Efficient Inhibition of Telomerase by Nickel–Salophen Complexes

Article abstract of DOI:10.1002/cmdc.201600171

Four nickel(II)–salophen complexes containing alkyl-imidazolium chains connected at the ortho or meta positions were prepared: N,N′-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (1), N,N′-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (2), N,N′-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N′-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (4). They protect G-quadruplex DNA (G₄-DNA) against thermal denaturation and show K_Avalues in the range of 7.4×10⁵to 4×10⁷m^?1for G₄-DNA models. Complex 4 exhibits an IC₅₀value of 70 nm for telomerase inhibition.

Full text of DOI:10.1002/cmdc.201600171

DOI: 10.1002/cmdc.201600171
Communications
Efficient Inhibition of Telomerase by Nickel–Salophen
Complexes
Laureline Lecarme+,[a] Enora Prado+,[a] Aurore De Rache+,[b] Marie-Laure Nicolau-Travers,[c]
Gis›le Gellon,[a] JØrôme Dejeu,[a] Thomas Lavergne,[a] HØl›ne Jamet,[a] Dennis Gomez,[c] Jean-
Louis Mergny,[b] Eric Defrancq,[a] Olivier Jarjayes,[a] and Fabrice Thomas*[a]
Four nickel(II)–salophen complexes containing alkyl-imidazoli-
um chains connected at the ortho or meta positions were pre-
pared: N,N’-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzyli-
deneamino)phenylenediamine (1), N,N’-bis(2-hydroxy-3-methyl-
fied, and G4-DNA has been associated with genomic and epi-
genetic instability as well as diseases and cancer phenotypes.[6]
Only recently has their presence in vivo been demonstrated.[7]
Perhaps the most documented biological function of G-quad-
ruplexes concerns telomere homeostasis.[8] Telomeres are spe-
cialized regions of 10–15 kilobases (at birth) for humans, which
are located at the end of chromosomes.[9] While they are
mainly composed of double-stranded DNA, there is a 3’-over-
hanging, single-stranded region of 100–200 nucleotides at the
terminus. This overhang contains several repeats of G3T2A se-
quences, which has the potential to fold into G-quadruplex-
es.[10] Successive DNA replication steps result in erosion of the
telomeres. Once one or more telomere(s) reaches a critical
length the cell no longer divides further and enters into senes-
cence. Telomerase adds copies of telomeric repeats at the
chromosomal terminus.[2] It is transcriptionally repressed in
normal cells, but is overexpressed in more than 85% of tumor
cells, wherein it prevents senescence.[11] It has been shown
that the presence of G-quadruplexes in the 3’-overhanging
region could inhibit telomerase activity.[2] There is thus a dual
goal for the development of selective G-quadruplex binders:
Firstly, they are potential anticancer drugs, in the sense that
they may restore the mortality of tumor cells;[12] secondly, they
are important tools for gaining deeper insight into the roles of
G-quadruplexes in the genomes.
3H-imidazol-1-iumbenzylideneamino)phenylenediamine
(2),
N,N’-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneami-
no)methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N’-
bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)-
methyl-3H-imidazol-1-iumphenylenediamine (4). They protect
G-quadruplex DNA (G4-DNA) against thermal denaturation and
show KA values in the range of 7.4”105 to 4”107 mÀ1 for G4-
DNA models. Complex 4 exhibits an IC50 value of 70 nm for te-
lomerase inhibition.
Since the seminal discovery of the antiparallel double helical
structure of DNA (B-form DNA) it has been shown that this bio-
logical macromolecule can adopt a wide variety of alternative
topologies such as A-DNA, Z-DNA, H-DNA and cruciform DNA.
Four-stranded G-quadruplex DNA (G4-DNA) has been the sub-
ject of intensive investigations over the past few decades.[1]
This topology requires guanine-rich sequences, such as those
found in the telomeric region of eukaryotic chromosomes, as
well as in promoters of some oncogenes.[2,3] G-quadruplexes
are structured into stacked planar G-quartets, each comprising
four guanines held together by Hoogsteen hydrogen bonds.[4]
A physiologically relevant cation (K+ or Na+) is sandwiched be-
tween two consecutive quartets and stabilizes the edifice. The
Several compounds that efficiently target G-quadruplexes
have emerged in the literature over the last decade.[13] The
most representative inorganic compounds that interact with
G4-DNA are metal complexes of porphyrin[14] and salophen[15] li-
gands, provided that they are functionalized by cationic arms.
We recently showed that nickel(II)–salophen complexes func-
tionalized at both para positions by alkylmethylimidazolium
groups[15e] bind strongly and specifically to G-quadruplexes.
