doi:10.1016/j.jmb.2003.09.018 J. Mol. Biol.

(2003) 334, 25–36

Drug Recognition and Stabilisation of the Parallel-

stranded DNA Quadruplex d(TTAGGGT)4 Containing
the Human Telomeric Repeat
Evripidis Gavathiotis1, Robert A. Heald2, Malcolm F. G. Stevens2 and
Mark S. Searle1*
1
School of Chemistry
University Park
Nottingham NG7 2RD, UK
2 dinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor
School of Pharmaceutical
Sciences, University Park
Nottingham NG7 2RD, UK
*Corresponding author
The NMR structure of the parallel-stranded DNA quadruplex
d(TTAGGGT)4, containing the human telomeric repeat, has been determined
in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl ]acri-
of telomerase at submicromolar levels (IC50 value of 0.33(^ 0.13) mM),
exhibiting a wide differential between telomerase inhibition and acute cel-
lular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic
potency through interaction with, and stabilisation of, four-stranded
G-quadruplex structures. RHPS4 forms a dynamic interaction with
d(TTAGGGT)4, as evident from 1H and 19F linewidths, with fast exchange
between binding sites induced at 318 K. Perturbations to DNA chemical
shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify
the 50 -ApG and 50 -GpT steps as the principle intercalation sites; a struc-
tural model has been refined using NOE-restrained molecular dynamics.
The central G-tetrad core remains intact, with drug molecules stacking at
the ends of the G-quadruplex. The partial positive charge on position
13-N of the acridine ring appears to act as a “pseudo” potassium ion and
is positioned above the centre of the G-tetrad in the region of high nega-
tive charge density. In both ApG and GpT intercalation sites, the drug is
seen to converge to the same orientation in which the p-system of the
drug overlaps primarily with two bases of each G-tetrad. The drug is
held in place by stacking interactions with the G-tetrads; however, there
is some evidence for a more dynamic, weakly stabilised A-tetrad that
stacks partially on top of the drug at the 50 -end of the sequence. Together,
the interactions of RHPS4 increase the tm of the quadruplex by , 20 8C.
There is no evidence for drug intercalation within the G-quadruplex; how-
ever, the structural model strongly supports end-stacking interactions
with the terminal G-tetrads.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: quadruplex DNA; quinoacridinium cation; telomerase
inhibition; NMR spectroscopy; molecular dynamics

Present address: E. Gavathiotis, Laboratory of
Molecular Biophysics, The Rockefeller University, 1230
York Avenue, New York, NY 10021, USA.
Abbreviations used: RHPS4, fluorinated pentacyclic
quino[4,3,2-kl ]acridinium cation; NOE, nuclear
Overhauser effect; NOESY, NOE spectroscopy; TOCSY,
total correlated spectroscopy; MD, molecular dynamics.
E-mail address of the corresponding author:
mark.searle@nottingham.ac.uk
Introduction
Telomeres are repetitive DNA sequences at the
ends of linear chromosomes that protect the
chromosome from recombination, end-to-end
fusion and nuclease degradation.1,2 In human
cells, the telomeric DNA is typically composed of
5– 15 kb of double-stranded pairs of tandem
repeats of the guanine-rich sequence TTAGGG
with a single-stranded 30 -end overhang necessary
to ensure complete chromosomal DNA replication.
With each cell division, telomeres shorten by

