI,
Section 8.2, Figure S8.3). These ligands were selectively bound
only to the 5’ G-tetrad, and this mode of interaction did not
disrupt the G4 structure. The compounds in class III carried a
nitro group: 46 and 51 were o-nitroaniline derivatives, and 40
was a click product with a nitro group on the aromatic ring and
served as a negative control for compound 38 in SAR (ESI,
Section 8.3, Figure S8.4). This interaction mode should be the
negative ΔT values in FRET, were also classified as non-binders
m
1
by H NMR as they did not induce any G4 signal perturbation.
In summary, we evaluated compounds of our fragment
1
library for their c-MYC G4 binding mode using H NMR
spectroscopy. Based on our results, it is apparent that the bis-
[
47]
Figure 9. (A) Schematic representation of the Pu24 oligonucleotide. Individual G-tetrads are color-coded. (B) Three distinctive classes of binding ligands
were identified by changes in the peaks of individual guanine NH groups within the amidic region of H NMR.
1
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amino compounds 17, 42, 49, and 50 that belonged to the
most active ligands according to FRET and PEA biology assay
also exhibited the strongest c-MYC binding mode (class I
binders). Additionally, most of the remaining compounds of the
bis-amino series belonged either to the binder class II or III,
both of which represent a weaker binding mode. On the other
hand, all compounds of the mono-amino series, including the
parent compound 7, were determined as non-binders. These
findings are in accordance with FRET and PEA results that
revealed mono-amino compounds as less active than com-
pounds of the bis-amino series.
Each lyophilizate was dissolved in 20 mM KH
PO , 70 mM KCl, NaN
2 4 3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
(
0.02%), pH 7.0 to the final concentration of 15 μM. Subsequently,
the solution was annealed at 95 C for 15 minutes and allowed to
gradually cool down to room temperature (RT), adopting the typical
‘propeller’ shaped conformation.
°
FRET
The dual-labelled oligonucleotides Fc-myc25T and FdsT (5’-FAM
and 3’-TAMRA) were purchased from Sigma-Aldrich®. Regarding Fc-
myc25T, the shorter sequence of the NHEIII1 region (Pu27 or c-MYC
G4) was used. This sequence contains only five G-tracts, enabling
folding in 1–4 and 2–5 c-MYC G4 conformers. The last GG was
removed to increase FRET efficiency. The sequence of the Fc-
myc25T oligonucleotide is:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
3. Conclusions
Fc-myc25T: 5’-FAM-TGGGGAGGGTGGGGAGGGTGGGGAA-TAMRA-3’
Stabilization of G-quadruplexes formed in the c-MYC promoter
region may be a viable strategy for anticancer therapy. We used
the 4-(4-methylpiperazin-1-yl)aniline molecule, identified in a
On the contrary, the FdsT used for control measurements contained
a 26-mer Q-sequence (ds26) forming a double-stranded hairpin:
FdsT: 5’-FAM-TATAGCTATATTTTTTTATAGCTATA-TAMRA-3’
[42]
previously published study as a c-MYC ligand, as a template
for the SAR-guided design of a small focused fragment library
suitable for click chemistry. The biochemical assays revealed
that the presence of a free amino substituent at the aromatic
ring was essential for stabilizing potency of this type of
molecules and that an additional amino group at position 2
further increased their activity. Moreover, the piperazine ring
All FRET melting experiments were performed in 10 mM Cacodylate
buffer (pH=7.2) containing 95 mM LiCl and 5 mM KCl. Oligonucleo-
tides were heat-denatured at 95°C for 10 minutes and cooled by
approximately 1 C per minute to RT. A 384-well plate was used,
with each well containing the dual-labelled oligonucleotide at a
final concentration of 0.2 μM in 20 μl. In competitive FRET experi-
ments with 0.2 μM of F-myc25T, ten-fold excess of non-labelled
ds26 oligonucleotide was added to the mixture to reach the final
concentration of 2.0 μM. All compounds dissolved in DMSO to a
concentration of 10 mM were loaded into the wells using an ECHO®
550 acoustic liquid handler with final concentrations of 100 μM
with 1% DMSO. Melting curves were measured on a LightCycler®
°
4
was amenable to N -alkynylation, which allowed subsequent
click reactions, with an optimal length of three methylene units.