They protect G-quadruplex DNA against thermal denaturation
and inhibit telomerase at the sub-micromolar level (IC50 =
700 nm).[15e] Furthermore, the alkylmethylimidazolium groups
confer high water solubility to the compounds. In this study
we investigated four ortho- and meta-functionalized complexes
(1–4, Figure 1). In particular, we found that one of them (com-
plex 4) exhibits a remarkable IC50 value of 70 nm for telomer-
ase inhibition.
biological relevance of G4-DNA has been established:[5]
A
number of natural G4-DNA binding proteins have been identi-
[a] Dr. L. Lecarme,+ Dr. E. Prado,+ G. Gellon, Dr. J. Dejeu, Dr. T. Lavergne,
Dr. H. Jamet, Prof. E. Defrancq, Dr. O. Jarjayes, Prof. F. Thomas
UniversitØ Grenoble Alpes, DØpartement de Chimie MolØculaire,
UMR-5250, 38041 Grenoble Cedex 9 (France)
Fax: (+33)476-51-4836
E-mail: Fabrice.Thomas@ujf-grenoble.fr
[b] Dr. A. De Rache,+ J.-L. Mergny
ARNA laboratory, Inserm U1212, CNRS UMR 5320, Institut EuropØen de
Chimie et Biologie IECB – UniversitØ de Bordeaux, 2 rue Robert Escarpit,
33607 Pessac (France)
[c] M.-L. Nicolau-Travers, Dr. D. Gomez
Institut de Pharmacologie et de Biologie Structurale,
205 Route de Narbonne, 31077 Toulouse Cedex 4 (France)
[+] These authors contributed equally to this work.
Complexes 1–4 were prepared by condensation of the ap-
propriate phenylenediamine[15e] with one equivalent of Ni(OA-
c)2·4H2O and two equivalents of either 3-(4-formyl-3-hydroxy-
benzyl)-1-methylimidazolium chloride (for the synthesis of
1 and 3), or 3-(3-formyl-2-hydroxybenzyl)-1-methylimidazolium
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600171.
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Communications
two cationic chains either at the meta or ortho positions (com-
pounds 1 and 2, respectively), it is clear that functionalization
at the meta position leads to more efficient G-quadruplex
binders (difference in DT1/2 of 11 8C for F21T). Thus, the connec-
tivity appears to be a crucial determinant for the affinity of
compounds featuring two cationic chains. The situation is
completely different for compounds with a third cationic chain
(3 and 4): The stabilization was already large for 1 (two side
chains) and could not be improved further by adding the third
arm (as in 3). The DT1/2 value was comparatively lower for 2,
and incorporation of the third arm (as in 4) was found to dra-
matically improve the thermal stabilization of the G-quadru-
plex. We further investigated the structural selectivity of 1–4
by comparing the DT1/2 values for various quadruplex topolo-
gies (Figure 2).[18] Herein we limit our discussion to 3 and 4,
which harbor three cationic arms. Using the F21CTAT and
FBom17T sequences (known as displaying an anti-parallel fold)
we found the DT1/2 value for 3 to be similar or slightly higher
than for 4 (differences of 0–38C). In contrast, the DT1/2 values
determined for the FTRF2RT, F25CebT, and FmycT sequences
(parallel folding) are much larger for 4 than for 3 (differences
of 4–108C). A similar trend was observed with the RNA se-
quence F21RT (parallel human RNA telomere). This indicates
that discrimination between folding types might be achieved
with this family of complexes.
Figure 1. Structures of complexes 1–4.
bromide (for the synthesis of 2 and 4), under basic conditions.
3-(4-Formyl-3-hydroxybenzyl)-1-methylimidazolium
chloride
was prepared from 4-(hydroxymethyl)-2-hydroxybenzalde-
hyde[16] after chlorination of the benzylic alcohol with thionyl
chloride, followed by nucleophilic substitution with the N-
methylimidazole. 3-(3-Formyl-2-hydroxybenzyl)-1-methylimida-
zolium bromide was obtained in a four-step synthetic proce-
dure from 2-hydroxyisophthalaldehyde[17] (Supporting Informa-
tion).
