Dynamic Generation of G-Quadruplex DNA Ligands by Target-Guided Combinatorial Chemistry on a Magnetic Nanoplatform

Article abstract of DOI:10.1021/acs.jmedchem.8b01459

Dynamic combinatorial chemistry (DCC) has emerged as a promising strategy for template-driven selection of high-affinity ligands for biological targets from equilibrating combinatorial libraries. However, only a few examples using disulfide-exchange-based DCC are reported for nucleic acid targets. Herein, we have demonstrated that gold-coated magnetic nanoparticle-conjugated DNA targets can be used as templates for dynamic selection of ligands from an imine-based combinatorial library. The implementation of DCC using DNA nanotemplates enables efficient identification of the lead compounds, from the dynamic combinatorial library via magnetic decantation. It further allows quick separation of DNA nanotemplates for reuse in DCC reactions. The identified lead compound exhibits significant quadruplex versus duplex DNA selectivity and suppresses promoter activity of c-MYC gene that contains G-quadruplex DNA forming sequence in the upstream promoter region. Further cellular experiments indicated that the lead compound is able to permeate into cell nuclei and trigger a DNA damage response in cancer cells.

Full text of DOI:10.1021/acs.jmedchem.8b01459

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Article
Dynamic Generation of G-quadruplex DNA Ligands by Target-
Guided Combinatorial Chemistry on a Magnetic Nano-platform
Snehasish Jana, Deepanjan Panda, Puja Saha, Dan Pantos, and Jyotirmayee Dash
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01459 • Publication Date (Web): 11 Dec 2018
Downloaded from http://pubs.acs.org on December 13, 2018
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Dynamic Generation of G-quadruplex DNA
Ligands by Target-Guided Combinatorial
Chemistry on a Magnetic Nano-platform
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Snehasish Jana†[a], Deepanjan Panda†[a], Puja Saha , G. Dan Pantoș and Jyotirmayee
[a]
[b]
Dash[a]*
[
a]
School of Chemical Sciences, Indian Association for the Cultivation of Science, Jadavpur,
Kolkata-700032, India, E-mail: ocjd@iacs.res.in; Fax:+91-33-2473-2805; Tel: +91-33-2473-
4
971, ext 1405.
[b]
Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY (UK)
ABSTRACT: Dynamic combinatorial chemistry (DCC) has emerged as a promising strategy for
template-driven selection of high-affinity ligands for biological targets from equilibrating
combinatorial libraries. However, only a few examples using disulphide exchange based DCC
are reported for nucleic acid targets. Herein, we have demonstrated that gold-coated magnetic
nanoparticle conjugated DNA targets can be used as templates for dynamic selection of ligands
from an imine-based combinatorial library. The implementation of dynamic combinatorial
chemistry using DNA-nanotemplates enables efficient identification of the lead compounds,
from the DCL via magnetic decantation. It further allows quick separation of DNA-
nanotemplates for reuse in DCC reactions. The identified lead compound exhibits significant
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quadruplex-vs.-duplex DNA selectivity and suppresses promoter activity of c-MYC gene that
contains G-quadruplex DNA forming sequence in the upstream promoter region. Further cellular
experiments indicated that the lead compound is able to permeate into cell nuclei and trigger a
DNA-damage response in cancer cells.
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INTRODUCTION
Target-guided synthetic (TGS) approaches have been considered as powerful strategies to
discover novel and selective small molecules for biological targets by bridging the gap between
chemical synthesis and screening assays.1-8 Dynamic Combinatorial Chemistry (DCC), a
thermodynamically controlled target-guided synthetic approach has been employed for the
identification of selective ligands in a cost- and time-effective manner.1-8 In DCC, simple
molecular building blocks assemble through reversible bond formation to generate a dynamic
combinatorial library (DCL) consisting of a mixture of continuously inter-convertible products.
In the presence of a template, the equilibrium of the system shifts towards the formation of those
DCL components that strongly bind to the template at the expense of weak binders. Thus, this
concept provides an unique platform for the in-situ generation of high-affinity binders, offering
new opportunities for bio-medicinal applications.9-14 DCC has also been effectively used for the
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development of hosts for small molecules, interlocked molecules,
and catalysts as well as
for the functionalization of liposomes, nanoparticles etc.14
However, only a limited number of studies have been reported for the development of small
molecule probes for proteins15-18 and nucleic acids.19-27 The Miller group first reported the
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application of DCC to generate ligands for double stranded DNA. The Balasubramanian group
used disulphide chemistry based DCC to identify selective binders for G-quadruplex targets.22-24
DCC has also been used for developing RNA binding ligands.25-27 To improve the identification
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of small molecule ligand(s) from DCL, Miller and co-workers have devised resin bound DCC28
and the Otto group has recently reported nanoparticle functionalized building blocks for DCC.29-
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Most of these studies employ the disulphide-exchange chemistry that has several advantages
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like water compatibility and very fast thiol-exchange property at mild pH. However, the
resulting disulphide leads may lose their druggability due to their instability in cellular
environment.32 On the other hand, imine-exchange based DCC using aldehyde and amine
building blocks produces the imine-leads, which could be reduced to stable amine products15, 20
for further biophysical and cellular studies.
In this study, we have employed gold-coated magnetic nanoparticle-conjugated G-quadruplex
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DNA (G -DNA nano-template), as templates for dynamic selection of G-quadruplex DNA
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ligands from aldehyde and amine building blocks. We envisaged that the G -DNA nano-template
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would accelerate the identification of high-affinity ligands by providing multiple templates on
the surface of gold coated magnetic nanoparticles, simplify the separation of ligands and enable
the isolation of DNA nano-template by magnetic decantation. The selective ligands have the
potential to exhibit anticancer activities by stabilizing the quadruplex nucleic acids.34-43
RESULTS AND DISCUSSION
Design and synthesis of aldehyde and amine building blocks. In our design, we have used a
carbazole aldehyde 1 and a series of amines 2a-j (Scheme 1) to develop specific ligand for G-
quadruplex DNA. Carbazole aldehyde 1 was selected as a building block as it can interact with
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the terminal quartets of G -DNA via π–π aromatic stacking interactions.