We subjected the scaffolds to ‘conventional’ and in situ click
reactions in the presence of Pu24. For the mono-amino click
products, the G4-stabilizing activity and their in situ formation
yield positively correlated with the increase in the number of
methylene units in the alkynyl chain. Similarly, in the bis-amino
series of N-alkynyl derivatives, we observed no change in
activity in compounds with one and two methylene units, but
we detected significant increases in activity in compounds with
4
80 Instrument II by monitoring FAM fluorescence (Syber green I
channel, with excitation wavelength at 465 nm and emission at
510 nm). Melting points were calculated in GraphPad Prizm 5 using
the asymmetric five parameter model. The ability of a particular
fragment to stabilize the secondary structure of the Pu25 (Fc-
myc25T) or ds26 (FdsT) oligonucleotide was expressed as a differ-
ence between the melting points of the sample and of a control.
Each test was performed in duplicates and repeated in three
independent experiments (n=3).
1
three methylene units. Using H NMR spectroscopy, we probed
the mode of interaction for the studied compounds. Interest-
ingly, the most potent bis-amino compounds, including the N-
alkynyl homologs and their click products, were destabilizing
the 5’ guanine tetrad and most likely stacking on the top of the
central tetrad. Our study suggests that the bis-amino derivatives
of the 4-(piperazin-1-yl)aniline template, including the click
products, appear to be potent c-MYC G4 stabilizers in FRET, PEA,
and NMR at low micromolar concentrations and may be
amenable to further therapeutic development.
DNA Polymerase Extension Assay (PEA)
In PEA, the movement of DNA polymerase on the template
produces a stop band if the G4 blocks are folded. Both the primer
and the template were purchased from Sigma-Aldrich® (www.sig-
maaldrich.com). The primer was labelled on the 5 – end with
using T4 polynucleotide kinase (TaKaRa ref: 2021 A) according to
the manufacturer’s instructions. P-30 colons were used to remove
unbound radioactivity. The labelled primer and the template were
[53]
3
2
P
mixed in water in a 1:1.25 ratio, heat-denatured at 95
°
C for 10
minutes and cooled approximately 1°C per minute to RT. The
Experimental Section
template concentration in the primer/template mixture was
12.5 μM. The polymerase stop reaction was performed using a
GoTaq® G2 DNA polymerase kit (PROMEGA ref: M784A); the reaction
was run in 1X Colorless GoTaq® Reaction Buffer. The final reaction
volume was 20 μl and contained 0.1 μM template (primer/template
mixture), 200 μM dNTP, and either compounds in DMSO solution or
DMSO as a control. The reaction started after 30-minute incubation
at 52°C (optimized temperature) by adding 2 μL of DNA polymer-
ase (1 U/μL) to reach 0.1 U per well and further incubated at 52°C
for 30 minutes. At the end, the reaction was stopped by adding 5x
loading solution (80% formamide, 10% glycerol, 20 mM EDTA pH 8,
Oligonucleotides
The HPLC-purified Pu24 oligo-deoxyribonucleotide mimicking c-
MYC G4 as well as the HPLC-purified ds26 oligo-deoxyribonucleo-
tide forming a double-stranded hairpin were purchased in lyophi-
lized form from Generi Biotech (www.generi-biotech.com):
Pu24: 5’-TGA GGG TGG GGA GGG TGG GGA AGG-3’
Ds26: 5’-TAT AGC TAT ATT TTT TTA TAG CTA TA-3’
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[54]
0,025% xylene cyanol and 0,025% bromophenol blue), heat-
yne to form 46 (ESI, Section 4, Scheme S-5). Then, subsequent
CuAAC reactions of 46 with 3-azido-N,N-dimethylpropan-1-amine
or 1-(2-azidoethyl)pyrrolidine were performed to form compounds
47 and 48 (ESI, Section 4, Scheme S-5). Both 47 and 48 were
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
denatured for 5 minutes at 95°C, subsequently cooled on ice and
kept at À 20 C until separation on a 15% AA denaturating gel
containing 40% of urea at 55°C, vacuum dried and visualized using
°
TM
[46]
phosphoimaging cassettes and scanned on the Typhoon FLA
5
subsequently reduced by hydrogenation on Pd/C to their bis-
500 at 100 μM resolution. Band densities were evaluated by Image
amino counterparts, 49 and 50. To avoid decomposition of the bis-
amino compounds, the respective hydrochloride salts were formed
immediately after quenching the hydrogenation reaction by adding
the HCl-ethanol solution.
Quant software and data were normalized using the following
formula:
Ligand rel: stop product ¼
Furthermore, 1-(4-nitrophenyl)piperazine was alkylated with 5-
[54]
iodopent-1-yne under basic conditions to form compound 39,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Ligand stop product=Ligand ðstop product þ full À length productÞ
which was then mixed with 1-(2-azidoethyl)pyrrolidine in the
Control stop product=
I
presence of catalytic amounts of Cu to afford the compound 40
Control ðstop product þ full À length productÞ
(ESI, Section 4, Scheme S-4). Lastly, compounds 41 and 17 were
prepared as described for 41 and 42: in a nucleophilic aromatic
substitution, 5-chloro-2-nitroaniline was mixed with 1-meth-
The data were determined as a quotient of stop band density of
the respective compound divided by the stop band density of the
parent compound 7, which was present on each gel. All
compounds were screened at 100 μM concentration with 5% of
DMSO.
[55]
ylpiperazine to form 51, and compound 17 was prepared from
5
1 by reduction of its nitro group by catalytic hydrogenation on
[55]
Pd/C. The decomposition of the bis-amino compound 17 was
avoided by immediate formation of hydrochloride salt (ESI,
Section 4, Scheme S-6).
Template Pu27: 5’-CTGCGATCCGTCAGTCCAACATGCTATACT-
-
GGGGAGGGTGGGGAGGGTGGGGAAGG-
In Situ Click Experiments
TTAGCGGCACGCAATTGCTAGCGTGAGTCG-3’
The reaction mixture for each in situ click experiment contained
three alkynes (1.0 mM each), two azides (1.5 mM each), Tris-HCl
Reverse primer: 5’-CGACTCACGCTAGCAATTGCGTG-3’
For control PEA measurement, the template strand containing the
Pu27 ’all-mut’ sequence incapable of G4 formation was used:
buffer (15 mM, pH 7.4), KCl (100 mM), CuSO (1.5 mM) and sodium
4
ascorbate (3.0 mM), with (experiment A) or without (experiments B
and C) the oligonucleotide Pu24 or ds26 (both 0.1 mM in Tris-HCl/
KCl buffer). In the control experiment (C), the respective oligonu-
cleotide was added to the reaction mixture only 1 minute before
quenching the reaction (for more detailed experimental setup, see
ESI, Section 6, Table S6.1-S6.3). After 2 hours of shaking at 300 rpm,
Pu27 ’all-mut’: 5’-CTGCGATCCGTCAGTCCAACATGCTATACT-
-GAAGAGAGTGAAGAGAGTGAAGAAGG-
TTAGCGGCACGCAATTGCTAGCGTGAGTCG-3’
2
2°C, trifluoroacetic acid (TFA, 40% in H O) was added to quench
2
the reaction by denaturing the DNA. The composition of each
reaction mixture was analyzed by LC–MS. The ratio of the integral
amount measured by LC–MS of each click product was calculated
between experiments A and B (A/B ratio) and between experiments
A and C (A/C ratio).
Chemistry
The mono-amino series (Figure 3A) was synthesized via an N-
alkylation reaction of the piperazine moiety at the 4-(piperazin-1-yl)
[54]
aniline with the respective alkynyl halides under basic conditions,
thereby preparing a homologous series of three 4-(piperazin-1-yl)
[45]
aniline-derived alkynes with different alkynyl chain lengths, 24,
LC–MS Evaluation of in Situ Click Experiments
2
5, and 26 (ESI, Section 4, Scheme S-1). Compounds from the bis-
amino series (Figure 3B) were synthesized as follows: firstly,
compound 41 was prepared by nucleophilic aromatic substitution
of 5-chloro-2-nitroaniline with piperazine under basic conditions,
then reduced by catalytic hydrogenation on Pd/C forming the
compound 42 (ESI, Section 4, Scheme S-2). Finally, 42 was N-
alkylated under basic conditions
Samples were injected onto a Luna Omega Polar C18 column
1
50x1.0 mm (Phenomenex) and separated by gradient of
acetonitrile in water (both mobile phases modified by 0.1% formic
acid). The separation was performed by an LC system (I-class,
Waters) coupled to Mass Spectrometer (Synapt G2, Waters) to
acquire m/z by positive electrospray ionization in the range of 100–
[
46]
[
54]
with the respective alkynyl
halide to obtain compounds 43–45 (ESI, Section 4, Scheme S-2).