To evaluate the potential of compounds 1–4 to stabilize G-
quadruplexes, a FRET–melting assay was performed by using
the F21T (human telomeric) sequence in K+ medium. All com-
pounds efficiently stabilize the quadruplex against thermal de-
naturation, with DT1/2 values of 20, 9, 19 and 198C in the pres-
ence of compounds 1, 2, 3 and 4, respectively, at 0.5 mm
(Figure 2). The DT1/2 values are lower in Na+ medium (Fig-
ures S1–S2), similar to the results from para compounds.[15e]
Two experiments show that the compounds are rather selec-
tive for G-quadruplexes: 1) addition of a duplex DNA competi-
tor (10 mm) did not abolish stabilization (Figures S1–S2), and
2) DT1/2 values for the control duplex (FdxT) are lower than for
G-quadruplexes (Figure 2). For complexes that feature only
The association constants (KA) of 1–4 for quadruplex models
were determined by surface plasmon resonance (SPR). We
used a method based on the concept of template-assembled
synthetic G-quadruplexes.[19] Basically, three oligonucleotides
were assembled on a template that constrains them to adopt
different topologies: The G-quadruplex model A, formed by
four interacting parallel strands d(TTAGGGT); an intramolecular
G-quadruplex model B d(GGG(TTAGGG)3TT), which mimics the
human telomeric sequence; and a hairpin duplex C. As shown
in Table 1, all the complexes are selective for G-quadruplexes.
They exhibit a slight preference for the G-quadruplex A, where
the top quartet is more accessible (absence of loop). For in-
stance, a remarkably large KA value of 4Æ1”107 mÀ1 was deter-
mined for 4 in interaction with A. The results obtained on
model B, which is likely to be more biologically relevant,
merits several comments. The KA values are about 10-fold
larger for ortho-functionalized compounds (5.8”106 mÀ1 and
7.8”106 mÀ1 for 2 and 4, respectively) in comparison with
those functionalized at the meta position (7.4”105 mÀ1 and
8.8”105 mÀ1 for 1 and 3, respectively). The kinetic data
(Table S1), however, point to very similar rate constants for the
Table 1. Association constants (KA mÀ1) of 1–4 in interaction with G-quad-
ruplex and duplex DNA structures.
Complex
Intermolecular
Intramolecular
Hairpin
quadruplex A
quadruplex B
duplex C
1
2
3
4
4.1Æ0.4”106
6.3Æ0.7”106
2.7Æ0.4”106
4Æ1”107
7.4Æ0.8”105
5.8Æ0.7”106
8.8Æ0.8”105
7.8Æ0.8”106
1.5Æ0.2”105
7.6Æ1”105
Figure 2. Structural selectivity profile. The FRET thermal stabilization induced
by compounds 1–4 at 0.5 mm is represented for structures adopted by eight
oligonucleotide sequences (all quadruplexes except FdxT). Detailed informa-
tion related to the folding of the sequences is provided in ref. [18].
3.5Æ0.5”105
1.7Æ0.2”106
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association of 1–4 with quadruplex B (kon in the range of 2.4–
3.5”105 mÀ1 À1). This attests that the approach toward the
mRNA through a cap-independent mechanism was shown to
be altered by G-quadruplex structures. Although the G-quadru-
plex present on the IRES region was described as necessary for
IRES activity,[23] Cammas et al. have demonstrated that the sta-
bilization of the G-quadruplex within the VEGF IRES provokes
a marked inhibition of IRES-mediated translation of VEGF.[24]
Based on these data we investigated the interaction of the
most active compound 4 with G4-RNA structure in cellulo. We
used a dual luciferase reporter, in which the VEGF IRES region
has been cloned between the Renilla (RLuc) and firefly (FLuc)
luciferase genes. In this construct, FLuc translation is under the
control of the VEGF IRES. In this way, FLuc activity reflects IRES-
dependent translation. The FLuc activity is normalized against
RLuc activity, which results from a cap-dependent translation
mechanism and reflects the amount of mRNA present in the
cell. As a control, we used a modified form of the VEGF IRES
region, in which the G-stretches into the quadruplex-forming
sequence are mutated. As shown in Figure 4, 24 h treatment
s
upper tetrad by the complexes is neither limited by the con-
nectivity nor the number of side chains. In fact, the most nota-
ble differences between the complexes are the dissociation
rate constants: koff values of 0.30Æ0.05 and 0.18Æ0.03 were
found for meta-functionalized compounds 1 and 3, respective-
ly, whereas ortho compounds 2 and 4 showed koff values of
0.038Æ0.004 and 0.033Æ0.005 sÀ1, respectively. This large dif-
ference of koff clearly demonstrates that an ortho positioning of
the side chains leads to higher binding constants with B,
mainly through significant slowing of the dissociation process.
Finally, kinetic data with the hairpin duplex C shows that the
association is slower and dissociation faster (Table S2) with this
DNA topology.