The aldehyde 1 was
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prepared from commercially available carbazole via mono-iodination, N-alkylation followed by
Vilsmeier-Haack formylation45 and Suzuki coupling46 in good overall yield (Figure S1,
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Supporting Information, S.I.). The 10-membered amine library 2a-j includes aromatic and
aliphatic amines that could be involved in electrostatic and hydrogen bonding interactions with
DNA structures. The amines possess comparable reactivity to avoid biasness in the competition.
Scheme 1. (a) Aldehyde (1) and amine (2a-j) building blocks for DCC (b,c) 10 member
dynamic combinatorial imine (3a-j) library and the corresponding reduced imines (4a-j).
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(a)
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The aldehyde 1 and amines 2a-j building blocks would reversibly react to form imines 3a-j
under near physiological conditions. Subsequently, the imine(s) formed could be irreversibly
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reduced to stable amines 4a-j by NaBH CN providing an equilibrated mixture for analysis. The
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reversibility of the reaction is a key factor for developing a Dynamic Combinatorial Library
(DCL). To establish the reversibility of the system, aldehyde 1 was continuously stirred with an
excess of amine 2e in MOPS buffer (20 mM, pH 6.5) containing 100 mM KCl for 12 h. The
MOPS buffer was used to maintain a slightly acidic medium, which is optimal for imine
exchange reaction and the buffer system containing 100 mM KCl is suitable to maintain the four
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stranded conformation of the G -DNA in near physiological pH. An aliquot of the resulting
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mixture was analyzed by ESI mass spectrometry confirming the formation of the corresponding
imine 3e (Figure S2). The amine 2d was then added in excess to the reaction mixture and the
reaction was continued for another 12 h. The ESI mass analysis of the resultant mixture showed
the presence of both the imines 3d and 3e (Figure S3). The formation of both imines indicates
that imine 3e was converted to imine 3d via reversible exchange and thus, these building blocks
are suitable for DCC reaction (Scheme 2).
Scheme 2. Reversible exchange between imines 3e and 3d in MOPS buffer (20 mM, pH 6.5)
containing 100 mM KCl.
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DNA templated imine exchange DCC. The 10-membered amine library (2a-2j) was then
exposed with the aldehyde 1 to form a DCL and left to equilibrate for 18 h at 25 C in MOPS
buffer (20 mM, pH 6.5) with 100 mM KCl. Five-fold excess of each amine was used for the
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complete consumption of aldehyde 1. Then, NaBH CN was added to the reaction mixture and
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incubated for 6 h at the same temperature for switching the system towards irreversibility. The
HPLC chromatogram of this reaction mixture provided overlapping peaks and ESI mass analysis
of the HPLC fractions revealed the formation of all the corresponding amines 4a-j without any
considerable selection (Figure 3a).
The reaction was then performed in the presence of a G-quadruplex nano-template (G4-
AuMNp) and a duplex DNA nano-template (dsDNA-AuMNp) under similar reaction conditions.
We have taken a thiolated G-quadruplex forming sequence present in the promoter region of c-
MYC oncogene.48,49 The G -AuMNp was prepared by immobilizing thiolated G -DNA on gold-
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coated magnetic nanoparticles. Similarly, a control self-complementary duplex DNA
nanotemplate, (dsDNA-AuMNp) was prepared by attaching a thiolated dsDNA sequence on
gold-coated magnetic nanoparticles (Figure 1a). The G -AuMNp and dsDNA-AuMNp templates
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were characterized by using TEM, UV-Vis and Circular Dichroism spectroscopy. These studies
revealed that DNA functionalized nanoparticles were 10-15 nm in diameter (Figure 1b & 1d)
and they displayed absorption peaks near 260 nm along with the typical SPR peak of Au at 535
nm (Figure 1e-f). The G -AuMNp exhibited a positive peak at 260 nm and negative peak at 240
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nm in CD spectroscopy indicating that G -AuMNp maintains the parallel conformation of G-
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quadruplex structure (Figure 1c). The CD spectrum of dsDNA-AuMNp also showed a positive
signal at 275 nm and a negative signal at ~250 nm corresponding to the duplex DNA
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conformation (Figure 1c). Moreover, these DNA coated magnetic nanoparticles could be easily
dispersed in aqueous media and could also be isolated by using magnetic separation.
In order to perform DCC reactions with DNA nano-templates, the aldehyde and amine
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building blocks were incubated in the presence of G -AuMNp or dsDNA-AuMNp for 18 h in
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MOPS buffer (20 mM, 100 mM KCl), pH 6.5 at RT. To this mixture of interconvertible imines,
NaBH CN was added and the mixture was stirred for 6 h for the complete reduction of imines to
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the corresponding amines; thus, the system became irreversible (Figure 2, Figure S4).
Subsequently, amine bound DNA nano-templates were separated by magnetic decantation and
the unbound amines remained in the supernatant. The isolated DNA-AuMNps were washed three
times with MOPS buffer and the resulting suspension was heated at 65 °C to release the template
bound amine products. Then DNA-AuMNps were instantly separated by an external magnet and
the supernatant was characterized by HPLC and ESI-MS analysis to identify the lead amine
products.
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Figure 1. (a) Schematic representation of the preparation of G -AuMNp (Seq.: 5’-[ThiC6]-
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TG AG TG AG TG A G -3’) and dsDNA-AuMNp (Seq.: 5’-[ThiC6]-CA T GCA T G-3’).
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Absorption spectra of dsDNA-AuMNp and G -AuMNp in 20 mM MOPS buffer (pH 6.5,
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Significant changes in the composition of DCL were observed in the presence of G -DNA and
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dsDNA nano-templates (G -AuMNp and dsDNA-AuMNp) (Figure 3 and Figure S5). The
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HPLC retention times and the mass analysis of the products revealed that amine 4e (reduced
product of imine 3e, derived from 1 and 2e) was exclusively obtained in the presence of G-
quadruplex nano-template out of 10 possible amine products 4 (Figure 3f). In the presence of
dsDNA nano-template, five amine products 4g, 4h, 4i, 4f and 4b (reduced products of imines
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g, 3h, 3i, 3f and 3b, respectively) were identified (Figure 3b). These results indicate that amine
e is a selective lead for the G-quadruplex target. Furthermore, 4e was exclusively obtained by
giving a shorter reaction time (i.e. 30 min to 4 h) for the reduction step (Figure S6, S.I.).
Next, we performed the DCC using the same aldehyde and amine library and after 18
h equilibration period, the imine-bound G -AuMNps were separated and reduced by
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NaBH CN. The HPLC and ESI-MS results revealed that ligand 4e was exclusively obtained,
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indicating G -AuMNp selects the best binder (i.e. 3e) from the imine DCL due to higher affinity
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of imine 3e and then 3e was reduced to stable amine (4e) upon addition of NaBH CN (Figure
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S7, S.I.). In comparison, by using solution phase DCC with free quadruplex DNA, we observed
a complex mixture of compounds in the HPLC chromatogram (Figure S8), making lead
identification difficult.
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Figure 2. Schematic representation of imine exchange based dynamic combinatorial
chemistry using G -AuMNp template.
4
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Figure 3. HPLC analysis of the DCL (a) without any external template and using (b)
dsDNA-AuMNp and (c-f) G -AuMNp after 4 h, 6 h, 12 h, 18 h respectively. HPLC
4
chromatogram of pure compound (g) 4e (h) 4g and (i) 4h.
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Next, we carried out a time-dependent imine exchange DCC reaction of the same library in the
presence of G -AuMNp to optimize the time required for the amplification of 4e. For this
4
purpose, we collected aliquots of the reaction mixture after 4 h, 6 h, 12 h and 18 h and then
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reduced using NaBH CN. Following the similar procedure, the lead compounds were isolated
3
from each reaction mixture using magnetic separation and then analyzed by HPLC and ESI-MS
(
Figure 3c-e). The time-dependent HPLC analysis revealed that ligands 4e, 4g and 4h were
formed in a ratio of ~ 25:12:10 respectively after 4 h, indicating the corresponding imines 3e, 3g
and 3h were in equilibrium in the presence of G -DNA nanotemplates. As the time progressed,
4
continuous elevation in the LC response signal of 4e was noticed and the LC response signals of
4g and 4h were gradually decreased at 6 and 12 h time period. Finally, after 18 h, ligand 4e was
exclusively formed and by giving longer equilibration time up to 48 h, ligand 4e was obtained as
the sole product (Figure S9, SI), illustrating that the system has reached thermodynamic
equilibrium by 18 h. For further validation, the DCL was pre-equilibriated for 24 h and then
incubated with G -AuMNp for 18 h; this DCL also produced 4e exclusively. These observations
4
indicate that among the possible amine products, 4e exhibits higher binding affinity and
selectivity for the G -DNA over dsDNA (Figure 4) as other lead products 4g and 4h were also
4
formed in the presence of dsDNA nanotemplate. Furthermore, when we mixed the G -AuMNp
4
with all the synthesized amines (pure 4a-j), LC response of 4e and 4g was observed (Figure S10,
S.I.), indicating that the amplification of 4e in DCC is not mediated by mere affinity capture by
DNA templates. Here, the imine DCL evolves and self-corrects in the presence of G4-
nanotemplate to amplify its best binder at the expense of other weak imine products. It is also
interesting to mention that the quadruplex nano-template could be recovered and reused for five
DCC reaction cycles (Figure S11, S12, S.I.).
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Figure 4. Comparison of relative composition of the DCL in presence of G -AuMNp with
4
time interval of 4 h, 6 h, 12 h and 18h.
Validation of lead compounds by biophysical analysis. To further validate these findings, all
possible amine products (4a-4j) were synthesized (Figure S13, S.I.) and their ability to stabilize
the G-quadruplex DNA was investigated by Fӧrster Resonance Energy Transfer (FRET) based
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0
DNA melting assays using FAM and TAMRA labelled G-quadruplex and double stranded
DNA sequences at 1 μM ligand concentration (Table 1, Figure S14, S.I.). Interestingly, the lead
compound 4e showed high stabilization potential for G -DNA (ΔT = 23.4 °C) over dsDNA
4
m
(
ΔT = 0.6 °C). The other two lead two compounds 4g and 4h showed ΔT values of 13.6 °C
m
m
and 11.8 °C for G -DNA and ΔT values of 9.5 °C and 8.5 °C for dsDNA, respectively. Ligands
4
m
4a with aromatic hydroxyl group, 4b with aromatic nitro group, bromo-substituted amine
product 4c and 4d with tertiary propyl amine side chain, 4f with thiazole side chain and 4j with
neutral cyclopropane side chain exhibited weak stabilization potentials with low ΔT values for
m
G and duplex DNA. The cationic tertiary amine side chain containing compound 4i showed
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moderate stabilization potential for both dsDNA (ΔT = 7.2 °C) and G -DNA (ΔT = 5.3 °C). In
m
4
m
addition, to assess the selectivity of ligand 4e for G -DNA, we performed competitive FRET
4
experiments with 0.2 μM of labelled G -DNA (c-MYC) and 1 µM 4e with increasing
4
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concentrations of non-labelled calf-thymus duplex DNA (ctDNA) competitor. These results
showed that the ΔT value of c-MYC G -DNA with 1 µM 4e was not altered in the presence of
m
4
excess ctDNA (1-200 eq of c-MYC G -DNA) (Figure S15, S.I.), indicating ligand’s selectivity
4
for quadruplex DNA over duplex DNA. Circular Dichroism spectroscopy further revealed that
the characteristic CD signature for the native parallel topology of c-MYC G -DNA (a positive
4
peak at 260 nm and a negative peak at 240 nm) was retained in the presence of ligand 4e, even at
10 eq concentrations (Figure S16, S.I.). These suggest that 4e binds and stabilizes the
quadruplex DNA without perturbing its parallel conformation.
Table 1. ΔT values of ligands at 1 µM concentration for c-MYC G and dsDNA in 60 mM
m
4
potassium cacodylate buffer, pH 7.4.
ΔT (°C)
m
Ligands
c-MYC G4
dsDNA
4
a
0.7
1.9
4
b
3.4
2.4
0.44
3.79
0.58
1.15
9.5
4
c
3.3
4
d
5.0
4e
23.4
3.2
4
f
4
g
13.6
11.8
5.3
4
h
8.5
4
i
7.2
4j
3.5
1.1
T
m
4
of c-MYC G -DNA is 70.1°C and dsDNA is 65°C in the
absence of ligands. DNA concentration: 200 nM.
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The binding affinities of the lead compounds (4e, 4g and 4h) for G -DNA and dsDNA were
4
determined using fluorescence spectroscopic titrations. As shown in Figure 5, ligand 4e
exhibited emission maximum at 400 nm (휆푒푚 ) when excited at 300 nm (휆 ). Upon incremental
푚푎푥
푒푥
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addition of G -DNA, the emission intensity of 4e at 400 nm was enhanced by 4.4 fold and a
4
large red-shift of 50 nm of the emission maximum was observed eventually leading to a new
emission maximum at around 450 nm (Figure 5a, Figure S17, SI). The observed red shift arises
from the strong binding interactions of 4e with DNA G-quadruplex, which restricts the rotation
of the 4-methoxy phenyl ring relative to the carbazole moiety in the excited state. With dsDNA,
no significant change in the fluorescence intensity of 4e was observed indicating its excellent
selectivity for G-quadruplex DNA (Figure 5b). The titration of compound 4g (휆푒푚 = 397 nm,
푚푎푥
휆 = 300 nm) with G -DNA and dsDNA showed that the fluorescence intensity was decreased
푒푥
4
by 2.3-fold and 1.5 fold respectively (Figure 5c-d). The fluorescence intensity of 4h (휆푒푚 = 396
푚푎푥
nm, 휆 = 300 nm) was also decreased by 1.5 fold with both G -DNA and dsDNA (Figure 5e-f).
푒푥
4
Ligand 4e displayed an apparent K value of 1.08 ± 0.2 μM for G -DNA whereas 4g and 4h
d
4
showed higher K values for G -DNA (Table 2). These results are consistent with the results
d
4
obtained using DCC, suggesting that ligand 4e, exclusively formed in the presence of G4-
AuMNp shows selective binding affinity to G -DNA compared to other lead compounds 4g and
4
4
h formed by giving short equilibration time (4-12 h).
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Figure 5. Fluorescence emission spectra of lead compounds with G -DNA and dsDNA.
4
Conditions: [Ligand] = 0.25 µM; 100 mM Tris.KCl buffer, pH 7.4, Excitation wavelength
3
00 nm.
Table 2. Apparent dissociation constants (K ) obtained from fluorimetric titration and
d
DC values obtained from FID assay for the lead compounds for G -DNA and dsDNA in
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0
4
1
00 mM Tris.KCl buffer (pH 7.4).
K (μM)
d
DC (μM)
50
Ligands
G -DNA
dsDNA
N.d.
G -DNA
dsDNA
> 30
4
4
4e
4g
4h
1.08 ± 0.2
5.72 ± 0.1
7.17 ± 0.3
2.25
6.63 ± 0.3
7.03 ± 0.2
8.01
> 30
> 25.0
> 30
The binding selectivity of the lead compounds for G-quadruplex DNA over duplex DNA was
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1
further corroborated by using Fluorescence intercalator displacement assay (FID, Figure S18).
The FID assay was carried out using Thiazole orange (TO) as the fluorescent intercalator. For
each ligand, FID experiments were performed with both G -DNA and dsDNA and the binding
4
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affinities of ligands were evaluated in terms of DC values. In agreement with Fluorescence
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binding titrations, ligand 4e exhibited a significantly higher ability to displace 50% TO from G4-
DNA (DC = 2.25 µM) compared to dsDNA (DC > 30 µM) (Figure S18). The DC value of
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g was determined to be 8.0 μM for the G -DNA and > 30 μM for dsDNA. In comparison, the
4
5
0 % threshold limit was not acquired by 4h for the G -DNA and dsDNA even at a concentration
4
of 25 µM (DC > 25 μM) (Table 2). The FRET melting assay, fluorescence binding titrations
50
and FID studies establish a direct correlation between DNA-mediated amplification of selective
leads and their binding affinities for the DNA targets.
Biological activity of ligand 4e. XTT assay was carried out to evaluate the cytotoxicity of
these compounds against human carcinoma cell lines HeLa and A549 as well as in normal
human kidney epithelial cell line (NKE). Compound 4e and 4g showed significant cytotoxicity
towards cancer cell lines (IC50 values of 2-6 μM) whereas compound 4h exhibited weak
cytotoxicity with low IC values (Table 3). Interestingly, these three lead compounds displayed
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negligible cytotoxicity for the human normal cell line.
Table 3. Effect of compounds on the cell viability after 24 h exposure in HeLa, A549 and
NKE cell line.
Compounds
IC (μM)
50
HeLa
2.5
A549
6.4
NKE
> 50
> 50
> 50
4
4
4
e
g
h
2.0
4.3
11.5
> 25
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52
We further carried out dual luciferase reporter assay to study their ability to inhibit oncogenic
transcription by interacting with the G-quadruplex (Figure 6a). We used plasmid constructs
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containing wild-type c-MYC G-rich promoter sequence (WT c-MYC Luc) as well as mutant (G-
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to-A) c-MYC promoter sequence (Mut c-MYC Luc) upstream of the firefly coding gene (Figure
6
b). A control plasmid, pRL-TK (Renilla luciferase, linked to the non G-rich thymidine kinase
promoter) was co-transfected with MYC Luc constructs as a loading control. HeLa cells were
treated with each ligand at concentrations of 1 and 1.5 μM for 36 h after co-transfection. Ligand
4
e reduced the c-MYC promoter-linked expression by 31 % and 45 % at 1 and 1.5 μM
concentrations (Figure 6c).
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Figure 6. (a) Schematic representation of ligand-mediated downregulation of c-MYC gene.
