Discovery of new G-quadruplex binding chemotypes

Article abstract of DOI:10.1039/c3cc48616h

We report here on the discovery and preliminary evaluation of a novel non-macrocyclic low molecular weight quadruplex-stabilizing chemotype. The lead compounds, based on a furan core, show high G-quadruplex stabilisation and selectivity as well as potent

Full text of DOI:10.1039/c3cc48616h

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Discovery of new G-quadruplex binding
chemotypes†
Stephan A. Ohnmacht,a Ehsan Varavipour,a Rupesh Nanjunda,b Ingrida Pazitna,a
Gloria Di Vita,a Mekala Gunaratnam,a Arvind Kumar,b Mohamed A. Ismail,b
David W. Boykin,b W. David Wilsonb and Stephen Neidle*a
Cite this: Chem. Commun., 2014,
50, 960
Received 11th November 2013,
Accepted 29th November 2013
DOI: 10.1039/c3cc48616h
www.rsc.org/chemcomm
We report here on the discovery and preliminary evaluation of a
novel non-macrocyclic low molecular weight quadruplex-stabilizing
chemotype. The lead compounds, based on a furan core, show high
G-quadruplex stabilisation and selectivity as well as potent in vitro
anti-proliferative activity.
Quadruplexes (G4s) are higher-order nucleic acid arrangements
involving a core of p–p stacked guanine-quartets (G-quartets)
rather than the Watson–Crick base pairs of double-helical nucleic
Fig. 1 Structures of various non-polycyclic G-quadruplex ligands.
acids.1 G4-forming sequences are widely prevalent in eukaryotic requirement of most G4-binding ligands is the possession of
telomeric sequences as well as being over-represented in other side-chains terminating in cationic charge.6
genomes,2 notably promoter and 50-UTR sequences of genes
Few G4-binding small molecules have proceeded to in vivo
involved in cellular proliferation.3 The recent demonstration of evaluation in models of human cancer, and to date only one
the presence of G4s in human cells4 has added credence to the compound, Quarfloxin, has been evaluated in clinical trials.15 The
concept that G4s can be targets for therapeutic intervention, at the perceived lack of drug-like characteristics in many G4-binding
single gene or poly-gene levels.5 Appropriate small molecules can compounds may have hindered progress to the clinic. We report
serve to stabilise G4s and the resulting complexes can then act as here on a study to discover novel ligands with MW o400 Da that
impediments to telomere maintenance, transcription or transla- could be suitable starting-points for future drug discovery efforts.
tion, depending on the nature of the quadruplex target site.6
These effects have been shown in several target genes of relevance from the anti-parasitic drug discovery programme at Georgia State
to human cancer such as c-MYC7 and c-KIT.8 University16,17 (several hundred compounds), with highly diverse
Thirty-eight representative members of a large chemical library
A large number of small molecule chemotypes have been scaffolds and functional groups, were screened using a high-
reported as G4-binding ligands.6 The overwhelming majority throughput 96-well FRET (Fluorescence Resonance Energy Transfer)
are heteroaromatic with large flat surfaces, designed to com- assay.18 G4 stabilisation was initially evaluated using dual-labelled
plement the surface characteristics of a terminal G-quartet in a F21T (human telomeric 21-mer) and c-KIT2 (a tyrosine kinase
typical quadruplex structure. A second class of ligand is repre- oncogene) G4s, as well as a duplex DNA sequence (T-loop). The
sented by the cyclic polyoxazole natural product telomestatin.9 ten most active compounds were subsequently screened against an
A number of cyclic and acyclic analogues have been reported, expanded panel of fluorescently-labelled promoter G4-forming
some of which show potent biological activity.10 The acyclic sequences, with HSP90A, HSP90B (heat shock protein 90 promoter
compounds tend to be characterised by a crescent shape. For sequences),12 k-RAS21 (in the promoter of the k-RAS oncogene)19
example, pyridostatin11 and several series of phenyl- and pyridyl- and AR, a G4 recently identified in the promoter of the androgen
bis-oxazoles12–14 all selectively target G4s (Fig. 1). A more general receptor (involved in prostate cancer development).20
Six acyclic furan- and thiophene-based compounds (Fig. 2: 1–6),
representing two distinctive chemotypes were identified with high
(>15 1C) DTm values. A competition assay using unlabelled calf
thymus duplex DNA and compounds 1–6 examined the ability of
these compounds to differentiate between duplex and the F21T G4
a School of Pharmacy, University College London, London WC1N 1AX, UK.
