Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
Aim. To study the efficiency of tricationic porphyrin–imidazo[4,5-b]phenazine conjugate and its Zn(II) and Mn(III) complexes as G-quadruplex (G4) DNA ligands. Methods. FID (Fluorescent Intercalator Displacement) assay was used to evaluate the affinity of compounds for a model duplex and Tel22 quadru...
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nasplib_isofts_kiev_ua-123456789-1543452025-02-09T13:29:19Z Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling Взаємодія кон’югатів катіонного порфірину з імідазофеназином із ДНК-квадруплексом: метод FID та квантово-хімічне моделювання Взаимодействие конъюгатов катионного порфирина с имидазофеназином с ДНК-квадруплексом: метод FID и квантово-химическое моделирование Didan, Yu.V. Ilchenko, M.M. Negrutska, V.V. Dubey, L.V. Ryazanova, O.A. Dubey, I.Ya. Bioorganic Chemistry Aim. To study the efficiency of tricationic porphyrin–imidazo[4,5-b]phenazine conjugate and its Zn(II) and Mn(III) complexes as G-quadruplex (G4) DNA ligands. Methods. FID (Fluorescent Intercalator Displacement) assay was used to evaluate the affinity of compounds for a model duplex and Tel22 quadruplex DNA at various ionic strengths. Molecular modeling of the conjugate interaction with G4 DNA was performed using the Density Functional Theory (DFT) calculations with M06-2X functional and 6-31G(d) basis set. Guanine octet stabilized with K+ ion was used as a G4 model. Results. DC50 values and dissociation constants were determined for the complexes of three conjugates with duplex and quadruplex DNA. The structures and energetic parameters of G-octet complexes with Zn-metalated conjugate were obtained. Conclusions. All complexes have a strong affinity to the Tel22 quadruplex. The increase of ionic strength results in an increase in selectivity for quadruplex over duplex DNA and the decrease of binding affinity of the ligands. The structure of ligand–G4 complexes is determined by stacking interaction of porphyrin fragment with G-quartet, rather than an intercalative binging of the ligand. Мета. Дослідити ефективність трикатіонного кон’югату порфірин–імідазо[4,5-b]феназин та його Zn(II) і Mn(III) комплексів як лігандів G-квадруплексної (G4) ДНК. Методи. Метод заміщення флуоресцентного інтеркалятора (FID) використано для оцінки афінності сполук до модельної дуплексної ДНК та квадруплексу Tel22 за різних значень іонної сили розчину. Молекулярне моделювання взаємодії кон’югату з G4-ДНК проведено за допомогою обчислень теорії функціоналу густини (DFT) з використанням функціоналу M06-2X та базисного набору 6-31G(d). Як модель G4 використано гуаніновий октет, стабілізований іоном K+. Результати. Визначено значення DC50 та константи дисоціації комплексів трьох кон’югатів із дуплексною та квадруплексною ДНК. Розраховано структури та енергетичні параметри комплексів G-октету з Zn-металізованим кон’югатом. Висновки. Всі сполуки виявляють високу афінність до квадруплексу Tel22. Збільшення іонної сили веде до зростання селективності до квадруплексної відносно дуплексної ДНК та зниження афінності зв’язування лігандів. Структуру комплексів ліганд–G4 визначає передусім стекінгова взаємодія порфіринового фрагмента з G-квартетом, а не інтеркаляційне зв’язування ліганда. Цель. Изучить эффективность трикатионного конъюