Biaryl polyamides as a new class of DNA quadruplex-binding ligands

Article abstract of DOI:10.1039/b902359c

We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with significant cytotoxicity in several cancer cell lines; they form planar U-shaped structures that match the surface area dimensions of a terminal G-quartet in quadruplex structures rather than the grooves of duplex DNA.

Full text of DOI:10.1039/b902359c

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Biaryl polyamides as a new class of DNA quadruplex-binding ligandsw
Khondaker M. Rahman,a Anthony P. Reszka,b Mekala Gunaratnam,b Shozeb M. Haider,b
Philip W. Howard,a Keith R. Fox,c Stephen Neidle*b and David E. Thurston*a
Received (in Cambridge, UK) 4th February 2009, Accepted 8th May 2009
First published as an Advance Article on the web 5th June 2009
DOI: 10.1039/b902359c
We report a novel class of biaryl polyamides highly selective for
G-quadruplex DNA, and with significant cytotoxicity in several
cancer cell lines; they form planar U-shaped structures that
match the surface area dimensions of a terminal G-quartet in
quadruplex structures rather than the grooves of duplex DNA.
Previous attempts to devise quadruplex-binding acyclic
polyamides, based on a distamycin motif, have not resulted
in molecules with significant selectivity over duplex DNA.7
The molecules described here are from an 84-member
solution-phase library, the design of which allowed positioning
of the biaryl units at either end of the polyamide core in order
to introduce ligand diversity. The biaryl polyamides have two
different structural motifs. While 1–4 contain one biaryl unit,
5 and 6 are dimers of biaryl units (Fig. 1A).
Guanine-rich nucleic acid sequences can fold into four-stranded
G-quadruplex structures.1 These structures may be adopted by
telomeric DNA repeats as well as sequences in promoter and
other regulatory regions within human and other genomes.2
Stabilization of the 30-end single-stranded overhang of
telomeric DNA as G-quadruplex structures can inhibit the
telomerase enzyme complex (which is expressed in most cancer
cells) from synthesizing further telomeric DNA repeats, resulting
in selective cell growth inhibition. The prevalence of
quadruplex sequences in gene promoter regions suggests a
potential role for quadruplex structures in transcription,3 and
such quadruplexes have been characterized in the promoter
sequences of a number of proto-oncogenes and cancer-
associated growth factors,3 notably c-myc, c-kit, k-ras,
PDGF-A and bcl-2. In addition, G-quadruplex structures in
the 50-untranslated regions of gene transcripts have the
potential to modulate translation.4 Therefore, small molecules
that selectively bind and stabilize G-quadruplex nucleic acids
are of interest as a new class of anticancer agents. The majority
of quadruplex-binding molecules studied to date comprise
functionalized polycyclic aromatic systems such as acridines,
anthraquinones, indoloquinolines and porphyrins.5 A number
of these molecules have the ability to provide quadruplex-
related biological effects, although their physico-chemical
features are generally non-drug-like. More recently, a number
of ligands have been reported that have non-conjugated
groupings, whilst still maintaining the essential co-planarity
with the G-quartet motif.6
Our preliminary goal was to design molecules that have no
affinity for duplex DNA but have high affinity for DNA
quadruplexes. We used a distamycin scaffold as a starting
point and introduced biaryl building blocks in place of
pyrroles to switch preference from duplex to quadruplex.
A simple linear synthetic route was devised to prepare the
initial building blocks, and the points of diversity were
introduced using parallel chemistry. Our synthesis was based
on the application of HOBT–DIC-mediated amide coupling
and microwave-assisted Suzuki coupling8 to afford the building
blocks (Scheme 1). The pyrrole carboxamide 7 was coupled to
different 5-membered bromoheterocyclic acids to provide the
Suzuki substrates 8 in excellent yields. The Suzuki coupling
We report here a novel class of acyclic biarylz polyamides
1–6 which selectively bind to G-quadruplexes, and which offer
synthetic versatility and significant quadruplex selectivity.
a Gene Targeted Drug Design Research Group, The School of
Pharmacy, University of London, 29/39 Brunswick Square, London,
UK WC1N 1AX. E-mail: david.thurston@pharmacy.ac.uk;
Fax: +44 (0)20 7753 5964; Tel: +44 (0)20 7753 5931
b CRUK Biomolecular Structure Group, The School of Pharmacy,
University of London, 29/39 Brunswick Square, London,
UK WC1N 1AX. E-mail: stephen.neidle@pharmacy.ac.uk;
Fax: +44 (0)20 7753 5970; Tel: +44 (0)20 7753 5971
c School of Biological Sciences, University of Southampton, Bassett
Crescent East, Southampton, UK SO16 7PX
Fig. 1 A, Structures of biaryl polyamides. B, A molecular model of
compound 4 (blue) bound to a terminal G-quartet (yellow) taken from
a human intramolecular telomeric quadruplex–ligand complex crystal
structure, showing the characteristic U shape of the biaryl polyamides.
w Electronic supplementary information (ESI) available: Experimental
procedures for FRET, cell culture, CD spectra, NMR and MS data.
See DOI: 10.1039/b902359c
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Chem. Commun., 2009, 4097–4099 | 4097

