Xanthene and xanthone derivatives as G-quadruplex stabilizing ligands

Article abstract of DOI:10.3390/molecules181113446

Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric "bridged" form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data.

Full text of DOI:10.3390/molecules181113446

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Molecules 2013, 18, 13446-13470; doi:10.3390/molecules181113446
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Xanthene and Xanthone Derivatives as G-Quadruplex
Stabilizing Ligands
Alessandro Altieri 1,*, Antonello Alvino 1, Stephan Ohnmacht 2, Giancarlo Ortaggi 1,
Stephen Neidle 2, Daniele Nocioni 1, Marco Franceschin 1 and Armandodoriano Bianco 1,*
1
Dipartimento di Chimica, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, Roma 00185,
Italy; E-Mails: antonello.alvino@uniroma1.it (A.A.); giancarlo.ortaggi@uniroma1.it (G.O.);
daniele.nocioni@tiscali.it (D.N.); marco.franceschin@uniroma1.it (M.F.)
2
The UCL School of Pharmacy, University College London, 29-39 Brunswick Square,
London WC1N 1AX, UK; E-Mails: s.ohnmacht@ucl.ac.uk (S.O.); s.neidle@ucl.ac.uk (S.N.)
* Authors to whom correspondence should be addressed; E-Mails: alessandro.altieri@uniroma1.it (A.A.);
armandodoriano.bianco@uniroma1.it (A.B.); Tel.: +39-064-991-3229/3622 (A.A. & A.B.)
Fax: +39-064-991-3841 (A.A. & A.B.)
Received: 27 September 2013; in revised form: 21 October 2013 / Accepted: 23 October 2013 /
Published: 30 October 2013
Abstract: Following previous studies on anthraquinone and acridine-based G-quadruplex
ligands, here we present a study of similar aromatic cores, with the specific aim of
increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized
compounds include two and three-side chain xanthone and xanthene derivatives, as well as
a dimeric “bridged” form. ESI and FRET measurements suggest that all the studied
molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming
sequences of oncogene promoters. The dimeric compound and the three-side chain
xanthone derivative have been shown to represent the best compounds emerging from the
different series of ligands presented here, having also high selectivity for G-quadruplex
structures with respect to duplex DNA. Molecular modeling simulations are in broad
agreement with the experimental data.
Keywords: G-quadruplex; xanthene; xanthone; ESI-MS; FRET; telomere; c-myc; bcl-2

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1. Introduction
The first G-quadruplex binding ligand having an effect on telomerase activity was discovered in
1997 as a result of the collaboration between the Neidle and Hurley groups [1,2]. Particular attention
has subsequently been devoted to the stabilization of these structures involved in key biological
processes by small organic molecules, [3–5] especially in telomere regions [6–8]. However, the first
anthraquinone ligands show high levels of non-specific cytotoxicity, possibly due to redox cycling [9,10].
Subsequently, in order to solve this biological problem, a new ligand core was studied: the acridine
moiety [11]. The acridine core was chosen in part for its similarity with anthraquinones [12]. A small
library of 3,6-disubstituted acridines was synthesised: this showed substantial improvement with
respect to the previous series of anthraquinones, i.e., micromolar telomerase inhibition and lower
cytotoxicity. Neidle and co-workers also increased the number of side chains on the acridine core,
synthesising a library of 3,6,9-trisubstituted acridines [13]. The lead compound, BRACO-19 (Figure 1)
is one of the most studied G-quadruplex binding ligands to date, showing significant telomerase
inhibitory activity (telEC50 = 6.3 μM) [11].
Figure 1. Examples of anthraquinone and acridine derivatives known as DNA G-quadruplex
DNA G-quadruplex ligands.
O
O
N
N
NH
NH
N
Acridine 3-6 derivative
O
OH
HO
N+
O
O
N+
NH
NH
O
Anthraquinone 2-6 derivative
N
HN
O
O
N
N
H
N
N
N
H
BRACO-19
In this context we wished to study aromatic cores similar to those previously described so as to
induce quadruplex structures, and also possibly reducing the problems related to their unspecific
cytotoxicity. In particular, our efforts have been directed towards synthetic targets represented by
3,6 and 2,7-disubstituted xanthene and xanthone derivatives (Figure 2). This core, widely found in
Nature, represents a unique class of biologically active compounds possessing numerous bioactive
capabilities such as antioxidant properties [14]. These molecules constitute a restricted group of plant
polyphenols, biosynthetically related to flavonoids [15]. We developed this core with the specific aim
of increasing telomerase activity by rational design [16–18]. Molecular modeling studies predict that