E-mail: s.neidle@ucl.ac.uk; Fax: +44 (0)207 753 5970; Tel: +44 (0)207 753 5969
b Department of Chemistry and Center for Biotechnology and Drug Design,
Georgia State University, Atlanta, Georgia 30303-3083, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc48616h DNA at high duplex : G4 ratios. The compounds were also examined
960 | Chem. Commun., 2014, 50, 960--963
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examined by us,20 shows that 1–6 exhibit only moderate DTm
values with the AR G4, which are generally lower than with
other G4s. Compounds 1–6 produced slightly reduced but still
significant stabilisation with the c-KIT2 and k-RAS21 G4s, suggest-
ing that these compounds have the ability to act simultaneously on
multiple G4 targets (G4 poly-targeting). The stabilisation of a duplex
DNA sequence (T-loop) was not significantly affected by any of the
compounds at the biologically relevant concentration employed
here (1 mM). Compounds 1 and 5 in particular are highly selective
for G4 versus duplex DNA, as found in a series of competition
assays, where the G4 stabilisation ability of both compounds is
undiminished by adding calf thymus DNA in excess, at ratios up to
1 : 100/300. A control compound, 7, an established duplex DNA
minor groove binder, also showed significant G4 stabilisation,
Fig. 2 Structures of the lead compounds 1–6 identified in this study,
together with a control compound 7.
in a 96 h short-term sulforhodamine B (SRB) assay, to determine albeit with greater effects on the duplex DNA used (with a
their ability to inhibit cancer cell growth (Tables 1 and 2). All six sub-optimal sequence for this compound).
compounds showed potent G4 stabilising abilities, as judged by the
Circular dichroism (CD) was employed to qualitatively evaluate
large changes in DTm values for the selected G4s. In particular the the binding mode of the lead compounds and to examine induced
bis-phenyl-mono-furan compounds 1 and 5 had especially high DTm structural transitions in the telomeric G4. The CD spectra (Fig. 3
values, broadly comparable to those for established high-affinity and ESI†) show that compounds 1 and 3–6 produce very small
G4-binding compounds such as tetra-substituted naphthalene induced CD signals in a human telomeric quadruplex sequence.
diimides.21 The tri-furan compound 2 is consistently more effective Such weak induced CD signals are characteristic of quadruplex
in stabilising the G4s than the polyfuran compound 6. Compound 2 end-stacking compounds and the small differences in the CD
and 6, representatives of a tetrafuran second chemotype, were less signal patterns for different compounds indicate minor differences
selective at high duplex ratios and were inactive in the SRB assay, in the stacking geometries of the ligands at the terminal G-quartets.
possibly because of aqueous solubility and cellular uptake issues.
All the compounds exhibiting weak induced CD signals that
Switching from a furan (1, 5) to a thiophene (4), does slightly affect show a binding preference for an anti-parallel type quadruplex
G4 stabilisation and selectivity vs. duplex DNA, though not in vitro conformation as observed by decreases in the CD signal around
potency, which at least in the cell lines examined, is comparable to 260 nm. Compound 5 however behaves differently, inducing a
that of compounds 1 and 5.
much larger conformational transition in the telomeric quadruplex
Overall, F21T and the two HSP90 G4s have been most upon complex formation. Upon titrating 5, the CD intensity around
stabilised by compounds 1–6. Comparison with the behaviour 290 nm decreases with a subsequent increase in the CD signal
of a tetra-substituted naphthalenediimide compound previously intensity around 260 nm. This is most likely due to an induced
Table 1 FRET G4 stabilization (DTm at 1 mM in 1C) and calf thymus DNA (CT) competition data (the latter showing % retention of the F21T DTm at 1 mM).
hEsdi ꢀ 0.5 1C, from triplicate measurements. n/a: indicates unsuccessful curve fitting to the melting data. Compound 7, a negative control, is a para
analogue of the mono-furan compounds. Compound 8 is a tetra-substituted naphthalene diimide derivative21 used here as a G4 control
Compound Mol. Wt F21T c-KIT2 HSP-90A HSP-90B k-RAS21R AR
T-loop G4 : CT 1 : 1 G4 : CT 1 : 10 G4 : CT 1 : 100 G4 : CT 1 : 300
1
2
3
4
5
6
7
8
384.5
364.4
330.5
372.5
412.5
350.3
302.5
830.6
22.3 16.8
20.4 16.3
17.6 17.8
18.0 13.9
22.6 18.5
18.6 n/a
14.4 12.2
26.6 22.0
27.7
26.0
21.2
22.5
26.6
20.6
19.0
33.1
23.7
22.0
18.1
19.1
22.7
16.2
15.7
28.6
19.6
18.5
15.1
15.9
18.3
13.4
16.6
n/a
15.4 o2
13.8 o2
100
100
2.2 100
100
100
100
100
100
100
100
100
100
92.7
65.9
60.5
100
53.1
24.5
100
63.3
43.9
39.9
n/a
35.4
2.5
11.9
10.2 o2
14.7 o2
10.1 o2
100
100
100
9.9
15.9
3.4 100
4.9 100
n/a
27.2
Table 2 IC50 values of compounds 1–8 determined by a 96 h SRB assay (see the ESI for further details). hEsdi ꢀ 0.3 lM for compounds 1–6
A549
MCF7
ALT
Mia-PaCa2
Panc1
RCC4
786-O
WI38
1
2
3
4
5
6
7
8
0.3
>25
0.8
0.4
0.5
4.2
3.7
0.019
0.9
>25
0.9
1.4
1.2
15.6
11.7
0.070
1.0
>25
6.6
1.9
1.4
18.8
0.3
>25
2.0
0.7
0.5
7.0
8.90
0.011
0.5
>25
1.7
1.0
1.0
>25
10.9
0.003
4.9
>25
>25
3.3
3.9
>25
>25
3.0
>25
>25
5.6
4.8
12.8
>25
1.3
>25
>25
2.0
2.0
>25
>25
>25
0.063
0.560
0.320
0.230
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Several of the compounds showed low mM anti-proliferative
activity (Table 2) in a cancer cell line panel [A549 (lung cancer),
MCF7 (breast cancer), RCC4 and 786-O (renal cancer), Panc1 and
Mia-PaCa2 (pancreatic cancer), ALT (transformed WI38 lung
fibroblast cells characterised by Alternative Maintenance of
Telomeres) and WI38 (non-transformed lung fibroblast cells)].
