Journal of Medicinal Chemistry
Article
overnight. The test compounds were dissolved in a mixture of DMSO
(95% by volume) and 1 mM HCl (5% by volume) to give 3.12−
5.0 mM stock solutions. All solutions were stored at −20 °C and
defrosted and diluted immediately before use. Prior to use, the
compounds were diluted to 1 mM using DMSO. This was then further
diluted using suitable buffer to the appropriate concentrations. The CD
spectra were recorded in a strain-free 10 mm × 2 mm rectangular cell
path length cuvette. The data was obtained on an Applied Photophysics
Ltd. Chirascan spectrometer. The CD spectra were measured in the
wavelength region of 700−180 nm with the following parameters:
bandwidth, 1 nm; spectral range, 230−600 nm; step-size, 0.5 nm; time-
pep-point, 1.5 s. The CD spectra were collected and analyzed using the
Chirascan and Chirascan Viewer software, respectively. The following
CD spectra were recorded: (1) CD spectra of annealed HTelo DNA
(10 μM) in 50 mM tris-HCl and 150 mM KCl buffer (pH 7.4),
with 0−5 equiv of test compounds, (2) CD spectra of HTelo DNA
(10 μM) in Trizma hydrochloride buffer (0.1M, pH 7.4), annealed with
0−6 equiv of the test compounds, (3) CD spectra of nonannealed
HTelo DNA (10 μM) in Trizma hydrochloride buffer (0.1 M, pH 7.4),
with 0−6 equiv of the test compounds. FRET experiments were
performed using protocols previously described by us.6,7,11
Modified Telomerase Repeat Amplification Protocol (TRAP-
LIG).19 A 10 mM stock solution of each ligand was made in 100%
DMSO followed by a further dilution (1 mM) in distilled water with
1% HCl. The 1 mM stock solution was prepared freshly before use.
The TRAP assay was carried out in three steps with an initial primer
elongation step by telomerase, a subsequent removal of the primer
bound ligand, and a final PCR amplification of the telomerase products.
The first step of the TRAP assay was carried out by preparing a
master mix containing the TS forward primer (0.1 μg; 5′-
d(AATCCGTCGAGCAGAGTT)-3′, TRAP buffer (20 mM Tris-HCl
[pH 8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.05% v/v
Tween-20), bovine serum albumin (0.05 μg), and dNTPs (125 μM
each), protein extract (500 μg/sample) diluted in lysis buffer (10 mM
Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10%
glycerol, 5 mM β-mercaptoethanol, 0.1 mM AEBSF).
The PCR master mix was added to tubes containing freshly
prepared compounds at various concentrations and to the negative
control containing no drug. The initial elongation step was carried out
for 10 min at 30 °C, followed by at 94 °C for 5 min and a final
maintenance of the mixture at 20 °C. To purify the elongated product
and to remove the bound ligands, the QIAquick nucleotide purifi-
cation kit (Qiagen) was used according to manufacturer’s instructions.
The kit is especially designed for the purification of both double- and
single-stranded oligonucleotides from 17 bases in length. The kit
employs a high salt concentration buffer to bind the negatively charged
oligonucleotides to the positively charged spin-tube membrane
through centrifugation. An ethanol based buffer is then used to
wash any impurities away before elution of the DNA using a low salt
concentration solution. This was substituted with PCR-grade water in
our experiments. The purified extended samples were then subject to
PCR amplification. For this, a second PCR master mix was prepared
consisting of ACX reverse primer (1 μM; 5′-d(GCGCGG[CTTACC]3-
CTAACC)-3′), TS forward primer (0.1 μg; 5′-d(AATCCGTCGA-
GCAGAGTT)-3′), TRAP buffer, BSA (5 μg), 0.5 mM dNTPs, and 2U
of taq polymerase. An aliquot of 10 μL of the master mix was added
to the purified telomerase extended samples and amplified at: 35 cycles
of 94 °C for 30 s, 61 °C for 1 min, and 72 °C for 1 min. samples were
separated on a 12% PAGE and visualized with SYBR green staining.
Fluorescence from drug samples was normalized against a positive
control containing protein only. All samples were corrected for back-
ground by subtracting the fluorescence reading of negative controls.