(b) Firefly luciferase constructs containing either the WT or mutant c-MYC promoter G-
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rich sequence. (c) Histogram demonstrating effect of compounds on the c-MYC promoter
activity, determined by dual luciferase reporter assay. (d) Western Blot bands of c-MYC
and GAPDH proteins after 24 h treatment with ligand 4e at three different concentrations
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(1.0 μM, 1.5 μM and 2.0 μM). (e) Bar diagram showing dose-dependent downregulation of
c-MYC protein by 4e. (f) Confocal imaging of cells treated with 0.5 µM 4e. Nucleus staining
dye is NucRed647 and scale bar is 20 µm. (g) Ligand 4e induced DNA damage response in
Hela cells as indicated by the increase of Alexa Fluor 647-tagged anti-γ-H2AX stained cell
population in Flow cytometry.
However, the transcriptional activity of mutant plasmid construct was not inhibited with
increasing concentrations of 4e. Ligand 4g exhibited less pronounced transcriptional inhibition
(14 % at 1.5 μM) whereas 4h could not inhibit promoter activity in HeLa cells. The Western
Blot analysis confirmed that the major lead compound 4e could inhibit the MYC expression in
HeLa cells in a dose-dependent manner (Figure 6d-e). Ligand 4e repressed the MYC protein
expression by 27 % at 1.0 μM, 57 % at 1.5 μM and by 65 % at 2.0 μM concentrations without
affecting the expression of the house keeping gene, GAPDH. These results establish that 4e
could downregulate c-MYC expression at both transcriptional and translational level by directly
targeting its promoter quadruplex.54-58 Further investigations revealed that the ligand 4e localizes
into the cell as well as within the cell nuclei (Figure 6f). To investigate whether ligand 4e could
induce DNA-damage response in cancer cells, flow cytometric assays were carried out with
Alexa Fluor 647-tagged anti-γ-H2AX. As shown in Figure 6g, a significant increase in the γ-
H2AX foci (a significant marker for DNA damage response) was observed after treatment with
4
e for 24 h at 1 μM and 2 μM concentration. This indicates that ligand 4e triggers DNA damage
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response in cancer cells. Molecular docking studies further revealed that carbazole core of 4e
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stacks on the terminal G-quartet plane of G -DNA (PDB ID: 1XAV) by π -π interaction and the
4
aromatic cationic side chain interacts with DNA phosphate backbone via electrostatic
1
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interactions (Figure S19a, S.I.). Interestingly, imine 3e shows more preferred stacking
interactions on G-quartet by mostly exhibiting similar binding mode with G -DNA (Figure
4
S19b, S.I.). Collectively, these observations gave an understandable explanation regarding the
selectivity of 4e for G -DNA compared to other possible amine products.
4
Conclusions
In conclusion, we have demonstrated that a quadruplex nanotemplate can dynamically select
and amplify selective ligands for the quadruplex target from a pool of simple aldehyde and
amine building blocks. The present method allows efficient isolation and identification of
selective ligands for the quadruplex target as well as the recovery and reuse of DNA
nanotemplate. The lead ligand shows significant quadruplex-vs.-duplex selectivity and inhibits
gene expression by targeting its promoter quadruplex. The technique highlights quadruplex
selective ligands can be developed by using simple and straight-forward synthetic methods in
comparison to the previously reported ligands, often associated with synthetic complexity and
time-consuming purification and screening processes. In comparison to kinetically controlled
TGS approach, where the target selects and promotes irreversible bond formation between the
3
3
most potent reactive fragments to form the target selective products, the thermodynamically
controlled DCC method uses reversible bond formation to generate all possible products and the
system evolves and self-corrects in the presence of a target to amplify the best binder at the
expense of other products. Together, these results illustrate that nanotemplate guided DCC
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method could be potentially useful for the rapid generation of specific ligands for
biomacromolecular targets.
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EXPERIMENTAL SECTION
General information: All solvents and reagents were purified by standard techniques or used as
supplied from commercial sources (Sigma-Aldrich Corporation® unless stated otherwise). All
reactions were generally carried out under inert atmosphere unless otherwise noted. TLC was
performed on Merck Kieselgel 60 F254 plates, and spots were visualized under UV light.
1
13
Products were purified by flash chromatography on silica gel (100-200 mesh, Merck). H and C
NMR spectra were recorded on either Bruker ADVANCE 500 (500 MHz and 125 MHz), or
JEOL 400 (400 MHz and 100 MHz) instruments using deuterated solvents as detailed and at
ambient probe temperature (300 K). Chemical shifts are reported in parts per million (ppm) and
are referred to the residual solvent peak. The following notations are used: singlet (s); doublet
(
d); triplet (t); quartet (q); multiplet (m); broad (br). Coupling constants are quoted in Hertz and
are denoted as J. Mass spectra were recorded on a Micromass® Q-Tof (ESI) spectrometer with
0% methanol solution. The purity of the synthesized aldehyde (1) and the final synthesized
5
compounds (4a-j) were confirmed to be higher than 95% by performing HPLC with a dual pump
Shimadzu LC-20 AD system equipped with an 5.0 µm ODS2 reverse phase column (4.6 × 250
mm) using 300 nm detection wavelength.
Synthesis of AuMNp. An aqueous solution of HAuCl (5 mL, 2.0 mg/mL) was mixed into 20
4
mL of deionized water and boiled for 5 min. Then, 1 mL Fe O nanoparticle solution
3
4
(
synthesized by reported procedure)33 was added into the reaction mixture followed by the
addition of sodium citrate (1 mL, 80 mmol). The color of the solution gradually changed from
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brown to burgundy. The reaction mixture was refluxed under stirring for 5 min. After cooling,
the solution was sonicated for 10-15 min, and then gold-coated magnetic nanoparticles (AuMNp)
were collected using a magnet, washed 3 times and re-dispersed in pure water. Carbazole
aldehyde 1 was prepared from commercially available carbazole S1 (Figure S1).
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Synthesis of DNA coated AuMNp (G -AuMNp and dsDNA-AuMNp). All buffers and
4
solutions were degassed in order to avoid oxidative dimerization into disulfide dimers. Thiolated
DNA functionalized nanoparticles were synthesized by derivatizing 100 µL of an aqueous
AuMNp nanoparticle solution with 100 µL 5′-thiol capped DNA solution (oligonucleotide
concentration is 100 µM in 20 mM MOPS buffer, pH 6.5, 100 mM KCl). Thiolated DNA
sequences were pre-annealed in 20 mM MOPS buffer (pH 6.5, 100 mM KCl) by heating at 95 °C
for 5 min followed by gradual cooling to room temperature at a controlled rate of 0.1 °C/min and
then kept at 4°C for overnight.
