Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

Article abstract of DOI:10.1021/acs.jmedchem.7b01781

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.

Full text of DOI:10.1021/acs.jmedchem.7b01781

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Article
Targeting multiple effector pathways in pancreatic ductal
adenocarcinoma with a G-quadruplex-binding small molecule
Chiara Marchetti, Katherine G. Zyner, Stephan A Ohnmacht, Mathew Robson, Shozeb M. Haider, Jennifer P
Morton, Giovanni Marsico, Tam Vo, Sarah Laughlin-Toth, Ahmed A Ahmed, Gloria Di Vita, Ingrida Pazitna,
Mekala Gunaratnam, Rachael J Besser, Ana CG Andrade, Seckou Diocou, Jeremy A Pike, David Tannahill,
R. Barbara Pedley, T.R. Jeffry Evans, W. David Wilson, Shankar Balasubramanian, and Stephen Neidle
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01781 • Publication Date (Web): 22 Jan 2018
Downloaded from http://pubs.acs.org on January 23, 2018
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Targeting multiple effector pathways in pancreatic ductal
adenocarcinoma with a G-quadruplex-binding small molecule
†
‡
†
§
Chiara Marchetti , Katherine G. Zyner , Stephan A. Ohnmacht , Mathew Robson , Shozeb
†
ǁ,¶
‡
#
#
M. Haider , Jennifer P. Morton , Giovanni Marsico , Tam Vo , Sarah Laughlin-Toth ,
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†
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†
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Ahmed A. Ahmed , Gloria Di Vita , Ingrida Pazitna , Mekala Gunaratnam , Rachael J.
†
†
¥
‡,Δ
‡
Besser , Ana C. G. Andrade , Seckou Diocou , Jeremy A. Pike , David Tannahill , R.
7
ǁ,¶
#
‡,Φ
Barbara Pedley , T.R. Jeffry Evans , W. David Wilson , Shankar Balasubramanian and
†
Stephen Neidle *
†
UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London
WC1N 1AX, UK.
‡
Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way,
Cambridge CB2 0RE, UK.
§
Cancer Research UK Cancer Centre, UCL Cancer Institute, University College London,
London WC1E 6BT, UK.
ǁ
Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD
UK.
¶
Institute of Cancer Sciences, University of Glasgow, Glasgow G12 8QQ.
#
Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State
University, Atlanta, Georgia 30303-3083, USA.
¥
UCL Cancer Institute, University College London, London WC1E 6BT, UK.
Δ
Now at: Centre of Membrane Proteins and Receptors, University of Birmingham,
Birmingham, B15 2TT, UK.
Φ
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
C. M. and K. G. Z. contributed equally to this work
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Abstract
Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of
multiple signalling pathways. A novel approach to the treatment of PDAC is described,
involving the targeting of cancer genes in PDAC pathways having over-representation of G-
quadruplexes, using the tri-substituted naphthalene diimide quadruplex-binding compound
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,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)
benzo[lmn][3,8]
phenanthroline-1,3,6,8(2H,7H)-tetraone (CM03). This compound has been designed by
computer modelling, is a potent inhibitor of cell growth in PDAC cell lines and has anti-
cancer activity in PDAC models, with a superior profile compared to gemcitabine, a
commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to
analyse the effects of this quadruplex-binding small molecule on global gene expression. This
has revealed the down-regulation of a large number of genes, rich in putative quadruplex
elements and involved in essential pathways of PDAC survival, metastasis and drug
resistance. The changes produced by CM03 represent a global response to the complexity of
human PDAC, and may be applicable to other currently hard-to-treat cancers.
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INTRODUCTION
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Pancreatic cancers remain one of the most difficult to treat human cancers. Pancreatic ductal
adenocarcinomas (PDACs) comprise some 85 % of diagnosed cases and are classified into
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several sub-types on the basis of expression analysis of major mutated genes. PDACs
typically have extensive patterns of germline mutations, especially in DNA repair and
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,4
signalling pathways. The most common mutations in PDACs are in the KRAS, TP53,
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SMAD4 and CDKN2A genes, with a lower frequency of mutations in many other genes.
A
feature of PDACs is the distinctive pattern of changes in several gene pathways, such as those
involved in axon guidance and dysregulation of signal transduction via inhibition of Rho-
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,7
GTPases.
Only < 4 % of those diagnosed with stage III of PDAC survive for more than five
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, 9
years, a situation that has not significantly changed for more than 40 years. Surgery is an
option for < 15 - 20 % of cases, and chemotherapy for locally advanced inoperable or
metastatic disease is still largely palliative. The nucleoside analogue gemcitabine remains a
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widely used monotherapy regimen used to treat PDAC. Various formulations of
gemcitabine and combinations with, in particular, paclitaxel, have been developed: some
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have been in clinical trial and a small number have received regulatory approval but their
benefit is limited. The use of combination regimens of cytotoxic agents, as in the
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FOLFIRINOX (folinic acid + irinotecan + 5-fluorouracil + oxaliplatin) combination, is
restricted in use due to their toxicity. By contrast to other cancers, immunotherapy has not
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shown promising clinical results with PDAC, possibly due to its inherent
immunosuppression. Therapies based on the common mutations in pancreatic cancer, that
target hard-to-drug proteins such as KRAS, have not yet reached the clinic, although recent
13
results suggest promise for the future.
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Here, we report an alternative approach, of selectively targeting G-quadruplex (G4)
elements in the genomes of pancreatic cancer cells. The human genome contains a large
number of putative G4 sequences comprising several (typically four) short guanine tracts,
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separated by more general sequence loops (reviewed in ref. ). G4 sequences are over-
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represented in promoters and untranslated regions, particularly in genes associated with cell
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proliferation and cancer.
In principle, this over-representation in cancer-associated genes
gives G4 targeting a therapeutic advantage, which can be emphasised by stabilizing
individual G4 structures with an appropriate G4-selective small molecule. Visualisation of
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G4s in cancer cells
and tissues has further demonstrated their potential for selectivity.
Many studies have examined the effects of such compounds on G4s within individual target
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genes, arising from the concept that stabilization of a promoter G4 can result in inhibition of
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the expression of the gene involved. A wide range of chemotypes as G4 stabilizers have
been examined, although they tend to share common structural features of an extended
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aromatic or hetero-aromatic core with one or more cationic side-chains. Down-regulation of
“targeted” genes such as MYC, KIT, BCL2, VEGF and KRAS, has often been reported
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(
reviewed in ref ), although unequivocal demonstrations of direct cause and effect at the
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cellular level have rarely been made. The synthetic fluoroquinolone compound Quarfloxin
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(
CX-3543) has been evaluated in human cancer clinical trials and its G4-binding was
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reported as inhibiting RNA biogenesis. More recently another fluoroquinolone, CX-5461,
has been shown to be a G4-binding compound with selective effects in BRCA1/2-deficient
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cells; CX-5461 has recently entered clinical trials for patients with BRCA-deficient tumors.
We have adopted a distinct approach in comparison to the single G4 promoter
targeting strategy, in which we have not a priore assumed any particular promoter G4 targets.
Instead, we have used global genome transcriptome profiling to determine which genes are
affected by a rationally-designed G4-binding small molecule. This has enabled us to
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determine potential targets at the whole genome level, in two pancreatic cancer cell lines. We
find that pancreatic cancer cell growth is especially sensitive to CM03 and that it strikingly
inhibits tumor growth in PDAC animal models.
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Naphthalene diimides are a favoured chemotype for G4 binding, because of their high
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target affinity, synthetic accessibility and potential for chemical variability.
We have
previously disclosed several focussed libraries of naphthalene diimides, in particular tetra-
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0,31, 32a, 33
substituted analogues
(Fig. 1a) with molecular design based on crystallographic data
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for a series of G4-ligand complexes. We report here on a new lead compound, 2,7-bis(3-
morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino) benzo[lmn][3,8] phenanthroline-
1
,3,6,8(2H,7H)-tetraone (CM03, 633.34 Da; compound 4 in Scheme and Fig. 1c), which
contains two side-chains terminating in morpholino groups, a third side-chain terminating in
a protonated pyrrolidino group, and a lower molecular weight compared to the parent tetra-
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2a
substituted compound MM41 (831.08 Da; Fig. 1a). We report here that CM03 (i) has
potent growth inhibitory activity in vitro for pancreatic cancer cells, (ii) has anti-tumor
activity in vivo in PDAC animal models, (iii) down-regulates the expression of key genes
involved in maintaining the progression of PDAC and in drug resistance, and (iv) targets
genes which have high G4 propensity.
Altogether these results demonstrate that G4 targeting is a realistic and attainable concept
for potent G4-binding compounds, and has promise to lead to significant therapeutic
advantage in the treatment of PDAC.
RESULTS
Design and synthesis of the tri-substituted naphthalene diimide compound
The starting-point for CM03 design was the co-crystal structure of MM41 (Fig. 1a-b),
with an intramolecular human telomeric parallel G4. We reasoned that although the nature
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and structures of most potential target G4s are unknown, several features are common to all
G4s - a core of stacked G-quartets with small-molecule binding at one end of the core, as
observed in all such NMR and crystal structures to date. These crystal and docked structures
revealed that the chromophore of MM41 is bound slightly asymmetrically onto the terminal
G-quartet. Consequently, although three out of four of the substituent chains are each well
positioned at the mouth of a G4 groove, the fourth is not. This is due to the limited surface
area of the naphthalene diimide chromophore, so that the fourth side-chain cannot make
effective contacts with a G4 groove and is oriented away from the G4 surface (Fig. 1b),
consequently its contribution to overall binding is minimal. We hypothesised that a
naphthalene diimide with three substituents would bind with similar affinity, and has the
advantage of lower molecular weight and reduced overall cationic charge. CM03 (Fig. 1c)
was therefore conceived, and is the subject of the studies reported here. Docking with MM41
and CM03 reveals that the poses for the resulting G4 complexes are similar, even though the
CM03 side chains can access only three grooves, while the MM41 targets all four. CM03 is
predicted to have each side-chain positioned at the mouth of a quadruplex groove (Fig. 1d).
The aromatic chromophore is positioned slightly off-centre, since the side chains are not long
enough to interact symmetrically in each groove. The two morpholino side-chains serve to
maximize the binding onto the G-quartet platform.
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The synthetic approach to the trisubstituted series adapts previously published
procedures to simplify purification and increase yields of both intermediates and final
compounds (Scheme 1). 5,5-dimethyl-1,3-dibromohydantoin was used for the regio-selective
bromination of the starting material, enabling more efficient and convenient purification. The
separation of final products was achieved using column chromatography, giving a 26 %
overall yield for CM03. It has been formulated as a highly stable mixed chloride/formate salt
(pH 6.9), freely soluble in water and saline (>10 mg/mL).
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CM03 shows in vitro G4 binding together with selective and potent anti-proliferative
activity
CM03 and MM41 were evaluated for their ability to stabilize G4 binding, by means
of a Fluorescence Resonance Energy Transfer (FRET) protocol using a FAM/TAMRA
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donor/acceptor combination to measure the temperature (T ) of the helix-coil transition on
m
gradual heating. A human telomeric G4 sequence (F21T), together with promoter G4
sequences (from the HSP90, BCL-2 and KRAS genes), and a duplex control (T loop) were
screened against the two compounds (Fig. 1e). CM03 shows a consistent pattern of ~two-fold
(
range 1.98-2.25) lower melting stabilization temperature (ΔT ) compared to MM41, for all
m
tested G4s. The pattern of differences in ΔT values between individual G4s is closely
m
similar between the two compounds. Duplex DNA stabilization is significantly reduced for
CM03 compared to MM41.
Quantitative binding estimates as KD values were obtained from fluorescence
anisotropy titrations (MM41) and direct fluorescence titrations (CM03). The DNA human
telomeric G4 sequence 5'-d(GGGTTAGGGTTAGGGTTAGGG)-3' was used to enable direct
comparisons to be made with the modelling studies. Both compounds bind with nanomolar
affinity to this DNA G4, with a ~3-fold difference between MM41 (20 ± 2 nM) and CM03
(67 ± 4 nM).
Compounds were screened with a 96 h SRB (sulforhodamine B) assay for their anti-
proliferative activity in a panel of human cancer cell lines comprising lung adenocarcinoma
(A549), breast adenocarcinoma (MCF7), and PDAC (MIA PaCa-2 and PANC-1) cell lines,
as well as a fetal lung fibroblast-like non-oncogenic control cell line (WI-38) (Fig. 1f). Both
compounds show highly potent growth inhibition activity, particularly for the lung and
pancreatic cancer cell lines, which are responsive at low nM concentrations. The responses
are highly cell-line-dependent; CM03 shows > 20 - fold selectivity for MIA PaCa-2
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compared to MCF-7 cells. CM03 also shows greater selectivity than MM41 relative to the
non-oncogenic control fibroblasts (e.g. ~170 - fold vs ~21 for MIA PaCa-2 cells compared to
WI-38 cells for CM03 vs MM41 respectively). CM03 was further examined in additional
pancreatic adenocarcinoma cell lines (CAPAN-1 and BX-PC3) and again showed low nM
activity (IC 16 ± 4 and 19 ± 4 nM respectively). Gemcitabine is also active in these lines at
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low nanomolar levels, although less so in MIA PaCa-2 (IC 19 ± 5 nM) and more so in BX-
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PC3 cells (6 ± 2 nM). Taken together, these results show that CM03 has very potent growth
inhibitory activity in cancer versus “normal” cells, particularly for pancreatic
adenocarcinoma cells.
CM03 has in vivo anti-tumor activity
To determine if the in vitro activity of CM03 was paralleled in vivo we explored the
activity of CM03 in a mouse xenograft model of PDAC. We have previously reported that the
maximum tolerated dosage (MTD) for intravenous (IV) administration of MM41 is 25
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mg/kg. CM03 was well tolerated, up to a dosage of 45-50 mg/kg, as judged by a dose-
escalation MTD study. A therapeutic schedule of two CM03 intravenous (IV) doses was
explored using a subcutaneous MIA PaCa-2 pancreatic tumor xenograft model derived from
the human cell line used in the cell growth inhibition studies. A group size of eight CD-1
nude mice was used and dosing was conducted at 10 and 15 mg/kg, each twice weekly, over
a period of 4 weeks (28 days: 8 doses). Two further groups were given either MM41 or
gemcitabine at 15 mg/kg and received the same dose regimen. Over the course of the
experiment, CM03 showed a dose-dependent anti-tumor response. At the conclusion of
dosing on day 28, all four therapy groups showed significant tumor growth inhibition
compared to the vehicle control group (p < 0.001) (Figs. 2a, c) but notably 15 mg/kg CM03
resulted in the highest mean decrease in tumor volume (MTV) (-73%). From day 28 onwards,
whilst all treatment groups also showed significant reduction in MTV compared to the
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control (p < 0.05), the 15 mg/kg CM03 cohort consistently showed the greatest therapeutic
effect and no tumor regrowth was observed. Five out of the eight animals had tumor volumes
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that shrank to < 0.05 cm by day 62. No body weight reduction (Fig. 2b) or adverse effects
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such as tumor ulceration were observed in this group at any time during the study.
Histopathological analysis of vital organs from control and CM03-treated animals sacrificed
at the end of the experiments also showed no sign of organ damage (Fig. 2d). IVIS imaging
of treated mice was performed, taking account of the intrinsic fluorescence of CM03. This
showed significant accumulation of CM03 in the tumor xenograft on the flank of the animal
(Fig. 2e). Thus, in contrast to MM41 and gemcitabine, the higher-dose CM03 treatment
results in a statistically significant (p < 0.001) anti-tumor effect, which persisted long after
dosing had ceased, up to the termination of the xenograft experiment (62 days).
We also tested the higher dose of CM03 in a genetically engineered mouse model of
G12D/+
R172H/+
PDAC. The KPC (Pdx1-Cre; LSL-Kras
; LSL-Trp53
) mouse model is commonly
used for preclinical PDAC cancer studies as it develops tumors that exhibit many of the
genetic and pathological features of the human disease, including resistance to
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4
chemotherapy. Mice with late-stage disease, characterised by swollen abdomen, loss of
body conditioning and palpable tumor, were treated with CM03 intraperitoneally (ip) using
the 15 mg/kg twice-weekly dosing regimen employed for the xenografts. While the median
survival between treated and untreated animals is similar, we note that 3/7 mice survived
much longer than all of the untreated animals with two surviving more than twice as long
(Fig. 2f).
CM03 induces down-regulation of genes enriched in G4s
CM03 is a potent G4 ligand, as shown by the FRET and quantitative binding
experiments reported here. We therefore reasoned that the potent CM03 anti-proliferative
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activity on PDAC cells could be mediated through a G4-targeting mechanism. To test this
hypothesis, we determined whether transcriptome changes upon early CM03 treatment were
correlated with G4 presence in promoters and/or gene bodies. Two PDAC cell lines, PANC-1
and MIA PaCa-2 (both with activating KRAS mutations), were treated for 6 or 24 h with 400
nM CM03 (1/10 of the calculated GI at 24 h, Supplementary Data Fig. 1a), RNA was
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harvested and gene expression levels analyzed by RNA-Seq. The time and dose of CM03
treatment were selected to reveal initial but robust mRNA changes whilst minimizing chronic
effects due to cell toxicity. In both cell lines, the number of genes with altered expression (as
defined by > 0.5 increase in Log2 of fold change [Log FC], or < - 0.5 decrease; false
2
discovery rate [FDR] < 0.1) increased with duration of CM03 treatment. In PANC-1 cells
2
,165 genes at 6 h and 3,977 genes at 24 h were seen to change, while in MIA PaCa-2 cells
,306 genes changed at 6 h and 5,477 genes at 24 h, compared to untreated controls
5
(Supplementary Table 1). Suggestive of a common mechanism of action of CM03, the
differentially expressed genes were well correlated between the two cell lines at both
treatment times (6 h, r = 0.511; 24 h, r = 0.518) (Supplementary Data Fig 1b-h) with 13,044
expressed genes in common (Supplementary Table 2) and a high number of genes changed in
both cell lines (1,185 genes at 6 h, 1,917 genes at 24 h). Remarkably, the differentially down-
regulated gene set (considering each cell line individually or combined) was significantly
enriched (p < 1E-10) for putative G-quadruplexes (PQs), as defined by the G N G N
≥
3
1-7 ≥3+ 1-
3
5,36
G N G consensus sequence,
3≥ 1-7 ≥3
in promoters and gene bodies at both 6 and 24 h
7
treatments compared to up-regulated or unaltered genes (cell lines combined- Figs. 3a-b; cell
lines considered individually - Supplementary Data Figs. 2a-b and 3a-b). This association
was more marked for genes that were down-regulated to the greatest extent by CM03
(
(
Log FC ≤ 1.0, FDR < 0.05) after 6 h treatment (down strong > down, ratio of means)
2
Supplementary Table 3). To validate that the targeting of G4-containing genes is not a
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general feature of anti-proliferative agents, we also examined the effects of the first-line
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therapeutic agent for PDAC, gemcitabine, a potent anti-proliferative DNA synthesis
inhibitor whose mechanism of action is G4-independent and which remains a standard
therapy option in advanced PDAC. Both cell lines were treated with a range of gemcitabine
concentrations (Supplementary Data Fig. 5, Supplementary Table 4) for 6 or 24 h. Crucially,
no significant PQ enrichment was observed for the down-regulated genes in any gemcitabine
condition (Supplementary Data Fig. 2c-d and 3c-d; 300 nM gemcitabine is shown as an
example)). These results strongly support the hypothesis that CM03 specifically targets G4-
bearing genes and represses their expression.
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To gain insights into possible gene types and pathways targeted by CM03, we
performed a KEGG gene enrichment pathway analysis of the common down-regulated genes.
This revealed a total of 11 and 46 significant pathways (p-EASE ≤ 0.05) after 6 and 24 h
CM03 treatment, respectively. Many of these pathways, or genes within them, have been
previously associated with PDAC (Fig. 3c and d, Supplementary Data Figs 5, 6;
7
Supplementary Table 5, 6) such as axon guidance (14 genes, p = 5.75E-05) , the hippo
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39
signaling pathway (13 genes, p =1.15E-03) at 6 h; mTOR (12 genes, p = 1.07E-04) and
4
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VEGF signaling pathways (11 genes, p = 7.45E-04) after 24 h and insulin resistance,
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Rap1 and MAPK signaling pathways across both time points (Fig. 3e). This contrasts with
the pattern of the common up-regulated genes at 6 and 24 h (9 and 10 significant pathways
respectively) after CM03 treatment, with particular enrichment in cellular matrix adhesion
and major histocompatibility complex proteins pathways (Supplementary Data Fig. 7a). As
anticipated, 300 nM gemcitabine treatment affected strikingly different KEGG pathways
compared to CM03 (Supplementary Table 7). The most significant down-regulated
gemcitabine pathways (for both cell lines) were cell cycle after 6 h and ribosome-associated
pathways after 24 h, which is consistent with its known mechanism of action in inhibiting
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DNA synthesis, cell cycle progression and global translation. This contrasts with known
pathways seen to be up-regulated after gemcitabine treatment which includes NF-Kappa B
4
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signaling pathway (p = 1.61E-06) for PANC-1 and the Fanconi anemia pathway (p = 6.46E-
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6) for MIA PaCa-2. It is notable that PQs in promoters and gene bodies are highly
enriched within down-regulated KEGG gene pathways after CM03 treatment (Figs. 3f,
Supplementary Data Fig. 7b) compared to random sampling across the expressed genes or to
up-regulated genes (Figs. 3g and Supplementary Data Fig. 7c). Overall, these results robustly
support our contention that CM03 specifically targets critical pathways that lead to pancreatic
cell death via the inhibition of expression gene sets harboring a G4 structure.
CM03 treatment results in an increase in BG4 foci and DNA damage
Since many established G4-stabilizing ligands induce transcription and replication-
44
coupled DNA damage in the vicinity of G4-forming sequences , we investigated whether
CM03 treatment affects cellular G4 occurrence and/or induces a DNA damage response. To
do this, we treated asynchronous PANC-1 cells with CM03 and performed
18
immunofluorescence staining using the G4-specific antibody BG4 and an antibody against
4
5
the DNA damage and double-strand break (DSB) marker 53BP1 followed by 3D image
construction and analysis. As predicted, 400 nM CM03 treatment resulted in a time-
dependent significant (p < 0.05) increase in the number of nuclear BG4 foci (Figs. 4a and b)
(
393 ± 13 at 6 h and 444 ± 39 BG4 foci at 24 h) compared to controls (297 ± 3 BG4 foci).
This further confirms that CM03 has a direct action at G4 sites to increase G4 stabilization.
4
6
Consistent with the capability of unresolved endogenous genomic G4 sites and small
44
molecule-stabilized G4s to obstruct DNA replication, a two-fold increase in the number of
5
3BP1 foci was only apparent after 24 h treatment with CM03 (11 ± 2 compared to 21 ± 3
53BP1 foci) (Figs. 4a and c). To determine if CM03 induced DNA damage directly at G4
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sites, we calculated the extent of co-localization between BG4 and 53BP1 foci using object-
based image co-localization analysis. While the percentage of co-localization between BG4
and 53BP1 reproducibly increased by 50 % after 24 h 400 nM CM03 treatment (Fig. 4d),
only 1.4 % of total BG4 foci per nucleus were associated with DNA damage foci (Fig. 4e).
Nonetheless, our data strongly suggests CM03 stabilizes G4 structures leading to an
accumulation of nuclear G4 foci accompanied with the induction of DNA damage and
replicative stress.
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DISCUSSION
40
Whilst PDAC is predominantly (> 95%) driven by oncogenic KRAS, the
development of more targeted therapies against KRAS and other core PDAC associated pro-
survival pathways including: Ras-Raf-MEK-ERK and PI3K-AKt-mTOR have proved futile
5
-8
owing to the highly mutagenic nature of the disease and the ability of PDAC cells to
4
7
rapidly switch between phenotypic states, resulting in the evasion of therapy. As novel
alternative strategies for intervention in PDAC are urgently required, the targeting of G4s
with small molecules has been proposed as an innovative method for selective cancer
1
4
therapy. This is supported by recent G4-ChIP-seq and G4-IF studies identifying an
increased abundance of G4s in cancer versus normal cells, as well as G4 structure enrichment
1
6
33
at oncogenes. We have recently reported that the G4 ligand MM41 reduces tumor volume
by ~ 80 % in a PDAC xenograft model. MM41 binds strongly to the G4s in the down-
regulated genes KRAS and BCL-2, resulting in cell apoptosis, and suggested that G4 ligand
treatment is potentially a highly effective method for targeting PDAC. MM41 is a sub-
3
2
optimal drug candidate due to its higher molecular weight and four positive charges. We
present here CM03 as an improved rationally-designed derivative of MM41, and as a novel
lead candidate compound for potential PDAC therapy in humans. CM03 has comparable
affinity for a human telomeric G4 to the tetra-substituent compound MM41, indicating that
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the fourth side chain and increased cationic charge on MM41 does not make a major
contribution to affinity. Although affinities for CM03 or MM41 binding to (a single) G4 in
vitro may be difficult to directly relate to cell growth inhibitory properties, CM03 is as potent
in cells as MM41. Both compounds show good selectivity for cancer cell lines (especially
pancreatic) over ‘normal’ non-oncogenic fibroblasts but CM03 shows > 4-fold less activity
toward the WI-38 cell line. This enhanced selectivity is also apparent in vivo, where CM03
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th
has a higher level of tolerability (higher MTD) compared to MM41. This suggests that the 4
side-chain of MM41 may add to off-target toxicity burden; however, neither MM41 nor
CM03 show significant hERG liabilities (unpublished data), a common indicator of
cardiotoxic effects. The anti-tumor activity of CM03 results in sustained tumor growth arrest,
which continues long after dosing is ceased, and compares favorably with the growth delay
2
5
typically seen with experimental anti-tumor agents. The increased animal survival in a
subset of the autochthonous KPC mice to CM03 suggests that CM03 may be able to
effectively penetrate the tumor stroma microenvironment characteristic of human PDAC.
The RNA-seq data revealed that treatment with CM03 resulted in a striking global
differential down-regulation of genes containing putative G4s (PQs), which occurred as early
as 6 h after treatment. We have performed RT-qPCR on a small set of the genes down-
regulated by CM03 (Fig. 5a, b). The results confirm the RNA-seq analyses, that these PDAC-
pathway genes are transcriptionally down-regulated in both cell lines, and that they are not
significantly affected by gemcitabine (Fig. 5c, d). The RNA-seq results are consistent with
the hypothesis that G4 ligands can alter gene expression when PQs are targets in individual
2
3
genes.
Previously, global G4-motif specific changes in the transcriptome with
4
8
bisquinolinium compounds in HeLa cells have been reported using microarray analysis.
However, our work represents the first comprehensive analysis using state-of-the-art RNA-
seq methodology. This is the first demonstration that a G4-binding small molecule produces
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global down-regulation of G4-containing genes in human cancer cell lines, in striking
comparison with a non-G4 targeting drug, the modified nucleoside gemcitabine. Moreover, as
we identify G4 effects at much earlier times and smaller doses than earlier studies, this
further demonstrates that CM03 is a bona fide G4 ligand in cells, and by implication, in vivo
as well. The observed transcriptional effects of CM03 also align with the hypothesis that
down-regulation of gene expression is mediated through ligand-stabilized G4s, causing for
example steric hindrance and inhibition of RNA pol II in the transcription bubble (reviewed
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in ref ). The observations that transcriptional effects are closely aligned in two PDAC cell
lines, independently sequenced, gives added confidence to these conclusions.
KEGG pathway analysis shows that CM03 targets 42 pathways, many of which are
key to PDAC development and maintenance. The most significant down-regulated pathways
after 6 h were the hippo signaling, axon guidance and endocytosis pathways, which are all
deregulated in PDAC.
Hippo signaling acts through the core transcription factors, YAP and TAZ. In PDAC,
49
both YAP and TAZ are over-expressed and are essential for neoplastic progression.
YAP/TAZ also contributes to PDAC resistance to therapeutic approaches directly targeting
50,51
mutant KRAS.
Notably, CM03 down-regulates three prime effectors of YAP function,
TEAD2 and 4 (MIA PaCa-2 only) and TEAD3 (both cell lines) at 6 h. As TEAD 2/4 are
5
2
oncogenic in many cancers, CM03 may be suitable as a global cancer therapeutic agent.
CM03 also down-regulates the oncogenic Gli transcription factors Gli2/4, downstream of
6
TEAD and highly up regulated in PDAC. As Gli2 is essential for KRAS-driven oncogenesis
5
3
54
55
in vivo and plays a role in PDAC resistance to both gemcitabine and BET inhibitors, this
suggests CM03 may have a novel therapeutic potential.
For the axon guidance pathway, key PQs-containing genes down-regulated by CM03
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are PAK1 , ROBO3 and PLXNA1 , all of which are highly over-expressed in PDAC.
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While axon guidance is a neurodevelopmental process, many axon guidance genes are
deregulated in many cancers, including PDAC, and contribute to metastasis and tumor
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,59
growth.
PAK1 mediates pancreatic cancer cell migration, growth, promotes epithelial to
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mesenchymal transition (EMT) and is crucial in conferring gemcitabine resistance .
Expression of the SLIT3 receptor, ROBO3, increases with PDAC clinical grade and is anti-
7
correlated with patient survival. As ROBO3 promotes liver metastasis and tumor growth via
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Wnt signaling pathway this again suggests a more general CM03 therapeutic applicability.
,
Likewise, the SEMA3A receptor, PLXNA1, is up-regulated in PDAC and linked with poor
7
,58
patient prognosis due to altered PLXNA1-SEMA3A signaling.
PDAC tumors experience hypoxia and nutrient deprivation due to the dense
surrounding tissue stroma, thus PDAC heavily relies on endocytosis for scavenging of
6
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essential metabolites and receptors/ transporters recycling. A key endocytotic gene ARF6, a
member of the RAS superfamily of small GTPases involved in plasma membrane trafficking,
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cell motility, cytokinesis and cholesterol homeostasis, is strikingly down-regulated by
CM03 (by 6 h). Mutant KRAS induces ARF6, which acts as an oncogene in PDAC to drive
the constitutive activation of the ERK1/2 signaling and downstream effector MYC, to
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2
regulate the Warburg effect. ARF6 has many other roles including GLUT1 and CD95
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receptor recycling, non-canonical EGRF and Hedgehog (Hh) signaling and Hh-mediated
6
3
chemo-resistance. Furthermore, CM03 also targets IQSEC1, a guanine nucleotide-exchange
factor that is responsible for activating ARF6, and whose down-regulation inhibits PDAC
64
invasion by preventing internalization of E-Cadherin from the plasma membrane. Other
significant pathways with CM03 down-regulated genes (at 24 h) involved in altered PDAC
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metabolism include: fatty acid synthesis (oncogene fatty acid synthase (FASN)), glycolysis
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(
PCK2, PFKL and PFKFB4), autophagy (ULK1) and macropinocytosis (PAK1 and
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Other key genes down-regulated by CM03 (by 6 h) which also impinge on the above
pathways include the binding partners Par6 (PARD6A) and PRKCZ that are important for
tumor cell motility and metastatic potential in PDAC through activation of JAK/STAT
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signaling pathway. Par6 also binds PRKCi, another atypical protein kinase, which promotes
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PDAC transformation partly through activation of the Rac1-MEK/ERK signaling pathway.
Of note, PARD6A knockdown significantly reduces anchorage dependent growth and cell
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invasion of both PRKCZ and PRKCi non-redundant pathways. The PKCi-Par6 complex
68
also plays an oncogenic role in lung, ovarian and colon cancer. Together, this suggests that
CM03 could be effective in multiple cancer types. Other CM03 down-regulated genes whose
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DNA methylation status correlates with pancreatic cancer patient survival include PRDM16
rank 2), SHANK2 (rank 31), CBFA2T3 (rank 4) and TP73 (rank 20) (at 6h, Supplementary
Table 2).
For PDACs driven by mutant KRAS, drug resistance depends on a balance between
(
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PI3K/AKT/mTOR and MAPK/ERK pathways, and YAP1 and MYC up-regulation. It is
striking that CM03 treatment down regulates many elements of this mechanism (see Table 1).
For example AKT (AKT1), key effectors of mTOR (RPTOR, mLST8, AKT1S1), PI3K
pathway (PIK3R3 and PIK3R2) and MAPK proteins (MAPK9, MAPK11 and MAPK12) are
down-regulated in both PDAC cell lines by CM03 (by 24 h). In the non-canonical Hh-
mediated gemcitabine resistance pathway the transcript levels of Smo and Gli1 are also
5
4
down-regulated by CM03.
Tumor associated macrophages and cancer-associated
myofibroblasts in tissue stroma are also key drivers for chemo-resistance in PDAC and it is
notable that IGF2 and SHC2 are down-regulated by CM03 as these proteins have been
7
1
implicated in insulin receptor-mediated chemo-resistance. Collectively these findings
clearly demonstrate the ability of CM03 to simultaneously target several fundamental PDAC
tumorigenesis mechanisms.
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Curiously, while CM03 shows binding affinity to KRAS G4s in vitro (Fig. 1e), our
RNA-SEQ studies show that KRAS mRNA expression is unaffected by CM03 treatment in
either pancreatic cell line at the times and doses tested (Supplementary Table 1). It may be
that in a cellular context the KRAS G4 is rapidly resolved by unwinding helicases or that G4
binding proteins hinder drug accessibility. Thus, while the KRAS G4s may not be the
main/initial target of CM03, we cannot assume the accessibility of KRAS G4 for CM03
within the native epigenetic environment. Nonetheless our results suggests that simultaneous
targeting of multiple fundamental PDAC pathways (including important KRAS effector and
feedback loop signaling pathways and those involved in drug resistance) by G4 stabilization
will be an advantageous approach.
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CM03 also resulted in the induction of DNA damage in a replication-dependent
manner. While the number of BG4 foci increased upon CM03 treatment suggesting increased
G4 stabilization, only a small increase in BG4 and 53BP1 co-localization was observed. It is
tempting to suggest that CM03 does not cause an increase in damage at G4 sites, however,
the dynamics of whether a G4 structure can co-exist with DNA damage response proteins is
not well understood, or whether G4s persist after DNA damage response and repair have
been elicited. In support, many DNA repair pathway proteins are capable of unwinding and
72
destabilizing G4 and a recent study has elucidated the role of PARP3 negatively regulating
73
G4 in order to initiate DNA repair at DSB sites. Hence by 24 h the small number of co-
localizations could represent an intermediate repair state of DNA at a G4 site.
In summary, we have demonstrated that CM03 is a highly potent G4-binding small
molecule, with in vitro cell assays and in vivo models for human PDAC. Treatment with
CM03 successfully reduced tumor growth for an extended period in PDAC xenografts and
also prolonged survival in the KPC mouse model for this disease, at dosage levels that do not
cause observable damage to major organs yet enable selective tumor targeting. It is notable
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that the antitumor effects of CM03 in the xenograft model persisted until the termination of
the experiment at day 62 (34 days beyond the end of dosing). This is in striking contrast to
the anti-tumor response to gemcitabine, with tumor regrowth being apparent as soon as
dosing was stopped.
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Studies with PDAC cell lines suggest that the mechanism of action of CM03 is via the
G4-dependent down-regulation of genes in a number of essential pathways associated with
PDAC survival, metastasis and drug resistance, together with the activation of G4 replication-
dependent DNA damage. Furthermore, given the good bioavailability of CM03 and its ability
to target many oncogenic pathways essential to tumorigenesis in general, we suggest that
CM03 has the potential to be used as an effective anti-cancer therapeutic in a number of other
cancers in addition to PDAC. It is tempting to speculate that the selective tumor targeting is
associated with G4 binding, and future studies will address this question.
EXPERIMENTAL METHODS
Chemistry. The synthesis and characterisation of MM41 (4,9-bis((3-(4-methylpiperazin-1-
yl)propyl)amino)-2,7-bis(3-morpholinopropyl)
benzo[lmn][3,8]
phenanthroline-
3
2a
1
,3,6,8(2H,7H)-tetraone: molecular weight 831.08) has been previously reported. MM41
was analytically pure as shown by LC-MS and NMR methods and was formulated for
biological studies as the freely water-soluble formate salt.
The traditional synthetic route to highly substituted naphthalene diimides starts from
1
,4,5,8-naphthalene tetracarboxylic dianhydride, which is then reacted with
dibromoisocyanuric acid, leading to the mono- and di-bromo-naphthalene diimide as key
intermediates, followed by introduction of the amine chains at the imide and bay positions, in
that order. This synthesis results in a complex mixture requiring purification by preparative
HPLC at least twice to obtain the desired products in very low yield (less than 10%). The
critical aspect of the present synthetic scheme was preparation of the mono-brominated
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naphthalene diimide precursor; this is insoluble, so purification at this stage was not
attempted, nor was there any evaluation of the amount of mono-bromo derivative obtained.
Purification can be performed only after the N-N' imidation reaction, due to the aqueous
solubility that is imparted by the introduction of the basic morpholino side chains. Instead the
procedure was modified as shown in Scheme 1.
1
1
1
1
1
1
1
1
1
1
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All chemicals, reagents, and solvents were purchased from Sigma-Aldrich, Alfa Aesar,
Lancaster Synthesis, and Fluorochem (UK) and used without further purification. Solvents
were supplied by VWR and Fisher Scientific. Column chromatography was performed using
BDH silica gel (BDH 153325P). Chromatography was conducted under medium pressure in
glass columns or using a Biotage SP4 instrument in prepacked columns (FLASH Silica
columns (40-63 µm, 60 Å)). HPLC analysis was carried out with a Gilson apparatus
combining a 322 pump and an Agilent 1100 series detector, using a C18 5 μ (100 mm × 4.6
mm) column (41622271 (W), YMC, Japan), at a flow of 1 mL/min. Preparative HPLC was
carried out with a Gilson apparatus combining a 322 pump and a UV/vis-155 detector with
detection at 280 nm, using a C18 5 μ (100 mm × 20 mm) column (201022272) (W), YMC,
Japan, at a flow of 20 mL/min. Water and methanol with 0.1% formic acid were used as
solvents for HPLC. For the purification of compound CM03, the following method was used:
1
00% aqueous solution for 5 min after injection, then gradually decreased to 60% aqueous
solution over 25 min.
2
-Bromo-1,4,5,8-naphthalenetetracarboxylic acid dianhydride (2a) and 2,6-Dibromo-
1
,4,5,8-naphthalenetetracarboxylic acid dianhydride (2b). Naphthalene dianhydride (1,
from Sigma-Aldrich) (150 mg, 0.56 mmol) was slurried in sulphuric acid (1.5 ml) and the
suspension obtained was stirred for 5 min at room temperature to allow the complete
dissolution. 5,5-dimethyl-1,3-dibromohydantoin (88 mg, 0.308 mmol) was added slowly over
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a period of 1 h and the round bottom flask was tightly stopped to avoid the escape of bromine
from the reaction mixture. The solution was stirred at 80̊ C for 72 h and then poured onto ice
(30 ml). The yellow solid formed was filtered, washed with water (2 x 10 ml) and dried under
vacuum, yielding a mixture 2a and 2b. No NMR data were obtained due to solubility issues.
The resulting mixture was used without further purification in the next step.
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N,N'-Bis(3-(morpholino)propylamino)-2-bromo-1,4,5,8-naphthalenetetracarboxylic acid
diimide (3a). Compounds 2a and 2b (194 mg, 0.56 mmol) were suspended with sonication in
glacial acetic acid (1.5 ml) in a microwave reaction vessel. 3-morpholinopropyalamine (242
mg, 245 µL, 1.68 mmol) was added dropwise to the stirring mixture. The reaction tube was
o
sealed and treated for 30 min at 125 C in the microwave. The solution was then basified with
potassium carbonate and extracted with chloroform (3 x 5 ml). The organic phases were
collected, dried over sodium sulphate and evaporated. The residue obtained was purified
through column chromatography using as eluent a CH Cl /MeOH 96/40 mixture. Yield 3a
2
2
1
(
107 mg, 0.18 mmol, 35%) H NMR (CDCl ) δ 1.95-1.97 (m, 4H), 2.40-2.43 (m, 8H), 2.52-
3
2
1
3
1
.53 (m, 4H), 3.51-3.54 (m, 8H), 4.28-4.33 (q, 4H, J=8Hz), 8.77 (d, 1H, J= 8Hz), 8.82 (d,
1
3
H, J= 7.6Hz), 8.935 (s, 1H). C NMR (100 MHz, CDCl , TMS) δ 23.31, 23.44, 38.81,
3
9.11, 52.50, 55.51, 55.45, 65.37, 65.41, 123.97, 125.75, 125.95 (2C), 126.01, 126.84,
+
28.68, 128.76, 130.73, 131.71, 138.44, 161.17, 161.87, 161.98, 162.56. HRMS (ES )
+
calculated C H BrN O [M+2H] 600.1543. Found: 600.1536.
2
8
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4
6
2
,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)
phenanthroline-1,3,6,8(2H,7H)-tetraone (compound 4: CM03). Compound 3a (0.15 g,
.25 mmol), 1-(2-aminoethyl)pyrrolidine (0.06 mL, 0.51 mmol), and NMP (1 mL) were
suspended in a microwave reaction vessel. The reaction tube was sealed and treated for 25
benzo[lmn][3,8]
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o
min at 130 C in the microwave. After having been cooled to room temperature, the solvent
was concentrated and the crude mixture was purified by column chromatography, using as
eluent a mixture of CH Cl /MeOH/NH 95/5/0.4. Compound 4 was obtained as a red oil (118
2
2
3
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mg, 0.186 mmol, 75% yield). H NMR (400 MHz, CDCl , TMS) δ 2.019-2.040 (m, 8H),
3
2
4
8
.683-2.782 (m, 12H), 3.195 (t, 4H, J= 6.4Hz), 3.32 (t, 2H, J= 6.6Hz), 3.708 (t, 4H, J=
.8Hz), 3.764 (t, 4H, J= 4.6Hz), 4.009-4.024 (m, 2H), 4.22 (t, 4H, J= 7Hz), 8.237 (s, 1H),
1
3
.28 (d, 1H, J= 7.6Hz), 8.56 (d, 1H, J= 8Hz), 10.174 (t, 1H, exch D O, J= 4.2Hz). C NMR
2
(
100 MHz, CDCl ) δ 23.3, 23.6, 23.7, 38.2, 38.9, 40.1, 52.6, 52.8, 53.9, 53.9, 55.8 (2C), 65.6,
3
6
5.8, 100.7, 119.1, 119.5, 123.5, 124.9, 126.1, 128.0, 129.2, 131.4, 151.8, 162.8, 163.2,
+
+
165.9, 166.5. HRMS (ES ) calculated for (M+H) C H N O 633.3389, found 633.3401.
34
45
6
6
For the HPLC purity analysis of this compound the method used was: 100% aqueous solution
for 5 min after injection, to 60% w/v aqueous solution over 18 min as well as 100% aqueous
for 5 min after injection, to 60% w/v aqueous over 43 min. The purity for the final compound
was greater than 95% (HPLC, 280 nm) and was further checked by LC-MS.
CM03 was then converted to a mixed hydrochloride/formate salt in order to enhance its
aqueous solubility. At a concentration of 5 mg/ml, the pH of the CM03 salt solution was 6.95.
Molecular modelling. The crystal structure of the naphthalene diimide intramolecular
32, 74
telomeric G4 complexes
(PDB ids 2T5E, 4DA3 and 3UYH) were used as starting points
3
2a
to study G4-ligand interactions, in particular that with the compound MM41. The structure
of the native human intramolecular DNA quadruplex with parallel-stranded propeller-type
topology was extracted from the Protein Data Bank (www.rscb.org) and used for docking
with CM03. The final structures were subjected to 1000 steps of conjugate gradient energy
minimisation to relieve any structural distortions.
The docking protocol was consistent for all systems. The chemical structures of the
ligands MM41 and CM03 were sketched, built and docked using ICM-Pro software
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(
www.molsoft.com). Atom-centred charges were assigned using the ICFF force field. Grid
+
maps were made that encompassed the terminal quartets, central K ions and the loops.
Docking was done using the automated docking module in the ICM-Pro package, employing
the default parameters. The final docked conformations were chosen based on the strongest
binding energy between the docked ligands and the G4. The grids were large enough to allow
translation and rotation of the ligands. The top 20 plausible conformations of the ligands were
then analysed. The final docked models were chosen based on the largest interaction energy
between G4 and ligand. The positively charged ions were retained within the central axis of
the G4 channel and were treated as an integral part of the G4 structure.
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FRET studies. FRET DNA melting assays on MM41 and CM03 were performed using a
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fluorescence resonance energy transfer (FRET) assay. The labelled oligonucleotides had the
fluorophores FAM (6-carboxyfluorescein) as donor and fluorophore TAMRA (6-
carboxytetramethyl-rhodamine) attached as acceptor. The sequences used are:
human telomeric G4 (F21T) 5'-FAM-d(GGG[TTAGGG] )-TAMRA-3'
3
duplex sequence 5'-FAM-d(TATAGCTATA-HEG-TATAGCTATA)-TAMRA-3' (HEG
linker: [(-CH -CH -O-) ]))
2
2
6
HSP90a: 5'-FAM-d(GGGCCAAAGGGAAGGGGTGGG)-TAMRA-3'
HSP90b: 5'-FAM-d(GGGCGGGCCAAAGGGAAGGGG)-TAMRA-3'
Kras21 5'- FAM-d(AGGGCGGTGTGGGAAGAGGGA)-TAMRA-3'
Kras32 5'-FAM-d(AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG)-TAMRA-3'
Bcl-2 5'-FAM-d(GGGCGCGGGAGGAAGGGGGCGGG)-TAMRA-3'
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The FRET probe sequences were diluted from stock to the correct concentration (400 nM) in
a 60 mM potassium cacodylate buffer (pH 7.4) and then annealed by heating to 95 °C for 10
min, followed by cooling to RT in a heating block (3 - 3.5 h). Drug stock solutions (at 2 x
concentration) were prepared using 60 mM potassium cacodylate buffer (pH 7.4). 96-well
plates (MJ Research, Waltham, MA) were prepared by aliquoting 50 µL of annealed DNA
into each well, followed by 50 µL of the compound solutions. Measurements were made on a
DNA Opticon Engine (MJ Research) with excitation at 450–495 nm and detection at 515–
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45 nm. Fluorescence readings were taken at intervals of 0.5 °C in the range 30–100 °C, with
a constant temperature being maintained for 30 sec prior to each reading to ensure a stable
value. Final analysis of the data was carried out using a script written in the Origin 7.0
(OriginLab Corp., Northampton, MA) package and its advanced curve-fitting function for
derivation of ΔT values.
m
Quantitative G4 binding studies. Human telomeric DNA G4 was prepared using
oligonucleotides purchased from Integrated DNA Technologies (Coralville, IA, USA). The
DNA purity and sequence was verified by mass spectrometry. Desalted oligonucleotide
sequences were initially suspended in 150 mM NH OAc (pH 6.8) in nuclease-free water to
4
yield a final concentration of 1 mM and determined spectroscopically at 260 nm using the
nearest-neighbour method. The stock solution was aliquoted into 20 μL samples in 200 μL
PCR tubes. Using a thermocycler, samples were heated to 98 ºC for 10 min, slowly cooled for
1
5 min while remaining within the thermocycler, followed by an additional cooling session at
RT for 30 min and stored at 4 ºC before use.
Fluorescence anisotropy measurements were obtained on a Variant Cary Eclipse
Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) equipped
with a polarization attachment. The excitation and emission polarizers were fixed between
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the light source and detector, respectively. Measurements were converted into anisotropy
values by using Equations 1-2:
ꢁꢂꢂ−ꢃꢄ∗ꢁꢂℎ
ꢀ = ꢁ
(1)
ꢂꢂ+2∗ꢃꢄ∗ꢁ
ꢂℎ
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ꢁℎꢂ
ꢅꢆ = ꢁ
(2)
ℎℎ
r is the anisotropy value, I is the fluorescence intensity of the samples when excitation and
vv
emission polarizers are aligned in vertical positions, I is the fluorescence intensity of the
vh
samples when the excitation and emission polarizers are in vertical and horizontal positions,
respectively, I is the fluorescence intensity of compound when excitation and emission
hv
polarizers are aligned in horizontal and vertical positions, I is the fluorescence intensity of
vv
compound when excitation and emission polarizers are aligned in vertical positions, GF is the
grating factor of the polarizers. The GF value was introduced to the equation to correct for
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unequal sensitivity of the excitation and emission polarizers.
To determine dissociation constants, MM41 was added to a 3 ml volume of the
experimental buffer (50 mM KCl, 10 mM Tris-HCl, 1 mM EDTA in nuclease-free water at
2
5 ºC, pH 7.3) in a quartz fluorescence cuvette at a fixed concentration of 50 nM and G4 was
titrated into the cuvette. Oligonucleotide concentrations were calculated before each titration
experiment and fluorophore concentrations corrected after each addition, taking dilution into
account. Measurements were taken at a maximum emission wavelength of MM41 (λ = 670
em
nm) with excitation at λ = 630 nm. Both excitation and emission slits were at 20 nm. Each
ex
set of measurements were taken from a 50 sec average reading measurement (PMT = 630 V).
The dissociation constant (K ) of MM41 to G4 was obtained by fitting the change in
D
anisotropy value of the compound titration using the following equation:
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ꢘ
ꢔ +ꢖ+ꢗ− �(ꢔ +ꢖ+ꢗ) −4ꢖꢗ
ꢕ
ꢕ
ꢀꢇꢈꢉꢊꢋꢌꢊꢍ = ꢀꢎꢏꢐ + [(ꢀꢎꢑꢒ ꢓ ꢀꢎꢏꢐ
)
]
(3)
2ꢖ
rmin is the anisotropy value of free MM41, rmax is the anisotropy value of bound MM41, L is
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the total concentration of MM41, and D is the total concentration of G4 .
1
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Direct fluorescence measurements were performed using the same instrument and
experimental buffer. Both emission and excitation slits were set to 20 nm. Fluorescence was
measured in a quartz fluorescence cuvette with a starting CM03 concentration of 25 nM. The
amount of oligonucleotide needed for the experiment was calculated and titrated into the
cuvette. Changes in maximum emission intensity of CM03 (λem = 570 nm) were observed
and recorded upon binding to telomeric DNA. The curve fit equation is similar to Equation 3,
taking into account that fluorescence intensity values decrease upon titration (initial intensity
> final intensity). The results were fitted to obtain a dissociation constant (K ) using the
D
fluorescence quenching equation below:
ꢘ
ꢔ +ꢖ+ꢗ− �(ꢔ +ꢖ+ꢗ) −4ꢖꢗ
ꢕ
ꢕ
ꢙ = ꢙꢏꢐꢏꢚꢏꢑꢛ ꢓ [ꢜꢙꢏꢐꢏꢚꢏꢑꢛ ꢓ ꢙꢝꢏꢐꢑꢛ
ꢞ
]
(4)
2
ꢖ
Ainitial is the intensity measured for free CM03, Afinal is the intensity measured of bound
CM03, K is the dissociation constant of the complex, L is the total concentration of CM03,
D
and D is total concentration of G4.
Cell culture studies. All cell lines were purchased from ATCC and maintained in
appropriate medium supplemented with 10 % foetal bovine serum (Invitrogen, UK), 2 mM L-
glutamine (Invitrogen, Netherlands), and other components as specified by the suppliers. Cell
lines were maintained at 37 °C, 5% CO and routinely passaged. Drugs were dissolved in
2
H O and filtered through 0.22 µm pore-size filter units (stock 10 mM) before addition to
2
appropriate cell line media. Cellular growth inhibition was measured using the
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sulforhodamine B (SRB) assay in 96 well plates as described previously. 50% inhibitory
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concentrations (IC ) were determined by taking the mean absorbance at 540 nm for each
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drug concentration expressed as a percentage of the absorbance of untreated control wells.
Xenograft studies. All animal experiments were performed in accordance with the UK
Home Office Animals Scientific Procedures Act 1986 and the United Kingdom Co-
ordinating Committee on Cancer Research Guidelines for the Welfare and Use of Animals in
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Cancer Research and with the approval of the University College London Animal Ethics
Committee. Mice had access to food and water ad libitum.
The maximum tolerated dose (MTD) studies of MM41 and CM03 were performed in CD-
1
nude mice. The MTD of a single dose administered intravenously (IV) was examined and
repeat dosing was performed per week at different concentrations of MM41 (20, 25 and 30
mg/kg), and CM03 (10, 20, 30, 35, 40, 45, 50 and 60 mg/kg), with two mice per group.
Compounds were administered in saline. Mice were monitored visually for signs of adverse
effects, over periods of up to 72 h. The maximum tolerated dose was judged to have been
reached when all mice in a particular dose cohort survived in the absence of any observed ill-
effects, for the period of the experiment.
For therapy studies, female CD-1 nude mice (2-3 months old, weighing 20−25 g) were
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injected subcutaneously with 5 x 10 MIA PaCa-2 cells in the right flank (unsupplemented
3
RPMI + Matrigel). When the tumors were established (approx. 13 days, mean size 0.05 cm ),
the mice were divided into five treatment groups with eight mice/group. The MM41 and
CM03 samples were dissolved directly in saline to the required concentrations and
administered IV. Tumor size was measured weekly by calliper using the π-based ellipsoid
volume formula (length × width × height × π/6) every 3−4 days, and the mice were also
weighed and examined at the same time to determine any signs of toxicity from the drug.
Group 1: 15 mg/kg of gemcitabine (Sigma), twice weekly dose.
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Group 2: 15 mg/kg of MM41, twice weekly dose.
Group 3: 10 mg/kg of CM03, twice weekly dose.
Group 4: 15 mg/kg of CM03, twice weekly dose.
Group 5: saline only, twice weekly dose.
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The xenograft data was analysed using Student’s t test (GraphPad Inc.). Responses were
considered to be significant for those with probabilities (p) less than 5% (p < 0.05). Mice
3
were culled if there was any sign of tumor ulceration, if tumor volume exceeded 1.5 cm , or if
a weight loss over 20% of initial body weight was observed. Samples of liver, kidney and
spleen from each animal in the CM03 (15 mg/kg) and untreated control groups were excised
at the end of the therapy experiment, then fixed in 10% neutral buffered formalin for at least
4
8 h prior to processing and paraffin embedding. Tissue slices were stained with
haematoxylin and eosin, and assessed visually for any obvious differences compared with
controls, indicative of toxicity and tissue damage. For the imaging study mice were
anesthetised using Isoflurane and imaged using the IVIS pre-clinical imaging system from
Perkin Elmer. Images were captured after 4 h following intravenous administration of CM03
using Cy5.5 excitation for 5 sec.
G12D/+
R172H/+
KPC mouse model. KPC (Pdx1-Cre; LSL-Kras
; LSL-Trp53
) mice were
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described previously. Mice on a mixed background were bred in-house and maintained in
conventional caging with environmental enrichment, access to standard chow and water ad
libitum. Genotyping was performed by Transnetyx (Cordoba, TN, USA). Mice were
monitored 3 times weekly and randomized to receive either 15 mg/kg of CM03, or saline
control IP, twice weekly when exhibiting symptoms of PDAC (swollen abdomen, loss of
body conditioning resembling cachexia, reduced mobility). Pancreatic malignancy was
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