An assay for human telomeric G-quadruplex DNA binding drugs

Article abstract of DOI:10.1016/j.bmcl.2013.10.030

Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K⁺ or Na⁺.

Full text of DOI:10.1016/j.bmcl.2013.10.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6695–6699
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An assay for human telomeric G-quadruplex DNA binding drugs
Derrick Watkins b, Nihar Ranjan a, Sunil Kumar a, , Changjun Gong a, Dev P. Arya a,b,
⇑
a Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, United States
b NUBAD LLC, 900 B West Faris Road, Greenville, SC 29630, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase
activity and are potential cancer therapies. We have developed an assay for the screening of compounds
with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluores-
cent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence dis-
placement assay. The conjugation of the planar base stacking thiazole orange with the groove binding
neomycin results in high affinity probe that can determine the relative binding affinity of high affinity
HTG binding drugs in a high throughput format. The robust assay is applicable for the determination
of the binding affinity of HTG in the presence of K+ or Na+.
Received 8 August 2013
Revised 18 October 2013
Accepted 21 October 2013
Available online 1 November 2013
Keywords:
G-quadruplex
Telomere
Aminoglycoside
TO-neo
Ó 2013 Elsevier Ltd. All rights reserved.
High throughput screen
Over the last few decades, a number of different telomeric ends
have been identified including the human telomeres.1 The human
telomeric ends are non-coding DNA rich in tandem repeats of
(TTAGGG). In addition to single and double stranded DNA, the guan-
ine rich telomeres of chromosomal DNA can adopt unique
structures formed by the stacking of G-quartets called G-quadruplexes
(Fig. 1).2 Recent work on human telomeres has shown that the
human telomeric G-quadruplex (HTG) may be a target for emerging
cancer therapies. The interaction of telomeres with the enzyme tel-
omerase has been linked to cancer proliferation.3–5 However, the
formation of G-quadruplexes inhibits the activity of telomerase.5
Thus, work is under way to find compounds that can stop cancer
proliferation by stabilizing G-quadruplexes.
DNA.17 The ability of a drug to bind in the groove of quadruplexes
offers the potential for greater selectivity due to the ability to
‘sense’ differences in groove width as well as interactions with
the functional groups in the groove. Therefore, a higher selectivity
for G-quadruplex structures can be envisioned by targeting the
grooves HTG.
Our approach for increasing the affinity and specificity of HTG
binding ligands is the conjugation of aminoglycoside molecules
with various planar molecules. When the selectivity of groove
binding aminoglycosides is combined with the base stacking
surface areas of intercalating molecules with a HTG, ligands
with much a higher selectivity18 and a higher affinity19 for the
G-quadruplex can be designed.
The study of compounds that stabilize HTG is currently lacking
in two related areas. First, the number of compounds that bind
with high affinity and high specificity is limited. While ligands that
interact with different G-quadruplexes have been discovered in the
past decade, most of the G-quadruplex ligands contain planar aro-
matic moieties and stack to G-quadruplexes with moderate bind-
ing affinities.6–11 In order to increase the specificity of
compounds for HTG the approach of identifying ligands with high
selectivity for G-quadruplex grooves is needed.6–9
A related limiting factor in the discovery of HTG binding ligands
is the lack of a high throughput method of screening molecules
that bind in the groove of HTG. In order to develop a screen to
determine compound’s affinity for HTG, we have synthesized a
fluorescent probe by covalently conjugating thiazole orange with
neomycin (TO-neo) as shown in Scheme 1. The synthesis of TO-
neo (5) was achieved by coupling a thiazole orange derivative (3)
with Boc protected neomycin amine (4). The modified thiazole or-
ange derivative (3) was synthesized in three steps using a similar
procedure reported in literature.20,21 As displayed in Scheme 1,
3-methyl-2-(methylthio)benzo[d]thiazol-3-ium (1) was prepared
in one step by reacting 3-methylbenzo[d]thiazole-2(3H)-thione
with methyl iodide. In a separate reaction 1-(4-carboxybutyl)-4-
methylquinolinium (2) was synthesized by reacting 4-methylquin-
oline with 5-bromovaleric acid. The quaternary salts 1 and 2 were
reacted in the presence of triethyl amine to afford compound 3
which bears a carboxylic acid functional group. The Boc protected
Aminoglycosides have been shown to bind in the major groove
of a variety of nucleic acid targets.11–16 There is also strong evi-
dence that aminoglycosides bind in the grooves of G-quadruplex
⇑
Corresponding author. Tel.: +1 864 656 1106.
E-mail address: dparya@clemson.edu (D.P. Arya).
Present address: Department of Molecular Biophysics and Biochemistry, Yale
University, CT 06520, United States.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.030

