Stabilisation of human telomeric quadruplex DNA and inhibition of telomerase by a platinum-phenanthroline complex

Article abstract of DOI:10.1039/b709898g

Two new mono-substituted phenanthroline ligands and their platinum(ii) square planar complexes have been prepared; one of the complexes has been shown to induce a high degree of quadruplex DNA stabilisation and to inhibit telomerase. The Royal Society of

Full text of DOI:10.1039/b709898g

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Stabilisation of human telomeric quadruplex DNA and inhibition of
telomerase by a platinum–phenanthroline complex{
Julie E. Reed,abc Stephen Neidle*ab and Ramon Vilar*ac
Received (in Cambridge, UK) 29th June 2007, Accepted 26th July 2007
First published as an Advance Article on the web 13th August 2007
DOI: 10.1039/b709898g
structural and functional properties that metals can confer on a
molecule. We hypothesised that a metal coordinated to hetero-
aromatic multidentate ligands with a square planar geometry
could have important advantages over more ‘‘classical’’ quad-
ruplex DNA binders. The metal can play a major structural role in
organising the ligand(s) into an optimal structure for quadruplex
DNA interaction. Also, the electropositive metal can in principle
be positioned at the centre of the guanine quartet, increasing
electrostatic stabilisation by substituting the cationic charge of the
potassium or sodium that would normally occupy this site. In
addition, metal complexes tend to have interesting optical and
magnetic properties which could in turn be employed to probe the
interaction between small molecules and quadruplex DNA. As we
have recently shown,24 compounds based on planar nickel(II)–
salphen complexes are indeed excellent quadruplex DNA stabi-
lisers and telomerase inhibitors. Herein we present a new
quadruplex DNA stabiliser and telomerase inhibitor based on a
phenanthroline ligand and its square planar platinum(II) complex
(see Scheme 1).
Two new mono-substituted phenanthroline ligands and their
platinum(II) square planar complexes have been prepared; one
of the complexes has been shown to induce a high degree of
quadruplex DNA stabilisation and to inhibit telomerase.
The telomerase complex enzyme is responsible for the maintenance
of the integrity of the ends of chromosomes, preventing critical
telomere shortening by catalysing the synthesis of telomeric DNA
so that cells cannot reach crisis points of senescence and apoptosis.
Telomerase is up-regulated in 80–85% of tumour cells and is a
major factor in cancer cell immortalisation.1 Therefore, there is
considerable current interest in finding molecules that can inhibit
telomerase and potentially act as anticancer drugs.2,3 Human
telomeric DNA consists of the tandem repeat sequence TTAGGG
(having a length of 3–6 KB in cancer cells) with the 39 terminal
100–200 bases being single-stranded. Crystallographic4 and
NMR5–7 studies have shown that repeats of this sequence can
fold into higher-order guanine-quadruplex DNA structures.8–10
Since the substrate of telomerase is the 39-single-stranded overhang
of telomeric DNA, the stabilisation of quadruplex DNA structures
by small molecules can inhibit telomerase11 interfering with
telomere maintenance in tumour cells.12
Qualitative computer modelling was initially employed to
design small molecules with optimal binding properties to
stabilise quadruplex DNA (see Fig. 1). From these studies, it
was found that mono-substituted phenanthrolines and their
corresponding square planar metal complexes have the
appropriate structural features for a good overlap with guanine
quartets.
Over the past few years a wide range of planar heteroaromatic
molecules have been investigated as quadruplex DNA stabilisers
and evaluated as telomerase inhibitors.13–19 A few have also been
evaluated for anticancer activity.20,21 From crystallographic and
NMR spectroscopic studies, together with computer modelling, it
has been possible to develop a rational approach to designing
molecules with optimal properties for quadruplex DNA stabilisa-
tion. Some of the desired features these molecules should have are:
a planar p-delocalised system able to stack on the face of the
guanine quartet; a partial positive charge on the molecule
positioned at the centre of the quartet; substituents bearing a
positive charge to interact with the grooves and loops of DNA and
with the negatively charged backbone phosphates.
The modelling also suggested that the platinum(II) ion present in
the proposed metal complexes would be positioned at the centre of
the guanine quartet. Based on this design, the syntheses of L1 and
L2 and the corresponding platinum complexes 1 and 2 were carried
out as outlined in Scheme 1. All the compounds were fully
characterised by NMR and IR spectroscopies, mass spectrometry
and elemental analysis (see the ESI for full experimental details).