Sulforhodamine B Short-Term Cytotoxicity Assay. Human
cancer cell lines, breast (MCF7), lung (A549), pancreatic (MIA-Pa-
Ca-2, HPAC), renal (RCC4, RCC4-VHL, 786−0), and normal human
lung fibroblast lines (WI-38), were all purchased from American Type
Cell Culture (ATCC). Cell lines were maintained in appropriate
medium supplemented with 10% fetal bovine serum (Invitrogen, UK),
2 mM L-glutamine (Invitrogen, Netherlands), and other components
as specified by the suppliers. All cell lines were maintained at 37 °C,
5% CO2, and routinely passaged. Short-term growth inhibition was
measured using the SRB assay as described previously. Briefly, cells
were seeded at appropriate densities into the wells of 96 well-plates in
their corresponding medium and incubated overnight to allow the cells
to attach. Subsequently cells were exposed to freshly made solutions
of drugs and incubated for a further 96 h. Following this, the cells
were fixed with ice-cold trichloaceticacid (TCA) (10%, w/v) for
30 min and stained with 0.4% SRB dissolved in 1% acetic acid for
15 min. All incubations were carried out at room temperature except
for TCA fixation, which was at 4 °C. The IC50 value, the concentration
required to inhibit cell growth by 50%, was determined from the mean
absorbance at 540 nm for each drug concentration expressed as a
percentage of the control untreated well absorbance.
Crystallization Experiments. The HPLC-purified human telo-
meric DNA sequences were purchased from DNA Technology A/S
(Denmark) and used without further purification. Both sequences,
native d(AGGGTTAGGGTT) and the brominated d(AGGGTBr-
UAGGGTT) (where BrU is 5-bromo-2′-deoxyuridine-5′-monophos-
phate), were annealed at 3 mM (quadruplex concentration) before
use by incubation in a heating block at 80 °C for 15 min in 20 mM
potassium cacodylate buffer at pH 7.0 and left overnight to cool
gradually to room temperature. Stock solutions were prepared for
each of the metal-containing ligands by dissolving in 100% DMSO
(dimethyl sulfoxide) at a concentration of 20 mM. The stock solutions
were kept at −20 °C. The relevant stock solution was thawed
immediately prior to setting up the crystallization drops. The hanging-
drop vapor-diffusion method was used. The Crystalgen 24-well
SuperClear Plates (Jena Bioscience) and 22 mm circular siliconized
glass coverslips (Molecular Dimensions) were used in the setup. In
both instances, crystals grew as yellow plates after 3 weeks at 20 °C.
Crystals of the 11-quadruplex complex grew in the following initial
crystallization conditions: 0.6 mM quadruplex DNA, 0.6 mM ligand,
0.4% (w/v) PEG 2000, 20 mM potassium cacodylate buffer at pH 7.0.
This was equilibrated against a reservoir well solution of 60% (w/v)
PEG 2000. Prior to mounting the crystals, they were cryoprotected
in a solution containing 140 mM of each of potassium chloride,
sodium chloride, and lithium sulfate, as well as 12.5% (w/v) PEG 2000
and 25% glycerol.
Crystals of the 8-quadruplex complex grew from a 3 μL drop
containing 1.25 mM quadruplex DNA, 1.25 mM ligand, 0.83% PEG
(w/v) 10 000, 16.7 mM potassium chloride, 16.7 mM sodium
chloride, 16.7 mM lithium sulfate, and 20 mM potassium cacodylate
buffer at pH 7.0. This was equilibrated against a reservoir well solution
of 50% (w/v) PEG 10,000. No cryoprotection agent was required.
Data Collection, Processing, Structure Solution, Verification,
and Crystallographic Refinement. Data were collected for each of
the cocrystals of 8-quadruplex, 11-quadruplex, and 13-quadruplex on
beamline ID IO3 at the Diamond Light Source synchrotron. The
structure of the 11-quadruplex was determined using experimental
phases obtained using data collected to a resolution of 2.3 Å at three
wavelengths in a multiple anomalous dispersion (MAD) experiment
using the bromine atom as the anomalous scatterer. A single-flash
frozen crystal was used at 105 K. Diffraction data was processed and
scaled with the MOSFLM23 and SCALA20 software packages. The
space group was determined at this point as C222. The data set
collected at the peak wavelength 0.92060 Å was reprocessed with the
XDS software package24 and used for high-resolution refinement. The
structure of the 11-quadruplex complex was solved by a combination of
MAD and MR techniques. The position of the bromine atom was
initially determined by MAD phasing using the SHELXC/D/
E
25−27suite of programs utilizing the bromine K-absorption edge,
although otherwise the MAD maps were uninterpretable in terms of a
discrete quadruplex structure. The EPMR program28 was then used to
solve the rest of the structure by molecular replacement using a partial
model formed of the G-quartets only extracted from the native parallel
quadruplex structure (PDB ID 1K8P).29 Electron density maps were
calculated for each of the solutions produced by EPMR, but only one
solution confirmed the position of the bromine atom from the
MAD phasing and this resulted in interpretable maps. The loops were
built into subsequent electron density maps, which then revealed a
220
dx.doi.org/10.1021/jm201140v | J. Med. Chem. 2012, 55, 209−222