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
26
50 –200 bp because synthesis of the lagging strand
of DNA is unable to replicate the 30 -end overhang.
When the telomeres shorten to a critical length,
“normal” cells stop growing and enter a state of
senescence where end-to-end fusion and chromo-
somal instability leads to cell death. A cell can
escape from this normal cycle and become immor-
tal by stabilising (capping) the length of its
telomeres.3 – 5 This happens almost always under
activation of the enzyme telomerase.6
Telomerase is a ribonucleoprotein responsible for
maintaining telomere length by adding TTAGGG
repeats to the 30 -ends of chromosomes. The major
components of telomerase are an endogenous
RNA template (hRNA) of 11 nucleotides and a
reverse transcriptase (hTERT) that catalyses the
addition of telomeric repeats using the single-
stranded telomere as a primer.7 – 9 All human
somatic cells contain the hRNA template but lack
the hTERT.8 Telomerase is active in 85 – 90% of all
human tumours but not in normal somatic cells.6
It has been suggested that tumour cells have
unlimited proliferative potential as a consequence
of the activation of the telomerase mechanism.
Consequently, telomerase has become a high-
profile target for the development of novel anti-
cancer agents.10 – 13
It is well established that the endogenous RNA
template region of the telomerase requires a
single-stranded, non-folded telomeric DNA primer
for the effective addition of the telomeric repeats.7 – 9
However, G-rich telomeric repeats are able to
assemble into four-stranded quadruplex structures
consisting of guanine tetrads stabilised by mono-
Figure 1. (a) Structure of the G-tetrad; (b) RHPS4 with
the atom labeling scheme.
Drug Recognition and Stabilisation of d(TTAGGGT)4
valent cations (Naþ and Kþ) (Figure 1).14 – 17 Initially,
Zahler and co-workers showed that inhibition of
telomerase activity was possible through G-quad-
ruplex stabilisation by Kþ.18 Later, it was suggested
that formation of the G-quadruplex DNA is
involved in the dissociation of the primer from the
RNA template. Experimental data indicated that
incorporation of a 7-deaza-dGTP nucleoside ana-
logue of dGTP into the telomeric DNA primer
d(TTAGGG) resulted in telomerase inhibition, as
the nucleoside analogue lacks the N7 atom that is
essential for the formation of G-quadruplex
structure.19 Further, small molecules that bind and
stabilise the folded DNA quadruplex structure
selectively over duplex DNA are potent telomerase
inhibitors, with low levels of general cytotoxicity.
The distinct geometrical features of G-quadruplex
DNA such as four grooves and a channel of nega-
tive electrostatic potential can allow specific recog-
nition by small ligands, which can, in principle,
intercalate at GpG steps or stack on the terminal
G-tetrads at the XpG and GpX steps, or even bind
in the grooves.10 – 13
The high anti-tumour chemotherapeutic poten-
tial of ligands with the capability to stabilise
G-quadruplex has focused several research groups
on structure-based design approaches to the
development of molecules that interact with
G-quadruplexes. A number of compounds with an
extended aromatic ring system capable of p-stack-
ing with G-tetrads have shown the ability to inhibit
telomerase activity through the stabilisation of
G-quadruplexes. Recently, dibenzophenanthroline
derivatives20 and 3,6,9-tri-substituted acridine
inhibitors21 were reported to inhibit telomerase
action in tumour cell lines with IC50 values of up
to 28 nM and 60 nM, respectively. Inhibition by
these compounds is correlated to selective stabili-
sation of the human DNA quadruplex structure,
as demonstrated by fluorescence resonance energy
transfer and surface plasmon resonance exper-
iments, respectively. Tetra-(N-methyl-4-pyridyl)
and tetra-(N-methyl-2-pyridyl)-porphyrins,22,23
2,6-disubstituted anthraquinones,24 2,7-disubsti-
tuted fluorenones,25 and a dicationic piperidino
perylene tetracarboxylic diimide derivative
(abbreviated PIPER),26 have been reported to
inhibit telomerase activity with small IC50 values.
Absorption, circular dichroism and NMR
spectroscopy have demonstrated the ability of 2,6-
diamidoanthraquinone, (N-methyl-pyridyl) por-
phyrins and the perylene derivative (PIPER) to
bind and stabilise parallel or anti-parallel G-quad-
ruplex structures with associated inhibition of
telomerase activity.
Spectroscopic data suggest that such ligands
intercalate externally to the surface of G-tetrads
rather than intercalate between the stacks of the
G-tetrads. However, one study suggested inter-
calation of tetra-(N-methyl-2-pyridyl) porphyrin
between the G-tetrads on the basis of calorimetric
(ITC) and spectroscopic data.27 The same porphyrin
derivative was examined by an electrophoretic
Drug Recognition and Stabilisation of d(TTAGGGT)4
photocleavage assay,22 and NMR, UV spectroscopy,23
but these data are consistent with the ligands bound
externally to the G-tetrads. Molecular modelling
studies show that ligands are able to form stable
complexes by either intercalating or end-stacking
with G-tetrads, adding weight to this controversy.27,28
There has been a paucity of detailed structural data
available on drug–quadruplex DNA complexes due
to intractable NMR spectra arising from extensive
drug-induced line-broadening. NMR studies of the
PIPER–quadruplex complex show that the drug
forms either a sandwich complex bound between
the blunt ends of a quadruplex dimer formed from
d(TTAGGG)4, or intercalates by end-stacking at the
GpT step of d(TAGGGTTA)4.26 Very recently, the
first X-ray structure has been reported of a drug-
bound dimeric anti-parallel G-quadruplex contain-
ing the Oxytricha nova sequence d(GGGGTTTT
GGGG).29 The drug end-stacks with a terminal
G-tetrad and interacts with one of the diagonal
thymine loops.
Here, we describe the interaction and stabili-
sation of quadruplex DNA by a novel fluorinated
polycyclic quinoacridinium cation, RHPS4
(Figure 1), which shows enhanced binding to
higher-ordered DNA structures (triplex/quadru-
plex) over duplex and single-stranded DNA, as
measured by differential dialysis.30 RHPS4 has
been shown to induce telomere shortening with
an IC50 value of 0.33 mM, while decreasing tumour
cell proliferation of breast 21NT cells at concen-
trations as low as 0.2 mM.31 RHPS4 is weakly cyto-
toxic (mean GI50 value in the NCI 60 human
tumour cell panel is 13.18 mM), giving a thera-
peutic index (GI50/IC50) of 40.30 This activity does
not appear to be associated with Taq polymerase
and topoisomerase II inhibition, strongly
suggesting that RHPS4 is an inhibitor of telomerase
function. To investigate the drug – quadruplex
interaction, as part of a rational ligand design
approach, we have studied by NMR the RHPS4
complex with the intermolecular parallel-stranded
quadruplex d(TTAGGGT)4, formed from the
human telomeric repeat,32 and describe here a
detailed investigation of the structure and
dynamics of the binding interaction in solution.
Results and Discussion
Structure of the parallel-stranded DNA
quadruplex d(TTAGGGT)4
Although a number of intermolecular and intra-
molecular G-quadruplex structures have been
studied so far by NMR spectroscopy, few have
yielded to detailed structural characterisation in
the presence of bound ligands, largely as a conse-
quence of the considerable drug-induced line-
broadening effects. Here, we describe in detail the
interaction of RHPS4 with the parallel-stranded
intermolecular quadruplex d(TTAGGGT)4 con-
taining the human telomeric repeat. We have
27
previously determined the solution structure of
d(TTAGGGT)4 using nuclear Overhauser effect
(NOE)-restrained molecular dynamics (MD)
simulations.33 The structure consists of a kinetically
stable core of three stacked G-tetrads. Detailed
NOE analysis of inter-residue NOEs at the ApG
step reveals that the adenine bases stack on top of
the G-tetrads. NOEs between the 6-NH2 and H2
of adjacent adenine bases are consistent with a
partially stabilised A-tetrad with anti glycosidic
torsion angles and a well-defined hydrogen bond-
ing geometry involving intermolecular interactions
between 6-NH2 and N1. The 50 and 30 -terminal
thymine residues have a dynamic conformation
with no evidence for the formation of T-tetrads.
Differences in conformation and stability of the
A-tetrad are evident when compared with reported
NMR structures of the related sequences
d(AGGGT)4 and d(TAGGGT)4,34 suggesting that
the terminal thymine bases exert some influence
through base-stacking interactions.
Drug – quadruplex interactions by 1D 1H and
19
F NMR
RHPS4 was titrated into a solution of d(TTA-
GGGT)4 at 303 K and the binding interaction was
followed by 1D 1H NMR spectroscopy. The reso-
nances of guanine imino protons between 10 ppm
and 12 ppm are well resolved and prove to be
good probes of ligand binding (Figure 2). Titration
up to a drug to quadruplex ratio of 1:1 causes
dramatic line-broadening; however, the resonances
subsequently sharpen reaching a titration end-
point at a drug to quadruplex ratio of 2:1, at
which point a new set of much sharper resonances
are observed that are up-field shifted appreciably,
by up to 0.8 ppm. At 303 K the G4 imino proton
resonance remains exchange broadened but
sharpens considerably above 308 K. The change in
line-widths as a function of the bound ratio of
ligand shows clear evidence for a 2:1 binding
stoichiometry, with fast exchange between the two
partially filled binding sites resulting in extensive
line-broadening at ratios close to 1:1. The change
in line shape with concentration of ligand shows
classic second-order fast exchange behaviour,35
enabling the exchange rate ðkex Þ to be estimated
from quantitative analysis of line-widths as a func-
tion of the fraction of DNA bound. The G5 and
G6 imino proton resonances prove to be most
amenable to analysis, from which we estimate
kex ¼ 2800ð^1200Þ s21 ; assuming that the binding
affinities at the two sites are similar and inde-
pendent of each other. Previously, fluorescence
quenching studies have estimated the binding
affinity to the human intramolecular quadruplex
to be 2.2 £ 105 M.30
Studies of the effects of temperature on the line-
widths of the 2:1 complex show evidence for
further exchange broadening below 303 K with
changes in line shape consistent with the presence
of multiple bound conformations or orientations
28
Figure 2. The 1D 1H NMR spectra (10– 12 ppm region)
showing imino proton resonances of G4, G5 and G6 as a
function of RHPS4:d(TTAGGGT)4 ratio.
at each of the two principle binding sites. At
temperatures above 303 K, resonances sharpen
appreciably as the drug shifts into the fast
exchange regime. These conclusions are entirely
consistent with 1D 19F NMR studies of the 2:1
complex, which similarly show base-line exchange
broadening effects at 278 K but much sharper
signals above 308 K.
1
H NMR spectra at higher temperatures
(. 308 K) were generally of superior quality, with
well-resolved signals observed in the aromatic
region due to bound drug and bound quadruplex
in fast exchange between bound states. At 318 K, a
small subset of signals are apparent that arise
from single strands of DNA (, 10%), but these do
not interfere with resonance assignment. Thus, 2D
NMR data on the 2:1 RHPS4 –d(TTAGGGT)4 com-
plex were collected at this temperature. The effect
of RHPS4 on quadruplex stability is clearly evident
from the intensity of the guanine imino proton
resonances, which in the free quadruplex dis-
appear in a co-operative melting event at 333 K due
to complete dissociation of the quadruplex into
Drug Recognition and Stabilisation of d(TTAGGGT)4
single strands. However, the G N1-H imino reson-
ances are still visible in the drug complex at tempera-
tures up to 353 K, signifying a shift in melting
temperature and strong stabilisation of at least 20 8C.
NMR analysis of the 2:1 complex
NOE spectroscopy (NOESY) experiments at
several mixing times (100 – 400 ms) were recorded
on the 2:1 RHPS4 –d(TTAGGGT)4 complex at
318 K and 303 K in H2O and 2H2O solutions
together with total correlated spectroscopy
(TOCSY) data at 318 K. NOESY data showing H8/
H6-H10 and H8/H6-H30 NOE connectivities,
recorded at 200 ms mixing time and 318 K are
plotted in Figure 3. It is clear from the number of
DNA resonances that the intermolecular parallel-
stranded quadruplex retains its 4-fold symmetry
in the bound state. Chemical shifts were assigned
readily using standard protocols, while bound
drug resonances were correlated readily using a
combination of J-coupling (TOCSY) and through-
space (NOESY) connectivities. A number of methyl
to aromatic proton NOEs within the bound drug
are highlighted in Figure 4. The pattern of NOEs
between base H8/H6 and its own and 50 flanking
sugar H20 /H200 are characteristic of a right-handed
twisted backbone conformation. Sequential NOE
connectivities along the TTA segment of the quad-
ruplex appear weak, indicating dynamic inter-
actions between these nucleotides (see Figure 3) as
apparent also for the 50 -T and 30 -T nucleotides in
the structure of the unbound quadruplex.33 We see
no evidence of thymine imino protons even at
283 K, indicating that drug binding does not
stabilise or induce T-tetrad formation. In contrast,
Figure 3. Portion of the 200 ms NOESY spectrum of
the RHPS4:d(TTAGGGT)4 complex at 318 K highlighting
sequential connectivities between base H6/H8 (7.0–
8.5 ppm) and deoxyribose H30 (4.5– 5.2 ppm) and H10
(5.8– 6.4 ppm); intranucleotide NOEs are labelled.
Drug Recognition and Stabilisation of d(TTAGGGT)4
Figure 4. Portion of the 200 ms NOESY spectrum of
the 2:1 RHPS4 – d(TTAGGGT)4 complex recorded at
318 K highlighting a number of key NOEs. In the top
panel, NOEs labelled T1– T7 identify intranucleotide
connectivities within the base H6/H8 to deoxyribose
H20 /H200 sequential assignment pathway. Broken lines
highlight intense NOE cross-peaks between drug methyl
and drug aromatic protons used in the assignment pro-
cess. Thymine methyl to H6 NOEs are also assigned.
Key drug– DNA NOEs are labelled a –j as follows: a,
6-Me– G4H8, b, H5-G6H20 /H200 , c, H7-G6H20 /H200 , d,
H4-A3H20 /H200 , e, H5-A3H20 /H200 , lower panel; f,
8-Me– A3H8, g, 8-Me– G4H8, h, 8-Me –G6H8, i, 13-Me –
G4H8, and j, 13-Me– G6H8/T7H6. The peak labelled k
corresponds to drug 13-Me to H1/H12. The intensity
scale in the lower part of the Figure has been increased
by a factor of 2 in order to emphasise weak drug– DNA
NOE cross-peaks (f– j). The RHPS4 numbering scheme
is shown in Figure 1.
NOE cross-peaks between guanine bases are strong
and show that the G-tetrad stack is little perturbed
by drug binding. The differences in chemical shifts
of non-exchangeable protons between the bound
(2:1 complex) and the free quadruplex d(TTA-
GGGT)4, show that the thymine bases T1 and T2
are unperturbed by ligand binding. However, the
base H6 and CH3 protons of T7 shift by 0.2 ppm
and 0.35 ppm, respectively. Proton resonances of
the purine residues of the quadruplex show only a
small peturbation , 0.1 ppm; however, the imino
protons of G4 and G6 are ring current shifted by
up to 0.8 ppm (see Figure 2 and Table 1).
In addition, a total of 24 drug– DNA inter-
molecular NOEs were identified in the 2:1 complex
representing the time-averaged interaction of the
bound drug at the two sites. The fact that the two
binding sites are non-overlapping enables us to
partition these NOEs unambiguously between the
two intercalation sites. By far the largest number
29
of NOEs involve the base and sugar protons of G4
and G6. In contrast, no NOE to G5 is identified at
the centre of the stack of G-tetrads. The absence
of NOEs to G5 excludes any possibility of drug
molecules intercalated at the G4 – G5 or G5 –G6
step. NOEs are detected at the A3 –G4 step and
involve both purine nucleotides, indicating that
the ApG step is a primary intercalation site. A
number of NOEs to G6 show that the drug inserts
also at the G6 – T7 step; however, no NOE to T7
is observed unambiguously, despite the pertur-
bations to its chemical shifts. Two very strong
NOEs from the drug 13-CH3 to G4 NH and G6
NH show that end stacking on the G-quadruplex
is energetically very favourable. NOEs from drug
8-CH3 and 13-CH3 to G4 H8 and G6 H8 and from
drug H1, H5, H7, H9, H10, and H12 protons to a
number of DNA base and sugar protons position
the edges of the quinoacridinium ligand in the
grooves. A number of NOE cross-peaks between
RHPS4 and protons of A3, G4 and G6 are evident
in the portion of the NOESY spectrum shown in
Figure 4. The non-exchangeable proton sequential
connectivity pathway for H8/H6 –H20 /H200 is
shown. Taken together, the data strongly support
binding to the ends of the G-quadruplex stacking
primarily with G4 and G6 (see Figure 5).
A detailed analysis of the unbound quadruplex
showed clear evidence from guanine imino and
adenine amino proton NOE data that both guanine
and adenine nucleotides form hydrogen-bonded
tetrads. NOEs from adenine 6-NH2 to H2 are
consistent with intermolecular A-tetrad formation
and stabilising stacking interactions with the adja-
cent G-tetrad of G4.33 The latter are evident from
strong NOEs from the G4 imino to A3 H2 and H8.
MD refinement of d(TTAGGGT)4 shows that the
A-tetrad persists over long dynamics simulations.
The 6-NH2 group of A3 is also identified in spectra
of the complex at 318 K and is upfield shifted by
0.4 ppm. The data suggest that the amino protons
are partially protected against exchange broaden-
ing at higher temperatures through A-tetrad
formation, with chemical shift perturbations
arising from ring current effects from stacking
with the bound ligand. However, because of the
significant line-broadening effects of the drug on
both A3 H2 and H8, analogous NOEs to those
described above could not be identified to confirm
the integrity of a hydrogen bonded A-tetrad.
NOEs between A3 and G4, which were relatively
strong for the unbound quadruplex, are weaker,
reflecting the structural distortions caused by drug
intercalation at the ApG step. We investigate
further the stability of the A-tetrad and its contri-
bution to drug binding in MD simulations (see
below).
NOE-restrained MD modelling of the
2:1 complex
The structure of the RHPS4 – d(TTAGGGT)4 com-
plex has been refined using an NOE-restrained MD
30 Drug Recognition and Stabilisation of d(TTAGGGT)4

Table 1. Chemical shifts

H10 H20 H200 H30 H40 H50 /H500 H6/H8 Me H2 NH

A. 1H chemical shifts of the d(TTAGGGT)4 quadruplex at 298 K

T1 6.00 2.10 2.34 4.64 4.00 3.65 7.41 1.67
T2 6.25 2.06 2.34 4.74 4.06 3.93 7.33 1.78
A3 6.28 2.86 2.92 5.10 4.44 4.16/4.10 8.43 8.09
G4 6.01 2.67 2.91 5.05 4.49 4.27 7.95 11.50
G5 6.03 2.66 2.74 5.04 4.51 4.30 7.79 11.20
G6 6.27 2.57 2.70 4.91 4.52 4.27 7.70 10.92
T7 6.07 2.17 2.19 4.49 4.23 4.07 7.36 1.63
1
B. H chemical shifts for the DNA quadruplex of the 2:1 d(TTAGGGT)4 – RHPS4 complex at 318 K
T1 5.98 2.14 2.38 4.71 4.05 3.70 7.39 1.63
T2 6.11 2.00 2.30 4.79 4.42 4.15 7.32 1.71
A3 6.22 2.79 2.82 5.10 4.43 4.16 8.29
G4 5.94 2.59 2.86 5.00 4.46 4.21 7.85 11.05
G5 5.93 2.57 2.70 5.01 4.46 4.29 7.58 10.80
G6 6.30 2.68 2.71 5.10 4.47 4.27 7.74 10.11
T7 6.27 2.34 2.36 4.61 4.23 7.70 1.88
C. 1H chemical shifts for RHPS4 of the 2:1 d(TTAGGGT)4 –RHPS4 complex at 318 K
H1 7.24 8-Me 3.58
H2 6.87 H9 7.48
H4 7.33 H10 7.26
H5 7.25 H12 7.28
6-Me 2.36 13-Me 3.88
H7 7.09 MeOSO2 3 3.75

approach. The NMR data described represents a
dynamic complex in which the 4-fold symmetry of
the parallel-stranded quadruplex is retained by
virtue of the bound drug molecules exchanging
rapidly between the two binding sites, and
between different bound orientations. The time-
averaged 4-fold symmetry of the quadruplex struc-
ture in solution at 318 K complicates structure
determination because of the ambiguities in assign-
ing intermolecular NOEs from a dynamic structure
to specific nucleotides in a static, asymmetric
model. Based on the set of 24 drug– quadruplex
NOEs, drug molecules were docked at the A3 – G4
and G6 –T7 steps in a number of possible orien-
tations in order to satisfy the maximum number of
NOE restraints. The observation of NOEs from a
single DNA proton to several drug protons, appar-
ently remote from each other on the acridine ring,
can arise from the chemical shift degeneracy of
the four parallel strands. Thus, NOEs were manu-
ally assigned based on initial model building
studies (see Materials and Methods).
The majority of drug –DNA NOEs were satisfied
when the 8-CH3 and 6-CH3 groups of the ligand
are orientated towards the grooves and the
13-CH3 lies close to the central ion channel of

Figure 5. A representation of the

RHPS4 – d(TTAGGGT)4 complex
showing drug stacking on the ends
of the G-quadruplex at the ApG
and GpT steps. Potassium ions
(green) are bound at the GpG steps.
Drug Recognition and Stabilisation of d(TTAGGGT)4 31

Figure 6. Ensemble of ten structures taken from the

final 100 ps of the NOE-restrained dynamics simulation
showing the four strands in yellow, pink, red and blue.
The position of the drug molecules at the ApG (top)
and GpT steps (bottom) are shown. In each case, a mean
structure of the bound drug is shown in space-filling
format; the 50 -terminal thymine bases are disordered
and are omitted for clarity.

the quadruplex structure. The 1808 reorientation of

the ligand having the 13-CH3 pointing into the
groove of the quadruplex structure is in accord-
ance with only a few drug – DNA NOEs, while
most of the distances from the 8-CH3, 6-CH3 and
the drug aromatic protons to the quadruplex pro-
tons result in restraint violations with distances
over 5 Å. The restrained MD calculations show
that the NOE restraints are largely satisfied by one
preferred orientation of the drug at each of the
two sites, with small fluctuations about the mean
position. An ensemble of ten structures taken from
the last 100 ps of restrained dynamics are shown
in Figure 6, and five structures were chosen
randomly to illustrate fluctuations of the drug
from the mean position when stacked against
both the G4 and G6 tetrads (Figure 7(a) and (b),
respectively).
The drug does not adopt a centro-symmetric
stacking interaction with the adjacent G-tetrad but
is offset. The partial positive charge on the acridine
13-N appears to act as a “pseudo” potassium ion
and is positioned above the centre of the G-tetrad
in the region of high negative charge density gen-
erated by the carbonyl groups. The electron-
withdrawing effects of the two fluorine atoms
increase the positive charge density at the 13-N
position and enhance the electrostatic contribution.
In both ApG and GpT intercalation sites, the drug
is seen to converge to the same orientation in
which the p-system of the drug overlaps primarily
with two bases of each G-tetrad (Figure 8(c) and
Figure 7. Fluctuations in the conformation and orien-
tation of the bound RHPS4 molecule in the two drug-
binding sites taken from five randomly chosen structures
during the final 100 ps of the NOE-restrained dynamics
simulation; drug molecules (green) are stacked with the
G4-tetrad in (a) and the G6-tetrad in (b).
(d)). The core AGGGT structure, with drug
molecules end-stacking on the G-tetrads of G4 and
G6, are shown in Figure 8(a). Alongside is the
structure of the unbound quadruplex (Figure
8(b)), to illustrate the effects of the drug in distort-
ing and unwinding the DNA backbone at the
ApG and GpT steps. In both structures, all nucleo-
tides have anti glycosidic conformations with the
right-handed twisted helical backbone geometry
evident for each strand. The structure was
modeled with potassium ions inserted between
the planes of two G-tetrads, consistent with X-ray
structures of parallel and anti-parallel quad-
ruplexes.36,37 The bound Kþ ions impart considerable
rigidity to the G-tetrad core; the G-tetrads deviate
little from planarity, with only small fluctuations
32 Drug Recognition and Stabilisation of d(TTAGGGT)4