The alkyne derivatives of both families formed 1,4-disubstituted
triazole compounds by CuAAC.
1
500 m/z. The extracted ion chromatogram of the monitored peaks
was integrated by Quantlynx application of MassLynx 4.1 (Waters).
The alkynyl compounds from the mono-amino series, 24–26
1
Hit Validation by H NMR Spectroscopy
(
Figure 3A), were directly mixed with 3-azido-N,N-dimethylpropan-
I
1
-amine in the presence of catalytic amounts of Cu upon triazole
1
For the purpose of hit validation by H NMR, all tested compounds
[38]
formation. The CuAAC reaction products 33–35 showed sufficient
were dissolved in DMSO-d to the final concentration of 20 mM.
6
yields (ESI, Section 4, Scheme S-3). Similarly, CuAAC reactions were
The samples were formulated on one-to-one basis to avoid false-
positive hits caused by aggregation of ligands or misinterpretation
of complex spectra of mixtures. The stock solution of the target
[
38]
performed with 1-(2-azidoethyl)pyrrolidine to form compounds
6–38, with 2-azidoethan-1-amine to form compounds 27–29, and
3
with 3-azidopropan-1-amine to form compounds 30–32. All click
products were isolated in good yields (ESI, Section 4, Scheme S–3).
(
Pu24) was prepared to match the final concentration of 5 μM,
adding 10% D O for field locking, in an NMR buffer (pH=7.0)
2
containing 20 mM KH PO , 70 mM KCl, and 0.02% NaN . Individual
A direct CuAAC reaction of the bis-amino derivatives (Figure 3B,
2
4
3
ligand solutions were added to a G4 stock solution to match the
final 100x excess of ligand (500 μM) over target. Each tested sample
4
3–45) was not performed due to the low stability of the o-
phenylendiamine moiety. Instead, the nitro compound 41 was
was formulated as follows: 14 μL of the DMSO-d
solution
alkylated under basic conditions in the presence of 5-iodopent-1-
6
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containing the respective ligand in 20 mM concentration was
[11] M. L. Bochman, K. Paeschke, V. A. Zakian, Nat. Rev. Genet. 2012, 13, 770–
80.
[12] Y. Qin, L. H. Hurley, Biochimie 2008, 90, 1149–1171.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
7
added to 536 μL of 5 μM G4 stock solution containing 10% D O to
2
obtain final sample volume of 550 μM. This formulation introduced
[
[
[
[
[
13] A. T. Phan, V. Kuryavyi, H. Y. Gaw, D. J. Patel, Nat. Chem. Biol. 2005, 1,
67–173.
14] J. Seenisamy, E. M. Rezler, T. J. Powell, D. Tye, V. Gokhale, C. S. Joshi, A.
Siddiqui-Jain, L. H. Hurley, J. Am. Chem. Soc. 2004, 126, 8702–8709.
2.5% of DMSO-d from the compound‘s stock solutions. The peak
6
1
of the residual DMSO-d (δ=2.50 ppm) was used as an internal
6
reference standard. Therefore, there was no need to add additional
internal reference standard which could possibly interfere in the
15] A. Ambrus, D. Chen, J. Dai, R. A. Jones, D. Yang, Biochemistry 2005, 44,
1
STD H NMR experiment.
2048–2058.
16] D. Monchaud, M.-P. Telaude-Fichou, Org. Biomol. Chem. 2008, 6, 627–
All samples were spun down in Eppendorf Minispin Centrifuge at
636.