6696
D. Watkins et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6695–6699
Figure 1. (A) A diagram showing the arrangement of guanines in a G-quartet. Polymorphism of HTG in the presence of sodium and potassium ions (B) a Basket-type
conformation in the presence of Na+ (PDB: 143D) (C) a Propellar-type conformation in the presence of K+ with parallel strand topology (PDB: 3SC8), and (D) a Hybrid
(3 + 1)-type conformation with three parallel and one antiparallel strands (PDB: 2GKU).
N
S
(a)
N
S
S
S
N
(c)
1
S
N
OH
O
OH
(b)
O
N
N
3
2
NHR
N
O
NHR
O
HO
HO
S
HO
HO
NHR
RHN
O
O
(d,e)
NHR
NHR
RHN
N
S
O
O
+
3
H2N
O
O
OH
NHR
S
N
H
4
OH RHN
OH
O
OH RHN
OH
O
O
RHN
OH
O
OH
O
O
OH
R = Boc, 5a
RHN
R = Boc
O
R = H, 5.(6TFA)
Scheme 1. Reagents and conditions: (a) CH3I, 4 h, 50 °C, 84%; (b) 5-bromovaleric acid, 3 h, 110 °C, 38%; (c) Et3N, 50 °C for 2 h, then 1 h at room temperature, 24%; (d) DMF,
TBTU, DIPEA, room temperature, 65%; (e) TFA, DCM, 3 h, rt, 80%.
neomycin amine (4) was synthesized using previously reported
procedures.22,23 The coupling of 3 and 4 was achieved using
O-(benzotriazol-1-yl)-N,N,N0,N00 tetramethyluronium tetrafluoro-
borate (TBTU) as the coupling agent to afford Boc protected thia-
zole orange-neomycin conjugate (5a). The Boc protecting groups
in 5a were deprotected using trifluoroacetic acid to afford the
desired probe TO-neo (5) in 80% yield.
Structural information on HTG has been obtained with solution
studies by NMR2 and in the crystalline state using the sequence
d[AGGG(TTAGGG)3].24 HTG is polymorphic structures and can exist
as a parallel strand G-quadruplex25 or various mixtures of parallel
and antiparallel strand G-quadruplex (Fig. 1). The difference in the
structure of HTG is highly dependent on the cation present. When
formed in 100 mM sodium chloride and 10 mM sodium cacodylate,

