Reprogramming the mechanism of action of chlorambucil by coupling to a G-quadruplex ligand

Article abstract of DOI:10.1021/ja5014344

The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.

Full text of DOI:10.1021/ja5014344

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Reprogramming the Mechanism of Action of Chlorambucil by
Coupling to a G‑Quadruplex Ligand
Marco Di Antonio,†,‡ Keith I. E. McLuckie,‡ and Shankar Balasubramanian*,†,‡,§
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
‡Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, U.K.
§School of Clinical Medicine, University of Cambridge, Cambridge CB2 0SP, U.K.
S
* Supporting Information
(intrastrand) or between the two opposite DNA strands
(interstrand).2 Interstrand cross-links (ICLs) are the most toxic
adducts generated by Chl, since they block DNA replication
and elicit DSB formation leading to growth inhibition in highly
proliferating cells, but accompanied by significant toxicity in
healthy cells that can cause secondary malignacies.3 Moreover,
Chl has poor DNA recognition properties that can cause off
target alkylation. Substantial improvements in the molecular
recognition properties have been reported by conjugating Chl
to the A-T specific minor groove binder Distamycin4 or to
highly sequence-specific DNA binding hairpin polyamides.5
The enhanced DNA recognition properties of these com-
pounds enables treatment at lower doses. However, the drug’s
mechanism of action is not altered in these analogues, and
some of the side effects associated with ICL mediated toxicity
persist.
ABSTRACT: The nitrogen mustard Chlorambucil (Chl)
generates covalent adducts with double-helical DNA and
inhibits cell proliferation. Among these adducts, inter-
strand cross-links (ICLs) are the most toxic, as they stall
replication by generating DNA double strand breaks
(DSBs). Conversely, intrastrand cross-links generated by
Chl are efficiently repaired by a dedicated Nucleotide
Excision Repair (NER) enzyme. We synthesized a novel
cross-linking agent that combines Chl with the G-
quadruplex (G4) ligand PDS (PDS-Chl). We demon-
strated that PDS-Chl alkylates G4 structures at low μM
concentrations, without reactivity toward double- or
single-stranded DNA. Since intramolecular G4s arise
from a single DNA strand, we reasoned that preferential
alkylation of such structures might prevent the generation
of ICLs, while favoring intrastrand cross-links. We
observed that PDS-Chl selectively impairs growth in cells
genetically deficient in NER, but did not show any
sensitivity to the repair gene BRCA2, involved in double-
stranded break repair. Our findings suggest that G4
targeting of this clinically important alkylating agent alters
the overall mechanism of action. These insights may
inspire new opportunities for intervention in diseases
specifically characterized by genetic impairment of NER,
such as skin and testicular cancers.
Herein, we describe an investigation of the DNA cross-
linking properties and the mechanism of action of a novel Chl
analogue in which the alkylator is coupled with the potent G4
ligand pyridostatin6 (PDS, Figure 1).
G4s are four-stranded nucleic acid secondary structures that
can exist within guanine-rich DNA and RNA sequences via
stacking of hydrogen-bonded G-tetrads.7 G4 nucleic acids are
the subject of widespread investigation as targets for small
molecule intervention in biological studies.8 We have
demonstrated that G4s exist in human cells and can be
recognized and trapped by the small molecule ligands PDS and
carboxy-PDS.9 Given that G4s are preferentially formed during
DNA replication,9a cross-linking of these structures may
improve the specificity of alkylating agents toward highly
proliferative cancer cells.10 Selective cross-linking of G4s within
double-helical DNA can be achieved in vitro by combining G4
ligands with different alkylating agents.11 However, the
therapeutic advantages offered by covalent G4 targeting have
yet to be explored. We aimed to investigate the cellular
responses stimulated by selective G4 alkylation.
he nitrogen mustard Chlorambucil (Chl; Figure 1) is a
Tdrug employed in the treatment of various types of
malignancies.1 Chl possess two electrophilic sites and generates
covalent adducts with double-helical DNA, especially via
reaction with N7 of guanines, either within the same strand
To address this aim, we synthesized a novel G4 alkylating
agent by combination of Chl with the potent G4 ligand PDS
(PDS-Chl). We previously described the synthesis of a PDS
analogue bearing an alkyne functional group (1) and
demonstrated that such chemical modification and its further
chemical functionalization do not impair the G4 recognition
Received: February 10, 2014
Published: April 3, 2014
Figure 1. PDS and Chl structures.
