Controlled-folding of a small molecule modulates DNA G-quadruplex recognition

Article abstract of DOI:10.1039/b816861j

Differential recognition of diverse G-quadruplex structures can be achieved by controlling the folding of a small molecule. The Royal Society of Chemistry.

Full text of DOI:10.1039/b816861j

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Controlled-folding of a small molecule modulates DNA G-quadruplex
recognitionw
Sebastian Muller, G. Dan Panto-s, Raphael Rodriguez and Shankar Balasubramanian*
¨
¨
Received (in Cambridge, UK) 26th September 2008, Accepted 24th October 2008
First published as an Advance Article on the web 12th November 2008
DOI: 10.1039/b816861j
Differential recognition of diverse G-quadruplex structures can
be achieved by controlling the folding of a small molecule.
The human genome contains guanine-rich sequences which
have been shown to fold into G-quadruplexes in vitro. These
structures have been identified in biologically relevant regions
of the genome, such as the promoters of various proto-
oncogenes that include, among others c-kit,1 c-myc,2 K-ras,3
and at telomeres.4 These particular structures have been
Fig. 1 Molecular structure of the ligands.
associated with function and have thus emerged as motifs to
be targeted with small molecules.2,5 G-quadruplexes vary in
loop length and sequence, providing the opportunity for
differential molecular recognition. There is a need to design
small molecules that exhibit specificity with respect to duplex
DNA and also between different quadruplexes. Numerous
G-quadruplex binders have been reported that generally com-
prise an aromatic core with side chains to improve mole-
cular recognition.6 The specificity of such molecules has been
enhanced by varying the nature,6 position7 and stereo-
chemistry8 of the appendages, but the challenge to improve
specificity between different quadruplexes still remains.
Herein, we describe a new G-quadruplex ligand family for
which we demonstrated that controlled-folding of the main
scaffold can control specificity between the aforementioned
diverse nucleic acid structures.
rise to the expected bis-ureas. Two analogous series were
synthesised, one with a primary amine appendage a, and the
other with a pyrrolidine appendage b. This would allow us to
also assess whether the nature of the side-chain could influence
the nucleic acid recognition properties of differently folded
ligands.
To determine how the methyl substitution would affect
the folded state of the molecule, we performed molecular
modelling on molecules 1a, 2a and 3a using the PM3
semi-empirical method including a solvent model, followed
by 1D and 2D NOESY NMR experiments (see ESI).w
According to molecular modeling, 1a adopts the coiled
conformation as depicted in Fig. 2.
In contrast, 2b shows a twisted conformation, with one of
the quinolines pointing upwards; a conformation that was
supported by the presence of nOe cross peaks between the
hydrogens of the methyl substituent and one of the hydrogens
on one of the quinolines as shown in Fig. 3.
We designed a new class of small molecules (Fig. 1) based on
known structural features of classical quadruplex binding
ligands.9 We included two urea functionalities which link
two quinolines—moieties that have been shown to enhance
the binding of other quadruplex ligands—to a central benzene
core.5d,10 The urea bonds confer a degree of conformational
freedom to these molecules.11 Furthermore, we introduced
methyl substituents to the central benzene ring of some
derivatives to favour particular conformations as a con-
sequence of steric clash with the oxygens of the urea bonds.12
The molecules were synthesised using a short and high
yielding synthetic procedure (see ESIw). 2-Amino-quinolinone
was reacted with N-boc-ethanolamine or N-(2-hydroxyethyl)-
pyrrolidine under Mitsunobu reaction conditions11b to intro-
duce the amine side chains. The products were then reacted
with commercially available 1,3-phenylene diisocyanate,
2,4-toluene diisocyanate or 2,6-toluene diisocyanate giving
Molecule 3a was predicted to have a W-shaped conforma-
tion that drastically differs from the conformation of 1a and
2a. This was also supported by nOe cross peaks between the
hydrogens of the methyl substituent and one of the hydrogens
on both quinolines as depicted in Fig. 4.
The University Chemical Laboratory, University of Cambridge,
Lensfield Road, Cambridge, CB2 1EW, UK.