D. Watkins et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6695–6699
6697
HTG is dominated by a single basket type parallel/antiparallel
structure consisting of both lateral and diagonal loops (Fig. 1B).2
The dominant structure of the Na+ of HTG is monitored by its sig-
nature CD signal, with a maximum at 295 nm and minimum at
260 nm, characteristic of alternating anti and syn glycosidic confor-
mations along each DNA strand (Supplementary Fig. S1).
When the 22 base HTG is formed in the buffer containing
10 mM HEPES and 100 mM potassium chloride at pH 7.0, G-
quadruplex exists as a complex mixture likely to contain some
ensemble of structures such as basket type structure, the propeller type
parallel structure (Fig. 1C) and 3 + 1 hybrid structure (Fig. 1D).26,27
The K+ form of HTG has a broader CD spectrum in the 240–295 nm
wavelength range with signature positive peaks at 295 and 265 nm
and a negative peak at 235 nm (Supplementary Fig. S1).
are initially present. The initial measurements of the Na+ form have
large deviations for all concentrations of HTG and a modest fit of
the isotherm (Fig. 2a, R = 0.94). At 6 h, the deviations between
measurements at each concentration is significantly reduced and
the fit of the isotherm is significantly improved (Fig. 2b, R = 0.99).
The errors in measurements and fit of the isotherm remains con-
stant after 6 h for the Na+ form of HTG, indicating that the binding
equilibrium had been reached.
Similar to the Na+ conditions, the initial measurements in the
presence of potassium ions for each HTG titration point into
TO-neo were associated with large deviations and the fit of the
binding isotherm was less than ideal (Fig. 2d, R = 0.95). However,
the K+ structure does not reach equilibrium until 12 h (Fig. 2e,
R = 0.99).
The TO-neo binds with high affinity to both the Na+ and K+ forms
of HTG. A titration of the 22 base model of HTG into TO-neo results
in an increase in fluorescence as a function of HTG concentration
for both the Na+ and K+ forms (Fig. 2). The increase in the fluores-
cence can be attributed to stacking interactions of the thiazole
orange of TO-neo with the DNA bases of the G-quadruplex.
The most significant difference in the Na+ and K+ forms of HTG
is binding affinity for TO-neo. Analysis of the isotherm indicates
that in the presence of Na+ only, the binding affinity is
9.6 ꢀ 107 Mꢁ1. The binding affinity in the presence of K+ is
9.3 ꢀ 106 Mꢁ1. Thus the TO-neo has a 10-fold greater affinity for
the Na+ form of HTG than the K+ form of HTG.
While the isotherm of the Na+ and K+ forms of HTG are similar,
significant differences are present in the interaction of TO-neo with
the different structures. The binding ratio of HTG to TO-neo is
similar for the Na+ and K+ structures, with the Na+ having a HTG
to TO-neo binding ratio of 0.86 and the K+ with 1.15 ratio of HTG
to TO-neo. While these values appear similar, the difference in
the ratios is likely indicative of real differences in the interaction
of TO-neo with HTG in the presence of Na+ and K+ (see below).
In order to determine the binding affinity of HTG for TO-neo, the
binding isotherm was fit to a two state model at a 1:1 binding ratio
as previously described.28 The fitting was performed for titrations
measured at various time intervals for both the Na+ and K+ condi-
tions to determine if a stable equilibrium had been reached. In the
presence of both Na+ and K+, the equilibrium of HTG binding to
TO-neo is slow and the titration curve indicates multiple states
Despite the difference in the HTG structure in the presence of
Na+ and K+, TO-neo binds tightly to either structures and gives a
large change in fluorescence upon binding. In both the Na+ and
K+ conditions, the TO-neo binds with higher affinity than that of
thiazole orange or neomycin alone under similar conditions.17,29
Additionally, neomycin appears to dictate the binding ratio of 1:1
in potassium as opposed to the 2:1 binding ratio typically observed
between thiazole orange and G-quadruplexes.29 Thus, it was pro-
posed that TO-neo could be used to probe both structures of HTG.
In order to establish a high throughput assay for screening HTG
binding drugs a compound library of neomycin–anthraquinone
conjugated molecules were screened to identify the compound
with greatest affinity for HTG. The neomycin–anthraquinone
compounds have been previously identified as quadruplex binding
compounds,19 and compound DPA561 was identified from the
Figure 2. The HTG:TO-neo binding isotherms. The HTG was titrated (25–180 nM) into 100 nM TO-neo in 10 mM sodium cacodylate, 0.5 mM EDTA, 100 mM NaCl at pH 7.0
after incubating for (a) 0 h (b) 6 h (c) 12 h or in 10 mM HEPES, 100 mM KCl at pH 7.0 after incubating for (d) 0 h (e) 6 h (f) 12 h. Titrations were performed in a 96-well round
bottom Greneir black plate with an excitation wavelength of 485 nm and emission fluorescence measured at 535 nm from 100 reads, and all points of the isotherms were the
average of two wells. Time trials were determined from separate plates to insure minimal photo bleaching resulting from multiple measurements.