© 2014 American Chemical Society
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properties of the PDS scaffold.12 We took advantage of this to
generate a hybrid PDS-Chl by means of copper-catalyzed 1,3-
dipolar cycloaddition, reacting (1) with an azido Chl analogue
(2) (Scheme 1).
a
Scheme 1. Synthesis of PDS-Chl
Figure 2. G4-specific alkylation. PDS-Chl (0−5 μM) was incubated
with 0.2 μM oligonucleotides, for 1 h at 37 °C. Alkylated DNA
(ALKY) was separated from unreacted oligonucleotides on a 15%
denaturing PAGE. Oligonucleotides were detected by recording the 6-
FAM fluorescence signal (λex = 488 nm).
c-MYC, after 1 h incubation with PDS-Chl at 37 °C, by
nuclease digestion of the alkylated oligonucleotides to single
nucleobases that were purified and analyzed by LC-MS to
detect alkylated bases (SI). The MS fragmentation profile of
Chl adducts with nucleobases enabled the identification of the
alkylated sites after incubation of PDS-Chl with c-MYC.14 As
expected, PDS-Chl mainly reacts with water under these
conditions, as the bis-hydrated product was the most abundant
in the reaction mixture (Figure 3). The detected alkylated
nucleobases were the following: mono-adenine adducts at N1
and the exocyclic NH2, mono-guanine adducts at N7 and N1
(minor product), in addition to a bis-adduct with both adenine
and guanine (Figure 3).
Analysis of the relative base composition in the mixture
before and after treatment with different doses of PDS-Chl
(0.2−5 μM) confirmed alkylation sites. Incubation of c-MYC
with PDS-Chl confirmed dose-dependent alkylation of both
adenine and guanine (Figure S7). h-Telo showed alkylation of
adenine only; thus, the adenines in the loops of the telomeric
G4 are the only accessible sites for the alkylating agent (Figure
S8). This is in agreement with previous reports11c and is
consistent with the hydrogen-bonding of the guanine N7 lone
pair in G-tetrads leading to protection from alkylation.7
Importantly, no alkylation was observed upon incubation of
ds-DNA with up to 100 μM of PDS-Chl (Figure S9),
confirming a structure-specific reaction.
Finally, we sought to explore the therapeutic potential of
PDS-Chl within this overall strategy. We reasoned that selective
cross-linking of an intramolecular G4 structure would
predominantly cause intrastrand cross-linking. Intrastrand
cross-links are commonly formed in DNA after exposure to
UV light, as this triggers [2 + 2] cyclo-addition of adjacent
thymidine bases.15 Nature has evolved nucleotide excision
repair (NER) enzymes to overcome intrastrand cross-links and
repair T-T dimers.15 NER recognizes the damaged DNA
strand, excises it, and replaces it with freshly synthesized
DNA.15 Because of its importance to DNA repair after UV
exposure, deficiency in NER is responsible for several
photosensitivity diseases and is a significant cancer risk factor
and a common genetic signature of skin cancers.15 NER-
deficient cells are sensitized to the chemotherapeutic drug
cisplatin in which the cis-geometry of the drug imposes the
formation of covalent bonds with adjacent purine bases to
produce mainly 1,2- or 1,3-intrastrand cross-links.16 Similarly,
we postulated that our G4-specific alkylator PDS-Chl might
show selectivity toward NER-deficient cells by preferentially
inducing intrastrand cross-links via G4 structural recognition.
a
Conditions: (i) CuSO4·5H2O, sodium ascorbate, H2O/t-BuOH
(71%); (ii) CH2Cl2, TFA (99%).