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Molecules 2013, 18
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the xanthene moiety is at least comparable to the anthraquinone and acridine moiety in terms of
G-quadruplex binding affinity [19–21]. It is an inherently planar chromophore and also contains a
heterocyclic oxygen atom which could confer water solubility compared to the analogous
anthraquinone core, which is completely insoluble in water.
Figure 2. Xanthene and xanthone derivatives as G-quadruplex stabilizing ligands.
O
O
O
O
O
R
R
R
R
5
5
5
5
O
O
3 XA2-series
4 XO2-series
O
R =
N
N
N
N
R
O
O
O
R
4
4
8 HXO2-series
5
O
N
O
O
5
5
O
O
O
O
N
N
4
4
5
O
O
14 XA3c
11 Bridge Dimer
2. Results and Discussion
2.1. Design and Molecular Docking
We used a virtual screening computer-based technique for identifying promising compounds to
bind to a known G-quadruplex structure. Here we have adopted a docking screening method available
in the AutoDock suite of programs. This is a software suite for predicting the optimal bound
conformations of ligands to macromolecules [22–24]. Its use has been supported by a review by Trent
and co-workers [25] showing that AutoDock optimally balances docking accuracy and ranking. This
application and also the more general problem of modelling individual quadruplex structures have
some similarities to those of other nucleic acid modelling. There is also the added complexity of the
central ion channel and the intrinsic flexibility of the telomeric quadruplex itself. More recently,
however, enhancements in the performance of AutoDock combined with the increased availability of
high speed computers and computer clusters have allowed much larger computational experiments to
be undertaken, where entire compound libraries are screened against pharmaceutically-relevant targets.
We obtained the initial coordinates for the docking from the Protein Data Bank coordinates of the
crystal structure of the parallel 22-mer telomeric G-quadruplex (PDB ID: 1KF1) which shows a single
topology, the parallel fold [26].

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The corresponding intermolecular energy values were used to calculate the average binding
energies (and the relative standard deviations), reported in Table 1. Repeated experiments show good
reproducibility, suggesting that the number of structures generated was sufficient to be statistically
significant. The binding poses calculated for these compounds were then visually inspected to discard
all the ligands which were not able to form hydrogen bonds with any of the guanine bases and/or to
establish an electrostatic interaction with the backbone phosphate groups.
Table 1. Docking binding energies (kcal/mol) for different compounds with 1KF1.
The rmsd values for the whole complex are <4 Å.
Compound
XA2dma 3a
Binding Energy
−4.6
XA2pip 3b
−4.4
XA2mpz 3c
−4.5
XO2dma 4a
−5.3
XO2pip 4b
−5.0
XO2mpz 4c
−5.4
HXO2dma 8a
XO2pip 8b
−5.8
−5.5
XO2mpz 8c
−5.5
Bridge Dimer 11
XA3c 14
−7.0
−6.4
Anthraquinone 2,6 derivative
Acridine 2-6 derivative
BRACO-19
−4.1
−4.6
−7.0
There are several limitations to this methodology, mainly related to the inability of AUTODOCK
and other docking programs to fully account for the non-rigidity of the quadruplex DNA structure and
the likely significant polarisation effect of positive charges on the ligand molecule. Several attempts
have been made to try to solve these problems, and it is clear that the calculated binding energies must
not be considered as absolute values, but rather as indicative of relative ranking, which can be most
useful with a series of homologous molecules having similar chemical structure [27]. However, these
docking studies are in good qualitative agreement with the “threading intercalation” model proposed
for this type of molecule by Hurley and coworkers, in which the drug is stacked on the terminal
G-tetrad, stabilized by π-π interactions with the central aromatic core, while the side chains interact
with the G-quadruplex grooves [28].
The first series of xanthene-based G-quadruplex binding ligands XA2 (dma, pip and mpz; 3a,b and c)
and XO2 (dma, pip and mpz, 4a,b and c) have been the starting point of this work. Docking
experiments were performed with the xanthene core on a G-quadruplex monomeric structure. These
studies have shown good superimposition of the designed ligands with the terminal G-tetrad of the
G-quadruplex (Figure 3), similar to that shown by anthraquinone and acridine derivatives. The binding
energies calculated for molecules with a xanthenic core are in overall accord with those calculated for
known ligands (Table 1).