After standing for 16 h with AuMNps, nanoparticles were separated using magnet and washed
with 20 mM MOPS buffer containing 100 mM KCl (pH 6.5). The volume of the nanoparticle
solution was made up to 100 µL with the buffer. The HPLC purified thiol capped DNAs were
supplied by SIGMA Aldrich. The sequences of the DNAs are as follows;
G -DNA: 5’- [ThiC6] TG AG TG AG TG A G TG A -3’
4
4
3
4
3
4
2
2
4
dsDNA: 5’- [ThiC6] CA T GCA T G -3’
5
5
5 5
Reversible exchange of imines. In a 2 mL eppendorf, aldehyde 1 (20 μL, 10 mM) was taken in
1
mL of MOPS buffer (20 mM, 100 mM KCl, pH 6.5) followed by the addition of amine 2e (100
μL 10 mM). The mixture was stirred at rt for 12 h. An aliquot of the resulting mixture was
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analyzed by ESI mass spectrometry. Then the amine 2d was added in excess (100 μL 10 mM) to
the reaction mixture and the reaction was continued for another 12 h. The final reaction mixture
was also analyzed by ESI-MS.
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Procedure for G -AuMNp templated imine exchange. In a 2 mL eppendorf, 40 μL G -
4
4
AuMNp template (G -nanotemplate, ~ 20 μM G -DNA) was taken in 500 μL of MOPS buffer
4
4
(20 mM, 100 mM KCl, pH 6.5). Aldehyde 1 (5 μM) was then added to the mixture, followed by
the addition of 25 μM of each amine 2a-j. The resulting mixture was stirred at rt for 18 h. Then,
NaBH CN (10 mg) was added to the DCL for the reduction of imines and the resulting mixture
3
was stirred for another 6 h. Subsequently, G -AuMNp template was separated from the reaction
4
mixture using a magnet and washed twice with MOPS buffer to remove the unbound amines.
Afterwards, the nanoparticles were dispersed in MOPS buffer (50 L) and the dispersion was
then heated for 5 min at 65 °C and the DNA-linked nanotemplate (G -AuMNps) was separated
4
instantly. The supernatant containing the amine lead compounds were identified by HPLC and
ESI-MS spectroscopy.
Templated imine exchange using dsDNA-AuMNp. In a 2 mL eppendorf, 40 μL dsDNA
nanotemplate (dsDNA-AuMNp) was added to 0.5 mL of 20 mM MOPS buffer (100 mM KCl,
pH 6.5). Aldehyde 1 (5 μM) was then added to the mixture, followed by the addition of 25 μM of
each amine 2a-j building blocks. The resulting mixture was stirred at rt for 18 h. Then NaBH CN
3
(10 mg) was added to the DCL and the mixture stirred for another 6 h. After reduction, the lead
compounds for dsDNA was identified by following the above-mentioned separation technique.
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General procedure for the synthesis of reduced imines (4a-j). Carbazole aldehyde 1 (50 mg,
0
.13 mmol) was dissolved in EtOH (4 mL) in the presence of molecular sieves. The desired
amine 2 a-j (0.13 mmol) was added separately and the mixture was stirred for 24 h at room
1
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temperature (Figure S11). The resulting reaction mixture comprising imines was dried under
vacuum. Then MeOH (5 mL) and NaBH CN (20 mg) was added to the solid mass and stirred for
3
another 6 h at room temperature. The mixture was concentrated under vacuum and was purified
by flash column chromatography (from CH Cl (100%) to CH Cl /MeOH (10:1) to
2
2
2
2
CH Cl /MeOH/NH OH (10:1:0.5) to give the corresponding amines.
2
2
4
4-(((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)amino)phenol
(Amine 4a). Following the GP, the reaction of aldehyde 1 with amine 2a (35.3 mg) afforded 4a
1
(
61 mg, 65 %) as a black viscous liquid. H NMR (400 MHz, CDCl ): δ 8.51 (s, 1H), 8.19 (s,
3
1
H), 8.04 (m, 1H), 7.91 (m, 2H), 7.89 (m, 2H), 7.62 (m, 2H), 7.45 (m, 3H), 6.93 (m, 2H), 4.31
(t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.77 (s, 2H), 2.16 (t, J = 7.0 Hz, 2H), 2.13 (s, 6H), 1.92 (m,
1
3
2
1
4
H); C NMR (100 MHz, CDCl ): δ 157.9, 148.2, 144.5, 141.5, 135.2, 129.8, 129.3, 128.8,
3
28.6, 128.1, 127.7, 127.4, 127.1, 126.9, 124.1, 123.3, 123.2, 120.9, 109.7, 109.4, 56.5, 55.0,
+
7.1, 45.5, 41.1, 27.0; HRMS (ESI) Calcd for C H N O [M+H] 480.2572, Found 480.2575.
36 45
4
2
N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)-4-nitroaniline
(Amine 4b). Following the GP, the reaction of aldehyde 1 with amine 2b (25.3 mg) afforded 4b
1
(
51 mg, 69 %) as a brown viscous liquid. H NMR (400 MHz, CDCl ): δ 8.60 (s, 1H), 8.28 (s,
3
1H), 8.12 (m, 1H), 7.99 (m, 2H), 7.69 (m, 2H), 7.63 (m, 2H), 7.52 (m, 3H), 7.02 (m, 2H), 4.40
(t, J = 6.2 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.22 (s, 6H), 2.01 (m,
1
3
2
H); C NMR (100 MHz, CDCl ): δ 158.4, 148.5, 144.5, 141.5, 135.2, 129.8, 129.5, 128.8,
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2
2
2
2
2
2
2
3
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3
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28.6, 128.1, 127.7, 127.5, 127.4, 126.9, 124.1, 123.3, 123.2, 120.9, 109.7, 109.4, 56.4, 55.2,
+
8.2, 45.5, 41.1, 27.0; HRMS (ESI) Calcd for C H N O [M+H] 509.2474, Found 509.2462.