Compound (2) was prepared by chlorination of Chl using
thionyl chloride followed by coupling with 2-azidoethanamine
(see Supporting Information (SI)). Removal of the Boc groups
using TFA followed by HPLC purification afforded PDS-Chl in
good yields (Scheme 1).
We investigated the reactivity of PDS-Chl toward different
DNA structures using synthetic oligonucleotides fluorescently
labeled at both the 5′- and 3′-ends, with 6-FAM and TAMRA,
respectively. The tested oligonucleotides included the G4-
forming sequences (c-MYC and h-Telo), a double-stranded
DNA (ds-DNA) and a mutated c-MYC sequence (c-MYC-
MUT) no longer able to fold into a G4 conformation (SI).
Oligonucleotides were annealed at a final concentration of 200
nM in the presence of 100 mM KCl, buffered at pH 7.4 to
promote G4 formation. Annealed oligonucleotides were then
incubated with increasing doses of PDS-Chl (0.2−5 μM) for 1
h at 37 °C. The alkylated adducts were separated by
polyacrylamide gel electrophoresis (PAGE) under denaturing
conditions (8 M urea) and detected by fluorescence (6-FAM).
We observed formation of a new band only when PDS-Chl was
incubated with G4 forming sequences, with no alkylation
observed for ds-DNA and the mutated c-MYC sequence
(Figure 2).
The faster migrating band observed for the alkylated adduct
suggests the formation of a covalently locked secondary
structure that cannot be denatured. Compressed DNA
secondary structures occupy a smaller hydrodynamic volume
compared to their single-stranded counterparts, causing bands
to migrate faster than expected from their molecular weight.13
Imaging the same gel by exciting the second fluorescent tag
(TAMRA, λex = 583 nm) attached to the opposite 3′-end of the
oligonucleotides gave identical banding, which indicates that
strand cleavage is not the cause of the new band (Figure S6).
We explored the alkylation target sites of unlabeled h-Telo and
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Figure 3. Alkylated adducts with adenine and guanine detected by LC-MS analysis of nuclease digested c-MYC incubated 1 h with PDS-Chl.
To test this hypothesis, we applied PDS-Chl to SV-40 mutated
human fibroblasts genetically deficient in the expression of a
vital NER component XPA (XPA−, GM04312D) and
compared this to the wild type (WT) counterpart that
expresses XPA (XPA+, GM00637).17 These cells have been
widely used to characterize NER deficient sensitization to
drugs, since they comprise an identical cell line pair derived
from the same tissue of different patients (respectively, with
and without XPA deficiency) and differ only for the expression
of XPA.18 Using an impedance-based continuous cell-
monitoring approach (SI), we examined, in real time, the
effect on cell growth of PDS-Chl, PDS, and Chl to these cell
lines. We found that PDS-Chl impaired the cell growth of the
XPA− cells but showed no activity against XPA+ fibroblasts up
to 100 μM, the highest testable dose compatible with the
solubility of the compound (Table 1). Thus, the absence of
GM04312D cells for doses as high as 100 μM (GI50 > 100 μM)
when XPA activity is restored, further supporting the
hypothesis that the activity measured is NER-dependent. We
next investigated whether the cross-linking properties of PDS-
Chl were responsible for the growth inhibition activity
measured in XPA− cells. We generated the hydrolyzed
analogue of PDS-Chl by incubation of the compound in
water at 37 °C overnight; full conversion was ensured by HPLC
analysis (Figure S10). The hydrolyzed PDS-Chl derivative did
not impair XPA+ and XPA− growth for doses up to 100 μM,
supporting the hypothesis that the cross-linking activity of
PDS-Chl is essential to impair growth in XPA− cells. Given
these experiments suggest a preference for intrastrand cross-
linking by PDS-Chl, we next investigated whether the agent
generates ICLs. Since Chl generates DNA DSBs via ICLs, we
postulated that cells deficient in BRCA2 expression would be
sensitized to this compound, given the crucial relevance of this
gene in the DSB repair machinery, homologous recombina-
tion.8c We applied PDS-Chl, PDS, and Chl to HCT116 colon
carcinoma cells carrying either the WT BRCA2+/+ gene, or
where both copies have been removed (BRCA2−/−). We found
that Chl is 40-fold more active in BRCA2−/− cell lines (GI50
1.25 0.7 μM) as compared to BRCA2+/+ WT cells (GI50 50
2 μM). PDS is also more active against BRCA2−/− cells, as
previously reported.8c Importantly, we found that PDS-Chl
shows negligible activity against both BRCA2 positive and
negative cells up to a 100 μM dose (GI50 > 100 μM),
supporting the hypothesis that ICLs do not contribute to the
mechanism of action of this compound.
Table 1. GI50 Values of PDS, Chl, and PDS-Chl Measured in
XPA+ and XPA− Human Fibroblasts
GI50(XPA+) (μM)
GI50 (XPA−) (μM)
PDS
1.7 0.6
3.7 1.8
>100
0.8 0.1
Chl
22
21
4
5
PDS-Chl
NER activity sensitizes the cells to PDS-Chl, which become >5-
fold more sensitive to this compound compared to their WT
counterpart that expresses NER enzymes. By analogy with the
preferential growth inhibition exhibited by PDS-Chl for XPA−
cells, cisplatin showed a GI50 of ∼44 μM for XPA+ and ∼4.1
μM for the XPA−, therefore also showing a preference (10-
fold) for NER deficient cells.18 Conversely, both PDS and Chl
alone showed no preference for XPA− cells, and in fact, Chl
actually showed a preference for XPA+ cells (Table 1).
To rule out the possibility that differences in cell permeability
may bias the outcomes for cell lines derived from different
patients, we further evaluated PDS-Chl in GM04312D cells that
were supplemented with exogenous XPA cDNA, which restores
XPA expression and activity in the same cell line. We found that
PDS-Chl is no longer able to induce any growth inhibition in
Our findings suggest that tethering the nitrogen mustard Chl
to the G4 ligand PDS reprograms the mechanism of action of
this drug, preventing the generation of ICLs by selective cross-
linking of single-stranded DNA G4s. This alters the mechanism
of action of the nitrogen mustard moiety in PDS-Chl, resulting
in an agent that preferentially generates intrastrand cross-links
via selective G4 recognition, thus sensitizing NER-deficient
cells lines to this G4-selective alkylating agent.
Reprogramming a DNA cross-linker by means of G4-
directed reactivity may be a promising strategy to consider
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Mattinen, J.; Hakala, K.; Vilpo, J.; Hovinen, J. Chem. Res. Toxicol. 2003,
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for diseases where genetic impairment of NER is relevant, such
as skin, testicular, and drug-resistant cancers.
ASSOCIATED CONTENT
* Supporting Information
■
S
All experimental details and supplementary tables and figures
cited above. This material is available free of charge via the
Internet at http://pubs.acs.org.
(18) Stevens, E. V.; Nishizuka, S.; Antony, S.; Reimers, M.; Varma, S.;
Young, L.; Munson, P. J.; Weinstein, J. N.; Kohn, E. C.; Pommier, Y.
Mol. Cancer Ther. 2008, 7, 10.
AUTHOR INFORMATION
Corresponding Author
sb10031@cam.ac.uk
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Cancer Research U.K. for program funding and core
support. We thank Dr. Raphael Rodriguez and Dr. David
Tannahill for insightful discussions, Dr. Mike Booth for HPLC
support, Dr. Dan Le and Dr. Chris Lowe for proofreading of
the manuscript.
■
̈
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