The synthesis of both ligands was based on a previously
reported methodology for the formation of mono-substituted
phenanthroline amides.27,28 Complexes 1 and 2 were prepared by
reacting the deprotonated version of the corresponding phenan-
throline mono-amide with K2PtCl4. The products were isolated
and purified to yield [Pt(L1)Cl] (1) and [Pt(L2)Cl] (2) as orange/red
solids. The IR spectra of both platinum(II) complexes showed a
shift of the CO stretching frequency due to coordination (from
1648 to 1626 cm21 in 1 and from 1664 to 1626 cm21 in 2)
consistent with N-coordination of amidato groups. The 1H NMR
spectra of 1 and 2 show the expected peaks for L1 and L2
respectively, some of the resonances being shifted in comparison
to the free ligands. The formulations of both 1 and 2 were
confirmed by elemental analyses and ESI(+) mass spectrometry
Most of the molecules reported to date as quadruplex DNA
stabilisers and telomerase inhibitors are based on organic
heteroaromatic systems. In contrast, very few metal complexes
have been studied in this context17,22–26 in spite of the versatile
aDepartment of Chemistry, Imperial College London, London, UK
SW7 2AZ. E-mail: r.vilar@imperial.ac.uk; Fax: 44 (0)207 5941967;
Tel: 44 (0)207 5941139
bCRUK Biomolecular Structure Group, The School of Pharmacy,
University of London, London, UK WC1N 1AX
cInstitute of Chemical Research of Catalonia (ICIQ), Avgda. Pa¨ısos
Catalans 16, 43007 Tarragona, Spain
{ Electronic supplementary information (ESI) available: Full experimental
details and FRET results. See DOI: 10.1039/b709898g
4366 | Chem. Commun., 2007, 4366–4368
This journal is ß The Royal Society of Chemistry 2007

View Article Online
Scheme 1 Synthetic route for the preparation of phenanthroline ligands (L1 and L2) and the corresponding square planar platinum complexes (1 and 2).
(m/z = 567 a.m.u. corresponding to [Pt(L1)Cl + Na]+ and
657 a.m.u. corresponding to [Pt(L2)Cl + H]+).
To evaluate the effect of the metal on quadruplex DNA
stabilisation the corresponding free ligand (L2) was also analysed
(Table 1, second entry). In this case the DTm at 1 mM of L2 is 9 uC,
considerably lower than the DTm obtained with complex 2. This
suggests that the metal plays an important role in aiding
quadruplex DNA stabilisation. To evaluate the importance of
the ethylpiperidine substituent (in L2), L1 was also studied (Table 1,
first entry). This ligand is a poor quadruplex DNA stabiliser,
indicating that the piperidine substituent plays an important role
in increasing the strength of the interaction between planar
molecules, such as the phenanthrolines under study, and quad-
ruplex DNA.
The ability of L1, L2 and complex 2 to stabilise G-quadruplex
DNA (sequence: 59-FAM-d(GGG[TTAGGG]3)-TAMRA-39) was
then investigated by a FRET (Fluorescence Resonance Energy
Transfer) melting assay.29 The insolubility of complex 1 in water
and water–DMSO mixtures prevented us from evaluating its
binding properties towards DNA and therefore no data for this
complex are provided.
Complex 2 (see Table 1) induces a high degree of stabilisation
for quadruplex DNA with an increase in melting temperature
(DTm) at 1 mM of 20 uC. In contrast, at the same concentration of
metal complex, the DTm for a duplex DNA sequence (59-FAM-
The encouraging results obtained by FRET for complex 2
prompted us to investigate if this compound would also show
telomerase inhibition in the two-step TRAP assay. We have
substantially modified the assay to eliminate the possibility
of ligand being carried over to the second PCR step in the
assay,30 when it can then itself bind to PCR products and so
give false positive values for telomerase inhibitory activity.
Complex 2 showed 50% telomerase inhibition at 49.5 mM
concentration (see Fig. 2). Although this TelEC50 value is
greater than those observed for well-established telomerase
inhibitors such as BRACO-19 and tetra-N-methylpyridyl-
porphyrin (TMPyP4) (3.1 and 8.9 mM respectively using the same
modified TRAP assay) it shows that complex 2 is active as an
inhibitor for telomerase.
dTATAGCTATA-HEG-TATAGCTATA-TAMRA-39; Tm
=
60 uC in absence of complex) is negligible, suggesting a & 40-
fold selectivity for quadruplex vs. duplex DNA. Although the
stabilisation of quadruplex DNA by this compound is not as high
as that shown by the lead compound BRACO-19 (with a DTm
=
27.5 at the same concentration), it is worth pointing out that 2 is
more selective for quadruplex DNA vs. duplex DNA than
BRACO-19.