1
2.100 x g for 2 minutes to separate possible aggregates and to
17] B.-J. Chen, Y.-L. Wu, Y. Tanaka, W. Zhang, Int. J. Biol. Sci. 2014, 10, 1084–
remove air bubbles. The samples were transferred into NMR tubes
1096.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
and stored at 4 °C. The H NMR experiment was performed on a
[18] S. Maiti, N. K. Chaudhury, S. Chowdhury, Biochem. Biophys. Res.
Bruker Avance III™ HD 600 MHz spectrometer equipped with a
Commun. 2003, 310, 505–512.
[19] T.-M. Ou, Y.-J. Lu, C. Zhang, Z.-S. Huang, X.-D. Wang, J.-H. Tan, Y. Chen,
D.-L. Ma, K.-Y. Wong, J. C.-O. Tang, A. S.-C. Chan, L.-Q. Gu, J. Med. Chem.
Cryoprobe and a sample changer (for a detailed spectrometer
1
setup, see ESI, Section 8). The H NMR spectra were acquired using
2
007, 50, 1465–1474.
3
50/550μL samples of 5 μM Pu24 dissolved in NMR buffer at 293 K
[56–57]
[20] Y.-J. Lu, T.-M. Ou, J.-H. Tan, J.-Q. Hou, W.-Y. Shao, D. Peng, N. Sun, X.-D.
Wang, W.-B. Wu, X.-Z. Bu, Z.-S. Huang, D.-L. Ma, K.-Y. Wong, L. Q. Gu, J.
Med. Chem. 2008, 51, 6381–6392.
and the standard ‘stddiffesgp’
pulse sequence with excitation
sculpting and pulse-field gradients for water suppression. The
frequency of non-selective irradiation pulse was set to saturate the
well-separated signal of DNA (6824 Hz, amidic region), using a
50 ms shaped Eburp2.1000 pulse at a power of 40 dB. The
irradiated signals had to be sufficiently separated from the ligand‘s
signals to prevent their direct irradiation, which would generate
false positive hits. The spectra were acquired at a free induction
decay (FID) resolution of ~1 Hz. The typical experimental time of
each sample was 30 minutes, thus allowing measurements of the
whole library within one day.
[
21] Y. Ma, T.-M. Ou, J.-Q. Hou, Y.-J. Lu, J.-H. Tan, L.-Q. Gu, Z.-S. Huang, Bioorg.
Med. Chem. 2008, 16, 7582–7591.
[22] B. Dumat, G. Bordeau, E. Faurel-Paul, F. Mahuteau-Betzer, N. Saettel, M.
Bombled, G. Metgé, F. Charra, C. Fiorini-Debuisschert, M.-P. Teulade-
Fichou, Biochimie 2011, 93, 1209–1218.
[
23] A. Rangan, O. Y. Fedoroff, L. H. Hurley, J. Biol. Chem. 2001, 276, 4640–
4646.
[24] D.-L. Ma, V. P.-Y. Ma, K.-H. Leung, H.-J. Zhong, H.-Z. He, D. S.-H. Chan, C.-
H. Leung, Structure-Based Approaches Targeting Oncogene promoter G-
Quadruplexes, InTech, 2013.
[
25] W. Duan, A. Rangan, H. Vankayalapati, M.-Y. Kim, Q. Zeng, D. Sun, H.
Han, O. Y. Fedoroff, D. Nishioka, S. Y. Rha, E. Izbicka, D. D. Von Hoff, L. H.
Hurley, Mol. Cancer Ther. 2001, 1, 103–120.
Acknowledgements
[26] H. Mo, M. Vita, M. Crespin, M. Arsenian Henriksson, Cell Cycle 2006, 5,
191–2194.
2
[27] Y. Jiang, A.-C. Chen, G.-T. Kuang, S.-K. Wang, T.-M. Ou, J.-H. Tan, D. Li, Z.-
This work was supported by grants from GACR (17-04393S), the
Ministry of Education of the Czech Republic (LO1304, LM2015070
and LK11205), the European Regional Development Fund; OP RDE;
Project: “Chemical biology for drugging undruggable targets
S. Huang, Eur. J. Med. Chem. 2016, 122, 264–279.
[28] D. Dutta, M. Debnath, D. Müller, R. Paul, T. Das, I. Bessi, H. Schwalbe, J.
Dash, Nucleic Acids Res. 2018, 46, 5355–5365.