Figure 8. (a) Energy-minimised structure of the 2:1 RHPS4 – d(TTAGGGT)4 complex showing the AGGGT core with
RHPS4 intercalated at the A3-G4 and G6-T7 steps. G-tetrads are in blue, A-tetrad in pink and thymine bases in yellow;
(b) structure of the AGGGT core of the unbound quadruplex. Offset p-stacking of RHPS4 with (c) the G4 tetrad and
(d) the G6 tetrad.

in Hoogsteen N1 – O6 and N2 – N7 hydrogen bond
lengths. The observation that RHPS4 molecules
are bound on the ends of the G-quadruplex pro-
vides further evidence that the stabilising effects
of Kþ ions within the central channel preclude
drug intercalation at a GpG step because the energetic
cost of displacing a potassium ion is not suf-
ficiently compensated by stacking interactions
with the bound drug. In contrast, the adenine
nucleotides are less stable than the guanine bases
because only half of the surface area of the A-tetrad
is involved in stacking interactions with the p-sys-
tem of RHPS4. The thymine nucleotides at the 50
end of the quadruplex are much more dynamic,
showing appreciable conformational disorder even
with NOE restraints in place.
Unrestrained MD simulations and A-tetrad
stability
The stability of the A-tetrad and its time-depen-
dent interaction with RHPS4 was investigated
further through 1 ns of unrestrained MD simu-
lation of the complex. The structure of the purine-
rich core d(AGGG)4 remains largely unchanged
from the NOE-derived starting structure with
Drug Recognition and Stabilisation of d(TTAGGGT)4
Figure 9. Dynamic fluctuations in the conformation of
purine (G or A) tetrads measured from the N1– N6 dis-
tance of (a) the A-tetrad and (b) N2 – N7 distance of the
G-tetrad of G5 during 1000 ps of unrestrained molecular
dynamics simulation of the 2:1 RHPS4 – d(TTAGGGT)4
complex at 300 K in explicit solvent.
an RMSD over the dynamics trajectory of
1.73(^ 0.16) Å; this number rises to 2.63(^ 0.38) Å
when all heavy atoms of (TTAGGGT)4 are con-
sidered, reflecting the larger amplitude dynamics
of the terminal thymine bases. Variations in the
N1 – N6, N1 – O6 and N2 –N7 Hoogsteen hydrogen
bond distances for the A-tetrad and G-tetrads
were examined during the 1 ns simulation. Fluc-
tuations for the G-tetrads are in all cases small,
demonstrating a very stable compact confor-
mation. In contrast, the adenine N1 –N6 distance
shows more flexibility and suggests a significant
breathing of the A-tetrad structure, coupled with
buckling motions. The amplitude of the fluctu-
ations in N1 – N6 and N2 – N7 distances for A3 and
the core G5, respectively, are shown in Figure 9.
The average A-tetrad N1 –N6 distance is increased
by 0.35 Å compared with parallel simulations of
the unbound d(TTAGGGT)4 structure, suggesting
a small net destabilising effect of RHPS4 on the
integrity of the A-tetrad. The data suggest that
while RHPS4 binding stabilises the core G-quadru-
plex structure significantly, consistent with a Dtm
, 20 8C, stacking interactions with the drug distort
and partially destabilise the A-tetrad. In terms of
surface complementarity, it is clear to see why this
might be the case. In the structure of the unbound
quadruplex the core G-tetrad provides an excellent
template of similar surface area on which the
A-tetrad can readily assemble and hydrogen
bond. RHPS4 competes effectively with the
A-tetrad for these favourable interactions.
33
Conclusions
NMR studies of the perylenetetracarboxylic
diimide intercalator (PIPER) described by Federoff
et al.,26 and of the polycyclic quinoacridinium
cation reported here, show that drug molecules
are unable to disrupt the G-tetrad core by insertion
at a GpG step with displacement of stabilising
potassium ions. Instead, the drug forms complex-
stabilising interactions by stacking on the ends of
the G-quadruplex. Similar conclusions have been
drawn from modelling studies of a disubstituted
anthraquinone,28 and a number of tri-substituted
acridine derivatives with the human intramole-
cular quadruplex where the drug is proposed to
bind to the TTA cross-over loop while end-stacking
with a G-tetrad.21 The first crystal structure of a
drug –quadruplex interaction confirms this mode
of binding. Haider et al.29 report the complex of a
dimeric anti-parallel G-quadruplex formed from
the Oxytricha nova telomeric repeat d(GGGGTTTT-
GGGG) with a di-substituted aminoalkylamido
acridine. The drug binds within one of the
diagonal thymine loops and stacks on the terminal
G-quartet. The interaction is stabilised further by
specific hydrogen bonding interactions with
thymine bases in the loop; these interactions are
stable over 2 ns of MD simulation. The key struc-
tural feature to emerge from this study appears to
be the active role played by the flexible loop region
in binding and recognition. The NMR data
reported here for RHPS4, and for PIPER,26 empha-
sise the dynamic nature of the interaction, reflect-
ing the relatively simple architectural features of
the ligands so far investigated. NMR line shape
analysis suggests that RHPS4 exchanges on a
time-scale . 2000 s21, giving a life-time for the
bound state of , 0.5 ms at 303 K. Thus, although
the barriers to association and dissociation are
relatively small, the timescales involved are still
largely inaccessible to MD simulations. RHPS4 has
been identified as a potent inhibitor of telomerase
at submicromolar levels (0.33(^ 0.13) mM) using
the cell-free telomeric repeat amplification protocol
(TRAP). Encouragingly, RHPS4 exhibits a wide
differential between telomerase inhibition and
acute cellular toxicity, producing a marked cessa-
tion of cell growth with 21NT breast cancer cell
lines, and other cell lines possessing relatively
short telomeres.30,31 In addition, RHPS4 induces