Table 1 Stabilisation temperatures (determined by FRET) of quad-
ruplex and duplex DNA. The last column shows the concentration of
compound required to induce a DTm of 20 uC
Conc. (mM)
DTm = 20 uC
Fig. 1 Qualitative in silico docking of complex 2 with a guanine quartet
(using DS viewer Pro); a) a stick and ball model showing overlap of the
complex with a quartet of guanine nucelosides; b) a CPK model showing a
top view of the complex stacked on top of a guanine quadruplex
(structural details obtained from X-ray crystal structure of parallel
quadruplexes from human telomeric DNA4); c) a side view of the
quadruplex–complex interaction showing the piperidine side arm of
the complex fitting neatly into the pocket created by a TTA loop of the
quadruplex.
DTm at 1 mM (uC)
Compound
G4 DNA
dsDNA
G4 DNA
L1
0.5
9.0
20.0
27.5
0
0
0.5
14.5
.10.0
8.0
1.0
L2
2
BRACO-19a
0.7
a
Data reported in reference 29.
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 4366–4368 | 4367

View Article Online
Notes and references
1 N. W. Kim, M. A. Piatyszek, K. R. Prowse, C. B. Harley, M. D. West,
P. L. C. Ho, G. M. Coviello, W. E. Wright, S. L. Weinrich and
J. W. Shay, Science, 1994, 266, 2011–2015.
2 L. R. Kelland, Eur. J. Cancer, 2005, 41, 971–979.
3 J.-L. Mergny, J.-F. Riou, P. Maillieet, M.-P. Teulade-Richou and
E. Gilson, Nucleic Acids Res., 2002, 30, 839–865.
4 G. N. Parkinson, M. P. H. Lee and S. Neidle, Nature, 2002, 417,
876–880.
5 A. T. Phan and J.-L. Mergny, Nucleic Acids Res., 2002, 30, 4618–4625.
6 A. T. Phan, Y. S. Modi and D. J. Patel, J. Am. Chem. Soc., 2004, 126,
8710–8716.
7 Y. Wang and D. J. Patel, Structure, 1993, 1, 263–282.
8 S. M. Kerwin, Curr. Pharm. Des., 2000, 6, 441–471.
9 J. T. Davis, Angew. Chem., Int. Ed., 2004, 43, 668–698.
10 T. Simonsson, Biol. Chem., 2001, 382, 621–628.
11 D. Sun, B. Thompson, B. E. Cathers, M. Salazar, S. M. Kerwin,
J. O. Trent, T. C. Jenkins, S. Neidle and L. H. Hurley, J. Med. Chem.,
1997, 40, 2113–2116.
12 S. Neidle and G. Parkinson, Nat. Rev. Drug Discovery, 2002, 1, 383–393.
13 M.-Y. Kim, H. Vankayalapati, K. Shin-ya, K. Wierzba and
L. H. Hurley, J. Am. Chem. Soc., 2002, 124, 2098–2099.
14 C. Leonetti, S. Amodei, C. D’Angelo, A. Rizzo, B. Benassi, A. Antonelli,
R. Elli, M. F. G. Stevens, M. D’Incalci, G. Zupi and A. Biroccio, Mol.
Pharmacol., 2004, 66, 1138–1146.
15 R. J. Harrison, J. Cuesta, G. Chessari, M. A. Read, S. K. Basra,
A. P. Reszka, J. Morrell, S. M. Gowan, C. M. Incles, F. A. Tanious,
W. D. Wilson, L. R. Kelland and S. Neidle, J. Med. Chem., 2003, 46,
4463–4476.
Fig. 2 TRAP gel from compound 2 showing the characteristic ladders
by PCR amplification of the oligonucleotides generated by the activity of
telomerase on a TS primer. With increasing concentrations of 2 (values in
the figure are mM concentrations) a decrease in the intensity of the ladder
is observed (i.e. increase in telomerase inhibition).
Since the FRET assay showed large differences in quadruplex
DNA thermal stabilisation between the metal-free ligands and the
metal complex, the TRAP assay was carried out for both ligands
(again, complex 1 could not be evaluated due to its low solubility).
Both L1 and L2 show significantly less activity than 2: the
piperidine-substituted ligand L2 shows activity at approximately
200 mM while L1 shows no activity at the concentrations tested.
These results are in agreement with the FRET studies, strongly
suggesting a correlation between quadruplex DNA stabilisation
and telomerase inhibition.
16 A. Maraval, S. Franco, C. Vialas, G. Pratviel, M. A. Blasco and
B. Meunier, Org. Biomol. Chem., 2003, 1, 921–927.
17 I. M. Dixon, F. Lopez, A. M. Tejera, J.-P. Esteve, M. A. Blasco,
G. Pratviel and B. Meunier, J. Am. Chem. Soc., 2007, 129, 1502–1503.
18 G. N. Parkinson, R. Ghosh and S. Neidle, Biochemistry, 2007, 46,
2390–2397.
19 M. Franceschin, E. Pascucci, A. Alvino, D. D’Ambrosio, A. Bianco,
G. Ortaggi and M. Savino, Bioorg. Med. Chem. Lett., 2007, 17,
2515–2522.
20 A. M. Burger, F. Dai, C. M. Schultes, A. P. Reszka, M. J. Moore,
J. A. Double and S. Neidle, Cancer Res., 2005, 65, 1489–1496.
21 G. Pennarun, C. Granotier, L. R. Gauthier, D. Gomez, F. Hoffschir,
E. Mandine, J.-F. Riou, J.-L. Mergny, P. Mailliet and F. D. Boussin,
Oncogene, 2005, 24, 2917–2928.
22 D.-F. Shi, R. T. Wheelhouse, D. Sun and L. H. Hurley, J. Med. Chem.,
2001, 44, 4509–4523.
23 I. M. Dixon, F. Lopez, J.-P. Esteve, A. M. Tejera, M. A. Blasco,
G. Pratviel and B. Meunier, ChemBioChem, 2005, 6, 123–132.
24 J. E. Reed, A. A. Arnal, S. Neidle and R. Vilar, J. Am. Chem. Soc.,
2006, 128, 5992–5993.
25 D. P. N. Goncalves, R. Rodriguez, S. Balasubramanian and
J. K. M. Sanders, Chem. Commun., 2006, 4685–4687.
26 C. Rajput, R. Rutkaite, L. Swanson, I. Haq and J. A. Thomas, Chem.–
Eur. J., 2006, 12, 4611–4619.
27 E. J. Corey, A. L. Borror and T. Foglia, J. Org. Chem., 1965, 30,
288–290.
28 J. F. J. Engbersen, A. Koudijs, M. H. A. Joosten and H. C. Van
der Plas, J. Heterocycl. Chem., 1986, 23, 989–990.
29 J.-L. Mergny and J.-C. Maurizot, ChemBioChem, 2001, 2, 124–132;
C. M. Schultes, B. Guyen, J. Cuesta and S. Neidle, Bioorg. Med. Chem.
Lett., 2004, 14, 4347–4351.
In summary, two new mono-substituted phenanthrolines have
been synthesised and used as tridentate N,N,N-ligands to
coordinate to platinum(II). The interactions between these
compounds and DNA (both duplex and quadruplex) have been
studied by FRET methods. From these studies we have identified
a compound that induces high stabilisation of telomeric quad-
ruplex DNA (DTm = 20 uC at 1 mM) and high selectivity for
quadruplex vs. duplex DNA. In order to examine the ability to
bind strongly to telomeric quadruplex DNA, TRAP assays with
the two ligands and complex 2 have been carried out. These studies
and the FRET results indicate a significant correlation, with the
metal complex being a much more potent telomerase inhibitor
than the free phenanthroline ligands.
The Medical Research Council (UK) is thanked for a student-
ship to J.E.R. The ICIQ Foundation (Spain) is thanked for
financial support. We are grateful to Mekala Gunaratnam, Tony
Reszka and Christoph Schultes for help with the FRET and
TRAP studies, and to CRUK for programme grant support at the
School of Pharmacy (to S.N.). Johnson Matthey is thanked for a
loan of platinum.
30 J. E. Reed, M. Gunaratnam, R. Vilar and S. Neidle, to be published.
4368 | Chem. Commun., 2007, 4366–4368
This journal is ß The Royal Society of Chemistry 2007