[
29] S. Pelliccia, J. Amato, D. Capasso, S. Di Gaetano, A. Massarotti, M.
Piccolo, C. Irace, G. C. Tron, B. Pagano, A. Randazzo, E. Novellino, M.
Giustiniano, J. Med. Chem. 2020, 63, 2035–2050.
(ChemBioDrug)” (No. CZ.02.1.01/0.0/0.0/16_019/0000729). and the
Czech Academy of Sciences (RVO61388963). We thank Katarina
Psenakova and Carlos Melo for valuable comments and proof-
reading of the manuscript.
[30] A. Chauhan, R. Paul, M. Debnath, I. Bessi, S. Mandal, H. Schwalbe, J.
Dash, J. Med. Chem. 2016, 59, 7275–7281.
[31] W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radić, P. R. Carlier, P. Taylor,
M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. Engl. 2002, 41, 1053–
1057.
[
32] M. Whiting, J. Muldoon, Y.-C. Lin, S. M. Silverman, W. Lindstrom, A. J.
Olson, H. C. Kolb, M. G. Finn, K. B. Sharpless, J. H. Elder, V. V. Fokin,
Angew. Chem. Int. Ed. 2006, 45, 1435–1439; Angew. Chem. 2006, 118,
Conflict of Interest
1463–1467.
[
33] X. Hu, R. Manetsch, Chem. Soc. Rev. 2010, 39, 1316–1324.
The authors declare no conflict of interest.
[
34] S. K. Mamidyala, M. G. Finn, Chem. Soc. Rev. 2010, 39, 1252–1261.
[35] N. P. Grimster, B. Stump, J. R. Fotsing, T. Weide, T. T. Talley, J. G.
Yamauchi, Á. Nemecz, C. Kim, K.-Y. Ho, K. B. Sharpless, P. Taylor, V. V.
Fokin, J. Am. Chem. Soc. 2012, 134, 6732–6740.
Keywords: anticancer therapy · click chemistry · G-quadruplex ·
NMR · secondary structures
[
36] I. Glassford, C. N. Teijaro, S. S. Daher, A. Weil, M. C. Small, S. K. Redhu,
D. J. Colussi, M. A. Jacobson, W. E. Childers, B. Buttaro, A. W. Nicholson,
A. D. MacKerell, B. S. Cooperman, R. B. Andrade, J. Am. Chem. Soc. 2016,
138, 3136–3144.
[
[
[
1] S. Neidle, J. Med. Chem. 2016, 59, 5987–6001.
[
[
37] D. Bosc, J. Jakhlal, B. Deprez, R. Deprez-Poulain, Future Med. Chem.
2016, 8, 381–404.
38] M. Di Antonio, G. Biffi, A. Mariani, E.-A. Raiber, R. Rodriguez, S.
Balasubramanian, Angew. Chem. Int. Ed. 2012, 51, 11073–11078; Angew.
Chem. 2012, 124, 11235–11240.
2] J. B. Chaires, D. Graves, Quadruplex nucleic acids, SPRINGER, 2013.
3] N. Campbell, G. W. Collie, S. Neidle, Curr. Protoc. Nucleic Acid Chem.
2012, 50, 17.6.11-17.6.22.
[
4] S. Balasubramanian, L. H. Hurley, S. Neidle, Nat. Rev. Drug Discovery
2011, 10, 261–275.
[39] M.-H. Hu, X. Chen, S.-B. Chen, T.-M. Ou, M. Yao, L.-Q. Gu, Z.-S. Huang, J.-
[5] A. Siddiqui-Jain, C. L. Grand, D. J. Bearss, L. H. Hurley, PNAS 2002, 99,
11593–11598.
H. Tan, Sci. Rep. 2015, 5, 17202.
[
[
[
6] W. P. Tansey, New J. Sci. 2014, 2014, 27.
[40] D. Panda, P. Saha, T. Das, J. Dash, Nat. Commun. 2017, 8, 16103.
[41] P. Thirumurugan, D. Matosiuk, K. Jozwiak, Chem. Rev. 2013, 113, 4905–
4979.
[42] H. R. Nasiri, N. M. Bell, K. I.E. McLuckie, J. Husby, C. Abell, S. Neidle, S.
Balasubramanian, Chem. Commun. 2014, 50, 1704–1707.