delayed senescence in human breast MCF-7 and
lung A549 cells in vitro and acts synergistically, in
combination with taxol, to inhibit human tumour
xenografts in vivo (unpublished results). All of the
data point to RHPS4 exerting its chemotherapeutic
potency through interaction with, and stabilisation
of, four-stranded G-quadruplex structures. Our
NMR studies have revealed detailed structural
insights into the nature of the interaction, on the
basis of which we are currently rationally design-
ing substituted analogues of RHPS4 with added
side-chain functionality capable of interactions in
the grooves of the G-quadruplex.
34
Materials and Methods
Sample preparation
Preparation of d(TTAGGGT)4 followed standard
procedures. RHPS4 (3,11-difluoro-6,8,13,-trimethyl-8H-
quino[4,3,2,-kl ]acridinium methosulfate) was synthe-
sized as described with purity checked by 1H NMR.30
The RHPS4 – quadruplex complex was formed by titrat-
ing small aliquots of drug from a 5 mM solution into
the d(TTAGGGT)4 sample in 90% (v/v) H2O, 10% (v/v)
2
H2O, containing 100 mM KCl, 10 mM K2HPO4 and
1 mM EDTA at pH 7.0. The titration experiment was
carried out at 303 K, where the spectrum of the free
quadruplex is well resolved from the resonances of the
single-stranded d(TTAGGGT).
NMR experiments
1
H NMR spectra were recorded at 500 MHz and
600 MHz on Bruker DRX-500 and Avance600 spectro-
meters, and processed on R4600PC and R5000SC Silicon
Graphics O2 and Indy Workstations using Bruker
X-WinNMR v2.6 software and ANSIG software.38 Stan-
dard phase-sensitive 2D NMR pulse sequences were
used to obtain NOESY, TOCSY, double quantum filtered
(DQF)-correlated spectroscopy (COSY) spectra in 2H2O
and WATERGATE NOESY in 90% H2O, 10% 2H2O
solution. One-dimensional NMR experiments were
recorded over 16,384 data points with 128 transients,
spectral width of 20,000 Hz and a delay time of 1.5 s, at
temperatures between 278 K and 358 K. NOESY spectra
at various mixing times (400 ms, 300 ms, 200 ms,
150 ms, 100 ms) were recorded over 2048 data points in
t2 and 400– 512 points in t1 with 64 transients for each,
with a spectral width of 10,000 Hz and a delay time of
1.5 s, at temperatures of 303– 318 K. TOCSY using 75 ms
spin lock and DQF-COSY were recorded over 2048 data
points in t2 and 400–512 points in t1 with 48 transients
for each with a spectral width of 10,000 Hz and a delay
time of 1.5 s, at 318 K. WATERGATE-NOESY spectra at
300 ms, 200 ms, and 100 ms mixing times were acquired
using a sample in 90% H2O, 10% 2H2O solution collecting
2048 data points in t2 and 400– 512 points in t1 with 64
transients for each, and employed a 20,000 Hz spectral
width and a delay time of 1.5 s, at 303–318 K.
NOE restraints
A set of 668 NOE restraints (161 per strand and 24 for
both RHPS4 molecules) for non-exchangeable, exchange-
able and intermolecular drug –quadruplex protons
were determined from NOE cross-peak integration in
500 MHz NOESY spectra collected at 318 K. Volume
integrals were normalised to several fixed reference
distances within the DNA structure including deoxy-
ribose H20 – H200 (1.85 Å) and thymine CH3 – H6 (3.00 Å).
Distances were estimated from data at a number of mix-
ing times 100–200 ms in 2H2O and H2O solutions using
linear regression to extrapolate to 0 ms mixing time.39
Distances calculated from the extrapolation method
were assigned as strong (1.8– 3.0 Å) medium (2.5– 4.0 Å)
and weak (3.5– 5.5 Å). Interproton distances involving
exchangeable protons and also intermolecular NOEs
between drug molecules and quadruplex were given
wider restraints in the range of 2.5– 5.0 Å. Although the
experimental data show that the four strands are
equivalent, for the purposes of the structural modelling
Drug Recognition and Stabilisation of d(TTAGGGT)4
it was necessary to lift the 4-fold symmetry and assign
NOEs from drug protons to specific strands and model
a single conformation. A number of strong drug NOEs
from 13-CH3 to G4NH and G6NH were used initially to
guide the manual docking procedure. Orientations with
either the 8-CH3 or 13-CH3 of the drug in the central
cavity were examined and the violations of other NOEs
monitored. This procedure showed that having the
13-CH3 in the central cavity resulted in a better fit to the
data, particularly NOEs to a number of DNA deoxy-
ribose H1’s. Thus, in this orientation, all NOEs were sub-
sequently assigned to specific strands of DNA if the
distance in the initial model fell within 6.5 Å. The wider
restraint bounds used allowed for inherent uncertainties
in this approach. No hydrogen bonding restraints were
included for the A-tetrad during structure calculations
in order to examine the effects of drug binding on
A-tetrad stability and dynamics. Distance restraints
were checked against the energy-minimized structure
for large geometrical inconsistencies using MOLMOL.40
Structure calculations
Energy minimisations and restrained MD calculations
were performed on an Origin 200 Silicon Graphics Server
using the AMBER 6 suite† of programs employing the
AMBER 95 force-field with modifications41 and particle
mesh Ewald (PME)42 method for the treatment of long-
range electrostatics. The starting model of the RHPS4 –
quadruplex complex was generated by using an average
energy-minimised d(TTAGGGT)4 structure. Firstly, the
binding sites of the quadruplex were constructed by
moving apart the TTA segment and 30 -end thymine
nucleotides from the G-tetrads to a separation distance
of 6.5 Å, using the LEAP module of AMBER 6. This
manual generation of the binding sites resulted in
several violations of the phosphate backbone geometry,
which were corrected after a careful minimisation and