3
6
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4
2
0
1
2
3
4
5
6
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8
9
0
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2
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4
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4
-bromo-N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)-2-
methylaniline (Amine 4c). Following the GP, the reaction of aldehyde 1 with amine 2c (30 mg)
1
afforded 4c (61 mg, 79 %) as a brown viscous liquid. H NMR (400 MHz, CDCl ): δ 8.54 (s,
3
1H), 8.29 (s, 1H), 7.64 (m, 1H), 7.62 (m, 1H), 7.51 (m, 1H), 7.47 (m, 2H), 7.32 (m, 3H), 7.02
(m, 2H), 6.91 (m, 1H), 4.42 (t, J = 6.1 Hz, 2H), 3.88 (s, 2H), 3.81 (s, 3H), 3.73 (s, 3H), 2.29 (t, J
1
3
=
6.8 Hz, 2H), 2.24 (s, 6H), 2.05 (m, 2H); C NMR (100 MHz, CDCl ): δ 158.1, 144.5, 141.5,
3
135.2, 129.7, 129.1, 128.8, 128.4, 128.1, 127.6, 127.4, 127.3, 127.0, 126.9, 124.1, 123.2, 120.8,
1
20.5, 111.8, 110.2, 109.7, 109.3, 56.5, 54.9, 51.7, 45.5, 41.3, 26.9, 19.7; HRMS (ESI) Calcd
+
for C H N O [M+H] 556.1885, Found 556.1882.
3
6
45
4
2
4-(3-(dimethylamino)prop-1-yn-1-yl)-N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-
carbazol-3-yl)methyl)aniline (Amine 4d). Following the GP, the reaction of aldehyde 1 with
1
amine 2d (35.3 mg) afforded 4d (51 mg, 69 %) as a brown viscous liquid. H NMR (400 MHz,
DMSO-d ): δ 8.38 (s, 1H), 8.17 (s, 1H), 7.70 (m, 4H), 7.64 (m, 2H), 7.57 (m, 2H), 7.55 (m, 2H),
6
7.03 (m, 2H), 4.66 (s, 2H), 4.42 (t, J = 6.2 Hz, 2H), 3.80 (s, 3H), 3.16 (s, 3H), 2.32 (t, J = 7.0
13
Hz, 2H), 2.20 (s, 12H), 1.93 (m, 2H); C NMR (100 MHz, DMSO-d ): δ 158.3, 150.3, 139.8,
6
1
39.6, 133.7, 133.2, 130.9, 127.8, 125.5, 124.4, 122.8, 122.1, 118.8, 117.7, 116.2, 115.6, 114.4,
113.3, 109.7, 109.0, 83.7, 82.3, 63.6, 62.7, 55.8, 55.3, 52.2, 48.7, 44.7, 26.1; HRMS (ESI) Calcd
+
for C H N O [M+H] 545.3202, Found 545.3213.
3
6
45
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-(3-(dimethylamino)propoxy)-N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-
carbazol-3-yl)methyl)aniline (Amine 4e). Following the GP, the reaction of aldehyde 1 with
1
amine 2e (25.3 mg) afforded 4e (51 mg, 69 %) as a brown viscous liquid. H NMR (500 MHz,
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
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5
5
5
5
5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
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1
2
3
4
5
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DMSO-d ): δ 8.34 (s, 1H), 8.22 (s, 1H), 7.69-7.67 (m, 3H), 7.60 (d, J = 8.8 Hz, 1H), 7.53 (d, J =
6
8
3
2
1
1
.2 Hz, 1H), 7.48 (d, J = 6.3 Hz, 1H), 7.03-7.01 (m, 2H), 6.67-6.58 (m, 4H), 4.39-4.34 (m, 6H),
.78 (3H, s), 3.37 (t, J = 7.1 Hz, 2H), 2.86 (t, J = 5.6 Hz, 2H), 2.23 (s, 6H), 2.18 (s, 6H), 1.91 (m,
1
3
H), 1.82 (m, 2H); C NMR (125 MHz, DMSO-d ): δ 158.2, 149.9, 143.3, 142.4, 139.6, 139.5,
6
33.6, 130.9, 127.6, 125.7, 124.3, 122.6, 122.2, 119.2, 117.6, 115.4, 114.9, 114.3, 113.4, 109.5,
09.1, 66.1, 55.7, 55.3, 55.1, 45.4, 44.6, 44.3, 26.3, 26.0; HRMS (ESI) Calcd for C H N O
2
36 45
4
+
[M+H] 565.35358, Found 565.3535.
N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)thiazol-2-
amine (Amine 4f). Following the GP, the reaction of aldehyde 1 with amine 2f (32 mg)
1
afforded 4f (58 mg, 65 %) as a brown viscous liquid. H NMR (400 MHz, DMSO-d ): δ 8.38 (s,
6
1
H), 8.21 (s, 1H), 8.16 (m, 1H), 7.71 (m, 3H), 7.69 (m, 1H), 7.67 (m, 1H), 7.63 (m, 2H), 7.03
(m, 2H), 4.65 (s, 2H), 4.42 (t, J = 6.3 Hz, 2H), 3.81 (s, 3H), 2.30 (t, J = 6.9 Hz, 2H), 2.19 (s, 6H),
1
3
1
1
5
.94 (m, 2H); C NMR (100 MHz, DMSO-d ): δ 169.1, 158.2, 139.7, 139.4, 138.6, 133.6, 133.1,
6
30.9, 129.6, 127.6, 125.3, 124.2, 122.7, 121.9, 118.7, 117.6, 114.3, 109.5, 106.1, 63.5, 55.7,
+
5.1, 45.5, 44.5, 25.9; HRMS (ESI) Calcd for C H N O [M+H] 471.2140, Found 471.2141.