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evident when compared to distamycin, which has only very
modest quadruplex affinity and
duplex DNA.
a high preference for
Compounds 1, 3, 4 and 5 show greater G-quadruplex
stabilizing ability with the telomeric HT4 quadruplex
compared to the two c-kit ones. Compound 2 has a weak
though comparable stabilizing ability for all three quadruplexes,
whereas compound 6 shows distinctly superior stabilizing
ability for the two c-kit quadruplexes. Compound 3 has the
greatest selectivity in the series for the telomeric HT4
quadruplex, notably accompanied by no detectable duplex
stabilization. The difference between compounds 3 and 4,
which have high chemical structural similarity, is likely to be
due to the greater polarizability of the N-methyl imidazole
group in the former compared to the benzofuran group in the
latter, which would result in more effective p-stacking with a
terminal G-quartet in these quadruplexes. Compounds 5 and 6
are biaryl dimers with the identical first biaryl units having
distinct meta vs. para orientations. The DTm data show a clear
preference of 5 for the telomeric quadruplex compared to 6.
Surprisingly, the affinity of 6 for the c-kit quadruplexes is not
diminished, suggesting that this ligand is sensitive to the
structural differences between these three quadruplexes.
Molecular modelling has been used10 to rationalize these
results in the absence of structural data. Several distinct
polymorphic forms of the native human intramolecular
quadruplex have been identified,11 and it is not possible on
the basis of the biophysical data alone to define which is the
most appropriate one to use as a starting point. Instead
we have used a human quadruplex–ligand complex crystal
structure,12 focussing on maximizing ligand–G-quartet
overlap by means of a docking approach. The low-energy
conformations of the biaryl polyamides form planar U-shaped
structures (Fig. 1B) that match the surface area dimensions of
a terminal G-quartet but are too large to be accommodated
within the major or minor grooves of duplex DNA. The
inability of these molecules to bind to duplex DNA was
further confirmed by DNAse I footprinting experiments
against Hex A and Hex B sequences where they failed
to provide any footprints at concentrations up to 4100 mM
(ESIw).
Scheme 1 Key synthetic steps for the quadruplex-targeting biaryl
polyamides (Motif-1).
reaction between 8 and the aminoboronic/nitroboronic acids
proceeded well under microwave conditions with high yields
over two steps. The resulting analogues 9 were subjected
to coupling reactions with either commercially available
heterocyclic carbonyl chlorides or carboxylic acids to give
the Motif-1 biaryl polyamides in excellent yields (see ESIw
for synthesis of Motif-2 compounds).
The intermediates and each library member contained a
3,3-dimethylaminopropyl side chain which assisted in the
purification of these compounds by the ‘‘catch and release’’
method using sulfonic acid-based SCX resins.
Assessment of the G-quadruplex interaction of 1–6
was initially carried out using
a FRET-based melting
assay.9 Three different intramolecular DNA G-quadruplex
sequences were used: human telomeric G-quadruplex (HT4,
FAM-d(G3[TTAG3]3)-TAMRA) and two different c-kit
promoter G-quadruplexes (c-kit1, FAM-d(G3AG3CGCT-
G3AG2AG3)-TAMRA and c-kit2, FAM-d(G3CG3CGCGA-
G3AG4)-TAMRA).
[FAM-(TA)2GC(TA)2T6(TA)2GC(TA)2-TAMRA] was used
as control. Together, the melting data from both
A
DNA duplex hairpin sequence
a
G-quadruplex and duplex sequences enabled assessment of
the selectivity of the ligands for different G-quadruplex
sequences and selectivity for G-quadruplex versus duplex
DNA. The minor groove binding polyamide distamycin, with
established DNA duplex stabilizing ability,7 was used as a
control ligand.
Circular dichroism (CD) can be used to indicate whether
ligands bind to, and perhaps retain the overall topology of, a
quadruplex nucleic acid. CD titrations of 1–6 were conducted
for the telomeric quadruplex sequence in the presence of
100 mM KCl. Ligands 1–6 do not have any chiral centres
and so are CD inactive. The CD spectrum of the native
sequence showed the presence of mixed parallel and anti-
parallel structures with positive and negative peaks around
295 nm and 240 nm, respectively. The spectrum showed
significant changes on adding just one equivalent of ligand 3.
The data in Table 1 show that the biaryl polyamide
compounds 1–6 show significant selectivity towards
G-quadruplex compared to duplex DNA, and the best
