A Pt(ii)-Dip complex stabilizes parallel c-myc G-quadruplex

Article abstract of DOI:10.1039/c3cc40868j

A new G-quadruplex (GQ) stabilizer, [Pt(Dip)₂](PF ₆)₂ (Dip: 4,7-diphenyl-1,10-phenanthroline), is prepared by the microwave irradiation method. The complex can highly stabilize G-quadruplex, but has negligible interactions with duplex DNA. Aromatic anchors on the polypyridyl ligands bestow the stabilizer with a high binding preference towards parallel GQ.

Full text of DOI:10.1039/c3cc40868j

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A Pt(II)–Dip complex stabilizes parallel c-myc
G-quadruplex†
Cite this: Chem. Commun., 2013,
4
9, 4758
Jintao Wang, Kaihui Lu, Shuguang Xuan, Zaozhen Toh, Dawei Zhang* and
Fangwei Shao*
Received 1st February 2013,
Accepted 2nd April 2013
DOI: 10.1039/c3cc40868j
www.rsc.org/chemcomm
8
A new G-quadruplex (GQ) stabilizer, [Pt(Dip)
2
](PF
6
)
2
(Dip: 4,7-diphenyl- have been reported as G-quadruplex stabilizers. Pt(II) complexes
1
,10-phenanthroline), is prepared by the microwave irradiation can achieve a relatively high binding affinity to G-quadruplexes.
method. The complex can highly stabilize G-quadruplex, but has Nevertheless majority of the complexes show poor selectivity over
negligible interactions with duplex DNA. Aromatic anchors on the duplex DNA. The square shape di-ligand coordination to the Pt(II)
polypyridyl ligands bestow the stabilizer with a high binding cation provides the necessary planar plane for good p–p stacking with
preference towards parallel GQ.
G-quartets, but at the same time cannot circumvent the intercalation
to the base-pair stacking or minor groove binding to duplex DNA.
G-quadruplex (GQ) is a family of four-stranded guanine-rich motifs Furthermore, distinction among GQ topology remains a major
containing a core stack of planar guanine quartets wrapped by loops challenge for both inorganic complexes and organic stabilizers.
1
with various lengths and 3D orientations. Bioinformatics shows the
A porphyrin GQ binder, TMPyP4 (Fig. 1), which extends the
existence of potential GQ folding sequences (PGS) in many biological aromatic plane with four cationic pyridine anchors, achieves excellent
2
significant genomic regions. Human telomeres comprise large parallel GQ targeting and also down-regulates c-myc and inhibits the
9
numbers of such sequences at the chromosome ends, which protect proliferation of tumour cells. Crystal and solution structures show
3
the telomere from degradation and genomic instability. Besides, that the specific interactions between the side chains and GQ grooves
G-quadruplexes are found in the promoters of a wide range of genes make a pivotal contribution to the stabilization of parallel c-myc
10
that are important in cellular signalling pathways and are represen- quadruplex. Herein in order to improve the selectivity of the Pt(II)
tatives of all six hallmarks of cancer (for example c-myc, c-kit, and stabilizer not only to global G-quadruplexes, but among GQ poly-
4
bcl-2). The biological and therapeutic significance of G-quadruplexes morphism, we designed and prepared a Pt(II) complex (1) (Fig. 1) with
is well appreciated and the use of small molecules that promote the two Dip (4,7-diphenyl-1,10-phenanthroline) ligands. Firstly, complex 1
2
+
formation of and/or stabilize G-quadruplex structures has become an contains the [Pt(phen)
attractive approach towards anticancer drug discovery.
2
]
coordination core to provide aromatic
5
electron conjugation with suitable size for good p–p stacking on
A good G-quadruplex stabilizer should show excellent selectivity G-quartets and hence to achieve good stabilization to the G-quartet
over double-stranded DNA, since majority of genomic DNA exists in core, which constantly exists in all G-quadruplex structures. Secondly,
B-form double helical structures. Furthermore, G-quadruplexes are four phenyl groups might endow complex 1 with selectivity to
6
rich in sequence-dependent structural polymorphism. It is often topological GQ structures by interacting with the unique groove
observed that the same sequence can form multiple topological and loop arrangements in certain GQ topology, but at the same
structures under different buffer conditions. Hence good selectivity time, prevent the intercalation/groove binding to duplex DNA. The
2+
2
among GQ topological structures is necessary for stabilizer molecules neutral anchors in [Pt(Dip) ] can avoid the electrostatic interactions
to achieve disease specificity on therapeutic effects and to be utilized with the phosphate backbone in duplex DNA, which is presumably
as personal drugs. To date, several hundreds of small molecules that the reason for the nonspecific binding affinity of TMPyP4 to duplex
stabilize G-quadruplexes have been described in the literature and
their interactions with G-quadruplexes have been extensively
7
explored. Metal complexes, especially platinum complexes,
Division of Chemistry and Biological Chemistry, Nanyang Technological University,
2
1 Nanyang Link, 637371, Singapore. E-mail: fwshao@ntu.edu.sg,
zhangdw@ntu.edu.sg; Fax: +65 6791-1961; Tel: +65 6592-2511
Electronic supplementary information (ESI) available: Synthesis of complex 1,
†
1
H NMR spectra, CD spectra, G4-FID spectra, UV/Vis titration, PCR stop assay,
molecular docking and MTT assay. See DOI: 10.1039/c3cc40868j
Fig. 1 Structures of Pt(II) complexes and TMPyP4 as GQ stabilizers.