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4
2
N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)butan-1-
amine (Amine 4g). Following the GP, the reaction of aldehyde 1 with amine 2g (9.5 mg)
1
afforded 4g (46 mg, 69%) as a brown viscous liquid. H NMR (500 MHz, DMSO-d ): δ 8.35 (s,
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1
H), 8.22 (s, 1H), 7.69 (d, J = 7.4 Hz, 3H), 7.61 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H),
.49 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 8.3 Hz, 2H), 4.40 (t, J = 6.1 Hz, 2H), 3.96 (3H, s), 3.80 (s,
7
2H), 2.64 (t, J = 7.1 Hz, 2H), 2.18 (t, J = 6.5 Hz, 2H), 2.12 (s, 6H), 1.90 (m, 2H), 1.50 (m, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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6
7
8
9
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7
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9
0
13
1
.32 (m, 2H), 1.23 (t, J = 7.1 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H); C NMR (125 MHz, DMSO-d ):
6
δ 158.2, 139.9, 139.5, 133.6, 130.9, 127.7, 126.7, 124.4, 122.6, 122.1, 120.6, 117.6, 114.3, 109.6,
1
09.0, 56.0, 55.1, 52.7, 52.0, 47.7, 45.1, 30.5, 26.5, 19.8, 13.9; HRMS (ESI) Calcd for
+
C H N O [M+H] 444.3005, Found 444.3008.
2
9
38
3
N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)prop-2-yn-
1-amine (Amine 4h). Following the GP, the reaction of aldehyde 1 with amine 2h (7.2 mg)
1
afforded 4h (33 mg, 59 %) as a yellow liquid . H NMR (500 MHz, CDCl ): δ 8.25 (s, 1H), 8.11
3
(s, 1H), 7.65 (m, 3H), 7.45 (m, 3H), 7.02 (d, J = 8.5 Hz, 2H), 4.39 (t, J = 6.8 Hz, 2H), 4.08 (s,
2H), 3.88 (s, 3H), 3.50 (d, J = 2.0 Hz, 1H), 2.31 (t, J = 7.1 Hz, 2H), 2.24 (s, 6H), 2.04 (m, 2H),
13
1
1
4
.80 (s, 1H); C NMR (100 MHz, CDCl ): δ 158.6, 140.4, 140.0, 134.8, 132.0, 129.9, 128.2,
3
26.6, 124.9, 123.2, 123.1, 120.3, 118.4, 114.2, 109.0, 108.8, 82.3, 71.5, 56.6, 55.4, 52.7, 45.4,
+
0.9, 37.3, 29.7, 27.0; HRMS (ESI) Calcd for C H N O [M+H] 426.2545, Found 426.2542.
28 32
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N1-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)-N3,N3-
dimethylpropane-1,3-diamine (Amine 4i). Following the GP, the reaction of aldehyde 1 with
1
amine 2i (25 mg) afforded 4i (48 mg, 79 %) as a brown viscous liquid. H NMR (400 MHz,
CDCl ): δ 8.15 (s, 1H), 7.91 (s, 1H), 7.54 (m, 2H), 7.35 (m, 2H), 7.17 (m, 2H), 7.10 (m, 2H),
3
4
.30 (t, J = 6.1 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 2H), 2.55 (t, J = 6.0 Hz, 2H), 2.36 (t, J = 7.1 Hz,
1
3
2H), 2.21 (t, J = 6.1 Hz, 2H) 2.20 (t, J = 5.9 Hz, 2H), 2.14 (s, 12H), 1.95 (m, 2H); C NMR (100
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Page 29 of 44
Journal of Medicinal Chemistry
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MHz, CDCl ): δ 158.3, 148.4, 144.2, 140.6, 135.1, 132.4, 129.7, 129.1, 127.6, 126.7, 125.6,
3
1
24.4, 121.8, 120.5, 111.7, 109.6, 65.1, 57.0, 56.2, 55.1, 46.3, 45.5, 41.0, 29.8, 26.9; HRMS
+
(ESI) Calcd for C H N O [M+H] 473.3235, Found 473.3233.
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4
2
1
1
1
1
1
1
1
1
1
1
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5
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6
0
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7
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9
0
1
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7
8
9
0
1
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9
0
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0
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0
N-((9-(3-(dimethylamino)propyl)-6-(4-methoxyphenyl)-9H-carbazol-3-yl)methyl)cyclopropanamine
(Amine 4j). Following the GP, the reaction of aldehyde 1 with amine 2j (25.8 mg) afforded 4j
1
(50 mg, 69 %) as a yellow viscous liquid. H NMR (400 MHz, CDCl ): δ 8.25 (s, 1H), 8.08 (s,
3
1
3
H), 7.64 (m, 2H), 7.43 (m, 4H), 7.01 (m, 2H), 4.38 (t, J = 6.0 Hz, 2H), 4.04 (s, 2H), 3.87 (s,
1
3
H), 2.32 (t, J = 6.0 Hz, 2H), 2.24 (s, 6H), 2.23 (m, 1H), 2.04 (m, 2H), 0.43 (m, 4H); C NMR
(100 MHz, CDCl ): δ 158.8, 140.4, 140.2, 134.9, 132.2, 130.8, 128.4, 126.8, 125.1, 123.5, 123.2,
3
1
20.3, 118.5, 114.4, 109.1, 108.9, 56.8, 55.5, 54.2, 46.1, 45.5, 30.2, 27.0, 9.3; HRMS (ESI)
+
Calcd for C H N O [M+H] 428.2657, Found 428.2653.
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FRET-based DNA melting assay. FRET melting assay was carried out on a real-time PCR
apparatus (Roche LightCycler 480 II) using dual labelled DNA sequences. The DNA sequences
labelled with 6-FAM (donor fluorophore) at 5’-end and TAMRA (acceptor fluorophore) at 3’-
end were purchased from Eurofins Genomics. The fluorescently labelled oligonucleotides (G4-
DNA and dsDNA) were annealed in 60 mM potassium cacodylate buffer (pH 7.4) at a
concentration of 400 nM by heating at 95 °C for 5 min followed by gradual cooling to room
temperature at a controlled rate of 0.1 °C/min. Fluorescence melting curves were recorded using
a total reaction volume of 100 μL, with 200 nM labelled oligonucleotide in Tris-KCl buffer (pH
7
.4) in the absence and/or presence of 1 μM of each amine product (4a-4j). Measurements were
done with excitation at 480 nm and detection at 530 nm. Fluorescence readings were taken with
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