E-mail: sb10031@cam.ac.uk; Fax: +44 (0)1223 336913
w Electronic supplementary information (ESI) available: Further
experimental details, NOESY spectra, FRET-melting experiments
and HyperChems modeling. See DOI: 10.1039/b816861j
Fig. 2 Coiled conformation adopted by 1a.
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Fig. 5 DTm at different concentrations of ligand 1a: (pink) K-ras,
(red) c-kit1, (yellow) h-Telo, (green) c-kit2, (blue) c-myc, (black)
ds-DNA.
Fig. 3 Twisted conformation adopted by 2a. The nOe cross peaks are
indicated with arrows.
Fig. 6 DTm at different concentrations of ligand 2a: (pink) K-ras,
(red) c-kit1, (yellow) h-Telo, (green) c-kit2, (blue) c-myc, (black)
ds-DNA.
Fig. 4 W-Shaped conformation of 3a. The nOe cross peaks are
indicated with arrows.
These structural data support our hypothesis that confor-
mation can be controlled by the methyl substitution on the
benzene ring.
We next explored how distinct folded conformations of
these molecules influenced their ability to interact with
DNA. Small molecule–DNA interactions were evaluated,
using fluorescence resonance energy transfer (FRET) melting
experiments, that provide a measure of the ability of a
molecule to stabilise the folded form of a DNA structure.13
We evaluated the aptitude of the small molecules to stabilise
an array of different biologically relevant quadruplex forming
DNA sequences, occurring in the promoter regions of c-kit
(which includes two quadruplex forming sequences: c-kit1 and
c-kit2), c-myc, K-ras and the human telomeric quadruplex
h-Telo. Fig. 5–7 show thermal shift profiles in which the
increase in the transition temperature (DTm) is plotted as a
function of ligand concentration. Each DNA target exhibits a
different basal Tm value,14 thus one cannot directly compare
absolute DTm values for these targets. We found that different
folded molecules of the family induced distinct profiles,
suggesting that the folding of these ligands influences their
molecular mode of recognition. The ligands do not adopt a
planar conformation, and are thus unlikely to interact with the
quadruplexes via stacking, but may rather interact with the
loops of different G-quadruplexes. The selectivity observed
Fig. 7 DTm at different concentrations of ligand 3a: (pink) K-ras,
(red) c-kit1, (yellow) h-Telo, (green) c-kit2, (blue) c-myc, (black)
ds-DNA.
may be the result of the diversity observed between these loop
sequences and structures.15 None of the ligands showed any
detectable stabilisation of ds-DNA at any of the concentra-
tions measured, consistent with a high level of discrimination
between quadruplex and double-stranded DNA.
Compound 1a, that has a coiled conformation, showed very
high changes in melting temperature for all the quadruplexes
studied, as displayed in Fig. 5 and Table 1.
This suggests very high stabilisation potentials but little
discrimination between different G-quadruplexes. The stabili-
sation of the human telomeric quadruplex with a DTm of
27.3 K is in the range of the most potent ligands reported in the
literature; such as telomestatin and a quinoline macrocycle,
which show DTm values of 30.3 K and 33.8 K, respectively.10 It
is noteworthy, that this ligand showed the highest DTm that can
be measured by this method for all quadruplexes tested,14 which
is visualised by the flattening of the curves in Fig. 5. The
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Table 1 Concentrations of ligands 1a, 1b, 2a, 2b, 3a and 3b in mM at half maximal DTm (with DTm at 1 mM ligand in K shown in parentheses)17
1a
2a
3a
1b
2b
3b
K-ras
c-kit1
h-Telo
c-kit2
c-myc
ds-DNA
1.49 (16.7)
0.62 (34.0)
0.53 (27.3)
0.38 (16.2)
0.17 (17.7)
410.0 (0.0)
4.00 (4.0)
3.74 (6.6)
5.59 (2.8)
3.41 (4.2)
2.25 (6.2)
410.0 (0.0)
3.68 (2.7)
2.00 (16.0)
1.07 (19.8)
1.00 (16.7)
1.16 (9.3)
1.00 (7.5)
410.0 (0.0)
3.56 (9.4)
0.88 (20.1)
0.87 (20.0)
1.86 (6.1)
1.03 (7.1)
410.0 (0.5)
1.47 (20.8)
410.0 (0.7)
0.48 (25.9)
0.46 (14.9)
0.25 (13.8)
410.0 (0.4)
410.0 (0.0)
1.89 (3.9)
4.75 (1.0)
0.16 (14.1)
410.0 (0.0)
concentrations to reach this saturation were in the low micro-
molar range: 0.8 mM for c-myc, 2.0 mM for c-kit 1, 2.5 mM for
c-kit2, 2.8 mM for h-Telo and 5 mM for K-ras.