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Table 1 Pt(II) stabilizers increase melting temperatures (T
m
) of DNA structures
a
DT
m
(1C)
Complexes
c-myc
bcl-2
HT
ds26
1
2
18.9(ꢀ0.7)
14.5(ꢀ0.6)
9.5(ꢀ0.3)
6.3(ꢀ0.4)
8.0(ꢀ0.2)
ꢂ0.1(ꢀ0.1)
4.8(ꢀ0.2)
16.0(ꢀ0.2)
a
DT
m
= T
m
(5 mM DNA + 5 mM complex) ꢂ T
m
(5 mM DNA).
11
Fig. 2 Absorption and induced CD spectra of 1 in the presence of c-myc, bcl-2,
HT and ds26. [1] = 20 mM and [DNA] = 40 mM.
DNA via the cationic pyridine rings. Complex 2 with the same
aromatic planar core as 1, but no anchors, is also prepared as a
reference molecule.
12
Complex 1 was synthesized by the microwave method under high DNA (Fig. S4c, ESI†). The unique enhancement of MCLT absorption
temperature and pressure, since the conventional synthetic method, suggests that, besides the usual p–p stacking mode acquired by the
which was used to prepare complex 2 by simply incubating planar Pt(II)–polypyridyl complexes, Pt(Dip)
Pt(Phen)Cl and Phen in boiling water, cannot work for the synthesis native interactions with GQ c-myc, presumably from the contribu-
of 1, due to the low water solubility of Pt(Dip)Cl and the Dip ligand. tions from the interactions between the four phenyl anchors and GQ
More remarkably, the reaction time was shortened from overnight to groove/loops. Furthermore, in a polymerase chain reaction stop
2
may also harness alter-
2
2
16
less than 30 minutes and this is the first example of using microwave assay (PCR-stop), which demonstrates the inhibitory ability of
irradiation to assist the synthesis of the Pt(II)–polypyridinyl complex. GQ stabilizers on the replication of a template containing a G-rich
Thermal stabilization of G-quadruplexes in the presence of com- c-myc sequence, complex 1 exhibits a complete inhibition of c-myc
plexes 1 and 2 were studied by performing CD melting experiments. template extension at 15 mM, while no apparent polymerization
As shown in Table 1, complex 1 shows stabilizing effects only to GQ arrest is observed with 30 mM of complex 2 (Fig. S7, ESI†).
structures, with the highest T
case of GQ c-myc. No T enhancement of duplex DNA by complex 1 complex 1, CD spectra were used to observe the structural alterna-
is observed. Whereas, comparable T elevations in telomeric GQ tion of both DNA and the complex upon interaction with each other.
HT) and duplex DNA (ds26) are observed in the case of complex 2. The characteristic CD peak of GQ c-myc at 260 nm is enhanced
m
elevation, 18.9 1C, obtained in the
To better understand the origin of the topological selectivity of
m
m
(
This selectivity of complex 1 is further confirmed by G-quadruplex upon the binding of complex 1, but not in the case of binding of 2
fluorescent intercalator displacement assay (G4-FID) and competitive (Fig. S3a, ESI†). Fig. 2b shows the induced CD spectra (ICD) in the
13,14 G4
dialysis.
DC50 of 1 from G4-FID (this concentration of 1 is MLCT region (310–500 nm) of complex 1. Since absorption of DNA
needed to displace 50% thiazole orange (TO) from GQ c-myc) is in this region can be neglected, ICD spectra represent only the
ds
0
.44 mM, while DC50 cannot be achieved even with the addition of conformational changes of the complexes upon binding to various
0 fold excess of the complex (Fig. S5, ESI†). The efficient TO- DNA structures. In the presence of GQ c-myc, 1 exhibits a strong
displacement implies a stable p–p stacking between G-quartets and negative peak at 325 nm and a moderate positive peak at 425 nm,
, but not between duplex DNA and 1, since majority of TO molecules whereas both HT21 and ds26 induce a strong and a weak positive
1
1
interact with GQ and duplex DNA via p–p stacking. In the competitive peak at 425 nm and 345 nm, respectively. Upon addition of GQ
dialysis (Fig. S6, ESI†), 6.1- and 29.0-folds higher in bound concen- bcl-2, 1 generates a weak negative peak around 420 nm. The ICD
tration of complex 1 is observed on GQ c-myc than on duplex and spectra suggest that the interaction modes between 1 and GQ c-myc
single-stranded DNA, respectively. Hence, complex 1 shows excellent are distinct to those between 1 and GQ HT, bcl-2 or duplex DNA.