6698
D. Watkins et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6695–6699
screening of a small compound library (12 compounds) as the most
effective compound at displacing TO-neo (Fig. 3). DPA561 is a
addition of the compounds. Thus TO-neo, can be used to screen
compounds that bind to duplex DNA and quadruplex DNA, allow-
ing for a direct approach to determine compounds that discrimi-
nate between the two structures.
The TO-neo screen was standardized for HTG using DPA561.
The screen was performed using a fluorescence plate reader in a
96-well plate format. The concentration of TO-neo and HTG was
neomycin–anthraquinone conjugate and
a similar neomycin–
anthraquinone conjugate has been shown to bind with an affinity
of 1.25 ꢀ 107 Mꢁ1 19
.
As predicted, and as seen with the conjugation
of TO with neomycin, the conjugation of the groove binding
neomycin with the planar anthraquinone results in a compound
with much greater affinity for HTG than either compound alone.19
Thus, DPA561 was used as the positive standard to develop a high
throughput assay for high affinity HTG compounds.
0.1 lM in all wells, and experiments were performed with in
10 mM HEPES, 100 mM KCl at pH 7.0 or 10 mM sodium cacodylate,
0.5 mM EDTA, 100 mM NaCl at pH 7.0, with excitation wavelength
of 485 nm and an emission wavelength of 535 nm. The displace-
ment of TO-neo by DPA561 was measured as greater than a two-
fold decrease in the fluorescence intensity at a 2:1 ratio of
DPA561 to TO-neo (Fig. 3).
The TO moiety provides a strong fluorescent signal when bound
to HTG, and can be competitively displaced by HTG binding mole-
cules, resulting in a decrease in fluorescence. The fluorescent based
assay provides a platform that is readily adaptable to a high
throughput format to be used to identify compounds that bind
within the groove of the G-quadruplex, and determine the relative
binding affinity of compounds with high affinity for HTG. Several
fluorescent based assays have been developed to probe aminogly-
coside binding30 to a variety of nucleic acid structures, and recently
a fluorescent intercalator displacement assay using thiazole orange
(TO)31,32 was developed to screen for compounds that bind to
G-quadruplexes. However, these assays are limited in their ability
to discriminate against compounds that bind with high affinity
characteristic of a dual binding mode.
Another important aspect of compound screening for HTG bind-
ing is the discrimination between duplex DNA and quadruplex
DNA. To further develop the screening process we assessed the
ability of TO-Neo to bind to the duplex DNA sequence d(G4C4)2.
We found that a significant increase in the fluorescence intensity
occurs upon the addition of TO-neo (data not shown). Additionally,
the screening of the same compound library used in the HTG
screen shows a significant decrease in the fluorescence with the
The quality of the TO-neo displacement assay was determined
by the calculation of a Z0-factor using Eq. (1) for the displacement
of TO-neo from HTG by DPA561.
Z0-factor ¼ 1 ꢁ 3 ꢀ ðrp
þ
rnÞ=jlp
ꢁ
ln
j
ð1Þ
The final assay results were obtained using the average (
l
n) and
standard deviation ( n) from 48 wells of 0.1
complex as the negative control and from the average (
standard deviation ( p) 48 wells of 0.1 M complex mixed with
r
l
M TO-neo:HTG
l
p) and
r
l
0.2 l
M DPA561 as the positive control. A Z0-factor of 1 is ideal.
Z0-factor between 0.5 and 1 is considered excellent.33
Our results indicate that the assay is suitable for the detec-
tion of compounds that bind to HTG by the displacement of
the TO-neo probe in a high throughput format and is a func-
tional assay for both the Na+ and K+ structures. The difference
in cations present does not have an effect on the displacement
assay of TO-neo from HTG by DPA561. The Z0-factor for the
Na+ using DPA561 as a standard was 0.70, which was almost
identical to the 0.71 Z0-factor determined for the assay in the
presence of the K+.
Because the K+ is a more biologically relevant cation in the
vicinity of the telomeres, the formation of HTG in the presence of
K+ may be the more relevant form. However, because HTG is highly
polymorphic and the conformation is dependent on other factors
such as flanking sequence25 and crowding effects,34 the presence
of the Na+ form of HTG in biological systems cannot be ignored.
The ability to detect compounds with moderate to high binding
affinity for multiple forms is highly desirable. Therefore, the
TO-neo fluorescence based assay is a high throughput capable
screen for detecting high affinity dual mode binding ligands for
polymorphic HTG.
Figure 3. (a) Fluorescence change of TO-neo bound with HTG quadruplex or
G4C4duplex in the presence of Na+/K+ upon addition of neomycin-anthraquinone
conjugates (DPA 551–562). (b) Optimization of Z-factor using one of the best
binders DPA 561. Fluorescent TO-neo Displacement from HTG was performed in the
presence of Na+ (red) and K+ (blue). The displacement measurements were
performed in a 96-well round bottom Greneir black plate with an excitation
wavelength of 485 nm and emission fluorescence measured at 535 nm from 100
Acknowledgments
reads. All values were the average of 48 wells. The change in fluorescence (DF) was
calculated by subtracting the average of fluorescence measurement from 100 nM of
HTG:TO-neo complex from the average measurement of 100 nM complex with
200 nM DPA561 (insert). (For interpretation of the reference to color in this figure
legend, the reader is referred to the web version of this article.)
We thank the National Institutes of Health, NIH (GM097917) for
financial support. We also thank Gregory Jones, Clemson Univer-
sity, for his assistance with HPLC studies.

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Supplementary data (experimental procedures and synthesis
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in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
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