Ligand 2a, with its twisted conformation, did not show a
strong stabilisation potential for any of the quadruplexes
investigated at low ligand concentrations, but showed very
good stabilisation potential at higher ligand concentrations as
seen in Fig. 6.
serves as a new approach to achieve differential recognition
between G-quadruplexes.
We thank Cancer Research UK for programme funding and
for a studentship (S. M.), and Pembroke College for a Stokes
Junior Research Fellowship (G. D. P.). We are
thankful to Prof. J. K. M. Sanders for helpful discussions.
Notes and references
Much higher ligand concentrations were required to reach
saturation of the curves compared to 1a, making this ligand
less potent.16
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In contrast, 3a with its W-shaped structure, showed selective
stabilisation for a particular G-quadruplex. It stabilised the
c-myc quadruplex with a DTm of 14.1 K at a concentration of
0.3 mM as depicted in Fig. 7. Ligand 3a stabilised the other
sequences with a very low DTm at low ligand concentrations
(i.e. o1 mM). Similar ligand concentrations for 3a compared
to 2a were required to reach the maximum measurable
stabilisation for K-ras and c-kit2. However, ligand 3a was
more potent for h-Telo than 2a as it reached the maximal DTm
at ligand concentrations of 3 mM compared to 8 mM. It is
noteworthy that 3a does not stabilise the c-kit1 quadruplex at
any of the ligand concentrations used. To the best of our
knowledge, this is the first example of a small molecule that
exhibits significant stabilisation potential for one or more
quadruplexes, while showing no detectable stabilisation for
another. These results show that it is possible to achieve
excellent quadruplex discrimination by controlling the folding
of a particular molecular scaffold.
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Changing the primary amine of the side chain to pyrrolidine
did alter the quadruplex stabilisation potential. It decreased
the potency of ligands of type 1 but improved the molecular
recognition properties for molecules of type 2 and 3. The c-kit1
quadruplex was not stabilised by 3b, a feature displayed by the
ligands of the family possessing a W-shaped structure.
However, this molecule showed better molecular recognition
of the K-ras (DTm of 20.8 K), h-Telo (DTm of 25.9 K) and
c-kit2 (DTm of 14.9 K) quadruplexes and still showed good
stabilisation of c-myc with a DTm of 13.8 K. These results
show that it is possible to combine a controlled-folding
approach with side-chain variation to fine-tune the molecular
recognition properties of a particular scaffold.
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14 The Tm of the quadruplexes in 60 mM K+ in the absence of ligand
are: 45.9 1C (K-ras), 54.5 1C (c-kit 1), 72.7 1C (c-kit 2), 79.3 1C
(c-myc), 59.4 1C (h-Telo), 62.7 1C ds-DNA; the life cycler can
measure temperatures up to 94.0 1C.
15 As a recent example of a small molecule that interacts with
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In conclusion, we have described a novel small molecule
family that significantly stabilises an array of quadruplexes.
Some of the small molecules compare favourably to the most
potent ligands reported in the literature. We demonstrated
how controlling the ligand conformation has a dramatic effect
on the molecular recognition properties of a molecule, and
16 For the h-Telo, c-kit1 and c-kit2 quadruplexes the thermal shifts
exhibited a biphasic profile which may be indicative of a different
mode of binding at higher ligand concentrations.
17 The values are an average of three independent measurements.
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82 | Chem. Commun., 2009, 80–82