stabilizing selectivity to GQ structures over duplex DNA, while the The strong negative ICD in the Soret region indicates that p–p end-
reference complex 2 without the phenyl anchors can achieve only stacking and/or intercalating interactions could be the dominant
moderate selectivity between GQ and duplex (Fig. S2 and S6, ESI†).
interaction mode(s) and the reason for high affinity/selectivity
17
More remarkably, 1 exhibits enhanced topological selectivity to between 1 and GQ c-myc. In contrast, GQ HT and duplex DNA
GQ c-myc over HT and bcl-2 G-quadruplexes, compared to the can only induce positive ICD, indicating no stable stacking/inter-
reference molecule 2. In the presence of 1, the Tm of GQ c-myc is calating modes, but the external binding between the two structures
18
9.4 1C and 12.6 1C higher than those of GQ bcl-2 and HT, which and 1. Since intercalation to duplex DNA is achieved by 2 inducing
indicates the strong preference of 1 to stabilize GQ c-myc over the negative ICD spectra (Fig. S3b, ESI†), the four phenyl anchors on 1
other two GQ topology. A similar trend is absent in the case of 2, should be the cause for preventing 1 from stabilizing GQ HT and
which induces comparable enhancements in the T
m
of GQ c-myc duplex DNA via strong stacking/intercalating modes.
Molecular docking of complex 1 onto several GQ topological struc-
and bcl-2, though a moderate selectivity over HT is observed for 2. In
15
the DNA titration experiments (Fig. S4a, ESI†), an apparent hyper- tures, c-myc (PDB code: 1XAV), HT (PDB code: 143D) and bcl-2 (PDB
chromism of the MCLT band of complex 1 is observed upon the code: 2F8U), provides further explanation for the topological selecti-
addition of GQ c-myc and yields a K as high as 4.01(ꢀ0.6) ꢁ vity of complex 1. GQ c-myc folds into parallel GQ topology, which
B
6
ꢂ1
19a
1
0 M . Whereas, the titration of HT, bcl-2 and duplex DNA to the contains only side loops and four grooves.
Docking 1 on the
solution of 1 induces little alterations in the MLCT band (Fig. 2a), parallel GQ results in favourable stacking of the aromatic coordina-
suggesting that 1 is unlikely to form strong p–p stacking with GQ HT tion core on the G-quartet, while the four phenyl groups are well
and bcl-2, which would result in a similar hypochroism on MCLT accommodated in the four side grooves to lock the stabilizer into GQ
band to those observed in the case of complex 2 with duplex and GQ c-myc (Fig. 3A). The limited rotation of phenyl groups in GQ grooves
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Both in vitro and biological studies indicate that complex 1 with four
phenyl anchors shows excellent binding affinity to G-quadruplex and
selectivity over duplex DNA. Furthermore, significantly high binding
preference to parallel GQ, especially the c-myc promoter, is achieved
by 1, due to the distinct binding modes between 1 and different GQ
topologies. 1 exhibits a higher potency to various cancer cell lines
than the two reference molecules, 2 and TMPyP4. Hence, our results
suggest that appropriately extending aromatic anchors from the
aromatic Pt(II) coordination plane could be a promising strategy
for developing Pt(II) polypyridyl complexes as stabilizers for specific
GQ topology, if not necessary.
Fig. 3 Molecular docking of 1 (red) on parallel GQ (backbone and base
orientation: green), c-myc (PDB: 1XAV). (A) Side view; (B) top view.
We would like to thank the Nanyang Assistant Professor
Fellowship (M4080531) for the research fund support.
Notes and references
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3
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0
parallel GQ structures via p stacking between 3 -terminal purines.
6
7
Upon binding to the parallel GQ, the coordination plane in complex 1
becomes flatter (Fig. S8c and d, ESI†), which may result in the unique
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20
19b
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19c
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21
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9a
in the literature ). The results indicate that 1, as a stabilizer to parallel
GQ structure, can inhibit c-myc expression in cancerous cell lines.
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1
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the complexes after incubation for 48 hours are shown in Table S2
2
(
(
ESI†). 1 exhibits micromolar IC50 against all the cell lines (2–7 mM),
which can be twenty-fold more potent than 2 and TMPyP4.
In conclusion, we have synthesized [Pt(Dip) ](PF
novel microwave method with good yield and short reaction time.
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2
6 2
) by the
4
760 Chem. Commun., 2013, 49, 4758--4760
This journal is c The Royal Society of Chemistry 2013