[43] S. A. Latt, G. Stetten, J. Histochem. Cytochem. 1976, 24, 24–33.
7] N. Meyer, L. Z. Penn, Nat. Rev. Cancer 2008, 8, 976–990.
8] C. Y. Lin, J. Lovén, P. B. Rahl, R. M. Paranal, C. B. Burge, J. E. Bradner, T. I.
Lee, R. A. Young, Cell 2012, 151, 56–67.
[
9] T. A. Brooks, L. H. Hurley, Nat. Rev. Cancer 2009, 9, 849–861.
[
10] D. Yang, L. H. Hurley, Nucleos. Nucleot. Nucl. 2006, 25, 951–968.
ChemistryOpen 2020, 9, 1236–1250
www.chemistryopen.org
1249
© 2020 The Authors. Published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/open.202000261
ChemistryOpen
[
44] P. E. Pjura, K. Grzeskowiak, R. E. Dickerson, J. Mol. Biol. 1987, 197, 257–
[54] E. Hryhorenko, B. Sankaran, T. R. DeCory, T. Tubbs, L. Colt, B. M.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
2
71.
Remmerie, R. Salter, M. G. Donahue, Y. Gong, US 2014/0057303 A1
(27.02.2014).
[45] M. D. P. Risseeuw, D. J. H. De Clercq, S. Lievens, U. Hillaert, D. Sinnaeve,
F. Van den Broeck, J. C. Martins, J. Tavernier, S. Van Calenbergh,
ChemMedChem 2013, 8, 521–526.
[55] S. Ramachandran, S. Hameed, P. A. Srivastava, G. Shanbhag, S. Morayya,
N. Rautela, D. Awasthy, S. Kavanagh, S. Bharath, J. Reddy, V. Panduga,
K. R. Prabhakar, R. Saralaya, R. Nanduri, A. Raichurkar, S. Menasinakai, V.
Achar, M. B. Jimenéz-Díaz, M. Santos Martínez, I. Angulo-Barturen, S.
Ferrer, L. M. Sanz, F. J. Gamo, S. Duffy, V. M. Avery, D. Waterson, M. C. S.
Lee, O. Coburn-Flynn, D. A. Fidock, P. S. Iyer, S. Narayanan, V.
Hosagrahara, V. K. Sambandamurthy, J. Med. Chem. 2014, 57, 6642–
6652.
[
46] T. de la Fuente, M. Martín-Fontecha, J. Sallander, B. Benhamú, M.
Campillo, R. A. Medina, L. P. Pellissier, S. Claeysen, A. Dumuis, L. Pardo,
M. L. López-Rodríguez, J. Med. Chem. 2010, 53, 1357–1369.
47] S. Balasubramanian, S. Neidle, Curr. Opin. Chem. Biol. 2009, 13, 345–353.
48] A. R. Duarte, E. Cadoni, A. S. Ressurreicao, R. Moreira, A. Paulo,
ChemMedChem. 2018, 13, 869–893.
[
[
[
56] M. Mayer, B. Meyer, Angew. Chem. Int. Ed. 1999, 38, 1784–1788; Angew.
[49] Y. P. Kumar, P. Saha, D. Saha, I. Bessi, H. Schwalbe, S. Chowdhury, J.
Chem. 1999, 111, 1902–1906.
Dash, ChemBioChem. 2016, 17, 388–393.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
[
57] T.-L. Hwang, A. J. Shaka, J. Magn. Reson. Ser. A 1995, 112, 275–279.
[50] N. Ranjan, D. P. Arya, Molecules 2013, 18, 14228–14240.
[51] L. Yu, Q. Yang, J. Xiang, H. Sun, L. Wang, Q. Li, A. Guan, Y. Tang, Analyst
2015, 140, 1637–1646.
[
[
52] A. Anselmet, E. Mayat, S. Wietek, P. G. Layer, B. Payrastre, J. Massoulié,
FEBS Lett. 2002, 510, 175–180.
53] A. Galeone, ChemBioChem 2010, 11, 2050–2051.
Manuscript received: September 4, 2020
Revised manuscript received: November 2, 2020
ChemistryOpen 2020, 9, 1236–1250
www.chemistryopen.org
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