equilibration of the structure. The drug molecules were
docked manually between A3 – G4 and G6– T7 steps
with orientations guided, in part, by the set of drug –
quadruplex NOEs. The experimental data show that the
four strands of the quadruplex and the environment of
the bound drug molecules are averaged on the NMR
timescale, but this symmetry was removed for the pur-
pose of assignment of intermolecular NOEs. Each drug
molecule has two faces (A and B), that are equivalent in
terms of the possible pattern of NOEs that arise, result-
ing in four possible alternatives for the relative align-
ment of the two drug molecules (AA, AB, BA and BB).
These are indistinguishable from the viewpoint of the
NOE data. In addition, each one of these is associated
with the drug in four equivalent orientations defined by
the 4-fold symmetry of the quadruplex. We made the
assumption that the conformation and orientation of the
drug molecules in each site are decoupled from each
other completely. To simplify the structural analysis, we
chose one starting structure (AB) arbitrarily from the
four for refinement purposes, with the drug molecules
bound in the orientations shown in Figure 6. The
molecular structure of the RHPS4 was constructed and
minimised using the Macromodel software. To model
stacking interactions accurately, partial charges on
RHPS4 were calculated using the HF/6 – 31G(d)/RESP
methodology.43 Force-field parameters for the atom
† http://amber.scripps.edu/doc6/install.html
Drug Recognition and Stabilisation of d(TTAGGGT)4
types of the drug were adapted from comparable
standard parameters within AMBER.
The structure of the RHPS4 – d(TTAGGGT)4 complex
was solvated in a periodic TIP3 water box of approxi-
mate dimensions 60 Å £ 60 Å £ 60 Å, which extended
to a distance of 10 Å from any solute atom and contained
5000 water molecules. Two internal potassium ions,
generated using standard parameters for the AMBER
force-field, were positioned manually in the central
channel between adjacent G-quartets. The potassium
ions were positioned equidistant from the two adjacent
G-quartets to allow octahedral coordination with the
four polarized carbonyl oxygen O6 atoms. The quad-
ruplex system was neutralized with 20 potassium ions
placed at the most negative locations with the LEAP
module. All the potassium ions were treated as part of
the solvent. The drug– quadruplex system was allowed
to equilibrate fully before we performed restrained MD.
Minimisation was performed with 50 steps of steepest
descent and 5000 steps of conjugate gradient on the
water and counterions first with the d(TTAGGGT)4 coor-
dinates frozen, followed by a further 5000 steps on all the
components of the system. Next, 10 ps of unrestrained
MD was run at 100 K on the water alone with the DNA
and potassium ions constrained, followed by another
10 ps to allow the potassium ions to move. In the follow-
ing 5 ps of dynamics, the temperature of the system was
increased from 100 K to 300 K. At this point the set of
drug– quadruplex NOE restraints was introduced
gradually in the form of square well potentials with a
force constant of 30 kcal mol21 Å22 (1 cal ¼ 4.184 J) over
10 ps. In the next runs, each of 10 ps dynamics, the DNA
force constant is reduced gradually from 100 to 50, 25,
10, 5, and 2.5 kcal mol21 Å-2. Restrained MD continued
for 100 ps with the whole set of NOE restraints active,
followed by energy minimisation. Snapshots from the
100 ps simulation were extracted every picosecond. In
the final averaged energy-minimised structure, none of
the 668 NOE restraints showed violation .0.5 Å (no
restraint violation .0.3 Å were observed for the DNA)
giving overall a low-average restraint violation of 0.02 Å
and an overall restraint violation energy penalty of
only 36 kcal mol21. However, six out of 24 violations
of between 0.3 and 0.5 Å were observed for the drug–
DNA NOEs, with three of these corresponding to
very weak distance restraints of 5 Å, which have an
intrinsically high degree of uncertainty. The mean pair-
wise RMSD calculated over the final 100 snapshots was
1.26(^ 0.19) Å over all heavy atoms with an RMSD from
ideal covalent bond lengths of 0.024 Å and ideal bond
angles of 2.78. No back calculation was attempted to
compare the experimental NOE intensities with those
predicted from the structural models. The small number
of drug– DNA restraint violations may reflect, in part,
the fact that a significant number of these NOEs are
quite weak. Overall, the orientation of the drug in each
binding site is well-defined by the NOE restraints, result-
ing in small-scale fluctuations about the mean position,
suggesting a preferred mode of interaction with the
G-tetrad that is similar in both drug-binding sites (see
Figure 7).
A further 1 ns of unrestrained MD on the equilibrated
drug– quadruplex system was carried out in order to
assess the impact of the restraints on the structure of the
complex; in particular, fluctuations in the conformation
of the A-tetrad. The average structure and the RMSD
from the average structure, was calculated using the
CARNAL module of the AMBER suite. The co-ordinates
of the NMR structures of the 2:1 RHPS4:d(TTAGGGT)4
35
complex have been deposited in the PDB as 1NZM and
in the RCSB with ID code RCSB018391. Structures of the
unbound quadruplex have also been deposited with
PDB code 1NP9, and RCSB code RCSB018075.
Acknowledgements
We thank the EPSRC of the UK and AstraZeneca
for financial support to E.G. The Cancer Research
Campaign of the UK supports M.F.G.S. and
R.A.H. M.S.S. thanks the EPSRC and the School of
Chemistry for supporting NMR facilities.
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Edited by M. F. Summers