molecule in the series, 3, has no significant action on duplex
DNA at this concentration. The selectivity becomes more
Table 1 DNA G-quadruplex and duplex stabilization by compounds
1–6 and distamycin (Dist) in the FRET melting experiments. Melting
data are also available at higher ligand concentrations (ESIw)
DTm at 1 mM conc./1C
We observed
a
concentration-dependent enhancement
HT4
c-kit1
c-kit2
Duplex
Ligand
of the positive peak at 295 nm, appearance of a negative peak
around 260 nm, and disappearance of the negative peak
around 240 nm at the concentrations used in the study.
Titrations of 4 resulted in some concentration-dependent
changes in the CD spectra, although it is not possible in the
absence of other data to assign particular topologies from
them (Fig. 2A and B).
1
2
3
4
5
6
Dist
8.8
2.4
11.8
6.8
5.9
1.0
0.6
6.1
2.5
6.0
4.8
4.3
4.4
0.3
3.2
3.5
5.6
4.7
2.4
4.6
0.4
0.4
0.2
0.0
0.3
0.1
0.0
11.5
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Fig. 2 CD spectra of a 5 mM solution of G-quadruplex HT4 in Tris
buffer (pH 7.4) with 0–6 equivalents of ligands 3 (A) or 4 (B).
Table 2 IC50 (mM) values of the biaryl polyamides 1–6 against three
different cancer cell lines and a normal cell line (WI38) using the
sulforhodamine B assay
4 S. Kumari, A. Bugaut, J. L. Huppert and S. Balasubramanian,
Nat. Chem. Biol., 2007, 3, 218; S. Kumari, A. Bugaut and
S. Balasubramanian, Biochemistry, 2008, 47, 12664.
5 See recent reviews: C. A. De, L. Lacroix, C. Douarre, N. Temime-
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690.
Ligand
MCF7
A549
HT29
WI38
1
2
3
4
5
6
9.4
2.1
12.8
450
3.6
5.9
2.0
3.3
17.0
2.6
2.6
2.5
0.4
2.1
9.2
0.6
2.5
11.8
9.2
25.4
13.2
5.1
2.1
11.6
6 See for example: A. D. Moorhouse, A. M. Santos,
M. Gunaratnam, M. Moore, S. Neidle and J. E. Moses, J. Am.
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8 N. Miyaura and A. Suzuki, J. Chem. Soc., Chem. Commun., 1979,
866.
The short-term cell growth inhibitory activity of the biaryl
ligands was evaluated using the sulforhodamine B (SRB) assay
for four different cancer cell lines and a normal human lung
fibroblast cell line (WI38) (Table 2). The ligands generally
showed greater cell growth inhibition against human colorectal
carcinoma (HT29) and human lung carcinoma (A549) lines.
The aim when targeting cancer cells is to achieve selectivity
over normal cells. Although cell culture data can only be
indicative compared to in vivo results, the data here do show
that some ligands, in some cell lines, have significant selectivity.
Compound 3 has the best overall profile, especially for the
A549 and HT29 cell lines. This compound is also the
most effective at stabilizing a human telomeric quadruplex
(Table 1), although further studies will be required to
unequivocally demonstrate that it is targeting telomere
maintenance in tumour cells.
9 B. Guyen, C. M. Schultes, P. Hazel, J. Mann and S. Neidle, Org.
Biomol. Chem., 2004, 2, 981.
10 A terminal G-quartet from the crystal structure of a human
intramolecular telomeric G-quadruplex complexed with
a
naphthalene diimide ligand (PDB id 3CDM) was used as a starting
point to study plausible interactions with biaryl polyamides.
Automated docking studies were carried out using the AutoDock
program v4.0 (D. S. Goodsell, G. M. Morris and A. J. Olson,
J. Mol. Recognit., 1996, 9, 1). A total of 250 independent docking
runs were undertaken to enhance the reliability of the docking
process (R. Wang, Y. Lu and S. Wang, J. Med. Chem., 2003, 46,
2287). Cluster analysis was carried out on the docked results using
a root mean square (RMS) tolerance of 1.0 A. Fig. 1B shows the
ligand G-quartet overlap for the lowest-energy solution.
In summary, we report a new class of G-quadruplex ligands
with high selectivity over duplex DNA. The ligands described
here can be used as a template to attempt to generate
more-potent molecules while retaining this selectivity, as well
as for manipulating their selectivity to favour a particular type
of quadruplex. We note that there is high potential for diversity
in the terminal heterocyclic group, which should also be
amenable to optimization to enhance drug-like characteristics.
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Notes and references
12 N. H. Campbell, G. N. Parkinson, A. P. Reszka and S. Neidle,
J. Am. Chem. Soc., 2008, 130, 6722; G. N. Parkinson, F. Cuenca
and S. Neidle, J. Mol. Biol., 2008, 381, 1145.
z The term biaryl refers to two linked aromatic rings which can be
carbocyclic or heterocyclic, or a combination of both.
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 4097–4099 | 4099