The two poly-furan compounds 2 and 6 have low anti-
proliferative activity, even though both have G4-stabilising activity
comparable to the other four compounds in the group. This may be
due to cell uptake and nuclear localisation problems as well as
limited aqueous solubility; the lack of observed precipitation during
the SRB assay supports the former suggestions. Compounds from
the mono-furan and mono-thiophene series on the other hand
show activity in several cell lines in the low-mM range. The lung
cancer cell line A549 is slightly more sensitive to these compounds,
although we do not currently have a molecular explanation for this.
We cannot exclude the possibility of non-G4 targets being
involved in these cellular effects: experiments are currently under-
way to examine links with G4 affinity for these compounds.
G4 selectivity appears to be limited even though for the two lead
compounds at least, duplex DNA is less likely to be a target. This
suggests that they may be acting as poly-targeting agents, affecting
a number of genes and oncogenes involved in cellular proliferation
and also that the compounds do not have identical cellular targets.
It is notable that compounds 3 and 6 have high selectivity for
several of the cancer lines compared to the normal WI38 line and
have some activity in the ALT line, which maintains telomere
length by non-telomerase mechanisms. This suggests that telo-
mere maintenance rather than telomerase per se, is being targeted.
We report here that screening putative ligands using a HTS-FRET
assay against a panel of G4s with a duplex control sequence, has
resulted in the discovery of meta-substituted bisphenyl-monofurans
as a novel G4 stabilizing chemotype. A similar chemotype, with a urea
group replacing the furan ring, has been reported23 as having high G4
affinity. These compounds are structurally-simple, conformationally
flexible and chemically readily accessible with MW o400 Da. They
have G4 stabilisation ability comparable to those previously observed
with polycyclic heteroaromatic compounds21 (cf. compound 8
(4,9-bis((3-(4-methyl-piperazin-1-yl)propyl)amino)-2,7-bis(3-morpho-
linopropyl)benzo-[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone)
in Table 1), but with low duplex DNA affinity. They inhibit cancer
cell growth at low mM/high nM levels, suggesting that these or
related compounds may have potential as drug-like poly-quadruplex
targeting agents.
Fig. 3 Circular dichroism (CD) spectra for compounds 1 and 5 at differing
ligand : G4 ratios. F22T is a 22-mer analogue of the F21T sequence.
conformational transition from a hybrid to a more parallel-type
G4 form.
Analysis of the equilibrium conformation for compound 1
was undertaken with classical molecular mechanics (MM2 and
AMBER) force fields and by ab initio (STO-3G) calculations. The
MM2 analysis suggested that overall arrangement involved coplanar
rings. This somewhat implausible conclusion was at variance with
results from the AMBER force field and the ab initio calculations,
which concurred in suggesting a twisted conformation due to the
repulsive effects of the two furanoid methyl groups on the attached
phenyl rings. The barrier between planar and twisted conformations
is likely to be low so a qualitative analysis of plausible
G4-bound conformations for compounds 1 and 2 has been under-
taken. The NMR structure of an anti-parallel human telomeric
G4 complexed with a telomestatin derivative has been used as a
starting-point.22 Compounds 1 and 2 have similarities to the
overall shape and curvature of telomestatin (Fig. 4), albeit in an
acyclic manner. The NMR structure shows that the telomestatin
derivative is non-planar and its out-of-plane distortions comple-
ment the non-planarity of the G-quartet to which it is bound. The
qualitative low-energy structures proposed for compounds 1 and 2
incorporate such distortions, which still enable effective p–p
stacking onto the terminal G-quartet of this G4, as well as some
additional stabilisation from a thymine. The modelling show that
the meta position of the dihydroimidazole substituents in 1 and
the tetrahydropyrimidine substituents in 2, is crucial for shape
similarity to telomestatin. The para substitution in the control
compound 7 enables it to effectively bind to the minor groove of
duplex DNA, which is not possible for any of the six compounds
arising from the screen.
Synthesis and biophysical studies at Georgia State University
were supported by National Institutes of Health NIAID Grant AI-
064200 (WDW, DWB). Work at UCL was supported by a MRC
Confidence in Concept grant (SN).
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