The capacity of complexes 1–4 to inhibit the extension of te-
lomeric sequences by telomerase was evaluated by TRAP and
TRAP-G4 assays (Figure 3 and Figures S16–S18). In contrast to
Figure 3. Inhibition of telomerase activity by complex 4: A) TRAP-G4 assay
and B) TRAP assay. Increasing concentrations of complex (0, 1, 3, 10, 30, 100,
300, 1000 nm for lanes 1–8, respectively) were added to 100 ng telomerase
extract. Positions of TS, TS–G4, and internal control ITAS PCR products are in-
dicated in panel A). The IC50 values, corresponding to the concentration re-
quired to inhibit 50% of the amplification of the TS-G4 fragment, and selec-
tivity (in brackets) of the complexes are: 1: 2 mm (3.5); 2: 2 mm (5); 3: 0.7 mm
(10); 4: 0.07 mm (10).
Figure 4. In cellulo inhibition of VEGF mRNA translation by complex 4. IRES
activity was determined by transfecting HeLa cells with wild-type VEGF G-
quadruplex or VEGF G-quadruplex mutated bicistronic plasmids, and treat-
ment with complex 4 (40 nm). Error bars correspond to standard deviations
of the mean of four independent experiments with duplicates.
a more classical TRAP assay, the TRAP-G4 assay allows discrimi-
nation between quadruplex-based and catalytic inhibition of
telomerase. Furthermore, it provides access to the selectivity of
compounds for the quadruplex with respect to the duplex.[20]
The TRAP-G4 assay gave IC50 values of 2, 2, and 0.7 mm for 1–3,
respectively, indicating that these compounds efficiently inhibit
telomerase in vitro. Remarkably, complex 4 exhibits an IC50
value of 70 nm. Although we cannot exclude the possibility
that the compounds interfere with the PCR step, leading to an
underestimation of the IC50 value,[21] this value is the lowest re-
ported thus far for salophen complexes.[15] To compare 4 with
previously described compounds, we also performed a conven-
tional TRAP assay. In this assay the IC50 was found to be 30 nm,
approaching that of reference compounds such as telomesta-
tin.[22] From these experiments it is also evident that 4 is very
specific for quadruplexes, with a selectivity that reaches 10 in
the TRAP-G4 assay and 25 in the TRAP assay.
with complex 4 results in an 18% inhibition of the cap-inde-
pendent translation, in comparison with the untreated cells.
Furthermore, the mutations in the G-rich sequences decrease
the inhibitory effect. These results suggest that the inhibition
of cap-independent translation of VEGF IRES by complex 4 is
partially mediated by the G-quadruplex-forming sequences
present on VEGF IRES mRNA. They also confirm that com-
pound 4 is substantially internalized in the cells.
To gain insight into the binding mode of 4, we undertook
a docking study with the structure reported for the parallel
human telomere (PDB ID: 1KF1;[25] see the Supporting Informa-
tion for protocol details).[15e,26] Compound 4 is positioned over
the tetrad with significant overlap between the aromatic rings
of the complex and three guanines (stabilizing p-stacking in-
teractions; Figure S19b). A single cationic arm (the one con-
nected to the bridge) is inserted into one groove, while the pe-
ripheral cationic chains interact primarily with the loops. This
In addition to the inhibition of telomerase by the stabiliza-
tion of G-quadruplexes formed by telomeric sequences, the
stabilization of G-quadruplexes by small compounds also af-
fects other nucleic acid transactions such as transcription and
translation.[5] In humans, initiation of the translation of VEGF
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stands out from the others, with a remarkable IC50 value for te-
lomerase inhibition of 70 nm. These results, obtained with
polycationic nickel–salophen complexes featuring unprece-
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Acknowledgements
The authors acknowledge Prof. P. LabbØ (UniversitØ Grenoble
Alpes, DØpartement de Chimie MolØculaire, UMR-5250) and Dr. A.
van der Heyden (UniversitØ Grenoble Alpes, DØpartement de
Chimie MolØculaire, UMR-5250) for assistance and fruitful discus-
sions. This work was supported by grants from ANR QUARPDIEMS
(ANR-12-BSV8-0008-04), Labex ARCANE (ANR-11-LABX-0003-01),
PRES Toulouse, RØgion Mydi-PyrØnØes, and RØgion Aquitaine. The
Nanobio-ICMG platform (FR 2607) is acknowledged for the syn-
thesis and purification of oligonucleotides and SPR facilities.
[17] P.-H. Lin, M. Lecl›re, J. Long, T. J. Burchell, I. Korobkov, R. ClØrac, M. Mur-
ugesu, Dalton Trans. 2010, 39, 5698.
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Keywords: bioinorganic chemistry · DNA · G-quadruplexes ·
nickel · salophen
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Published online on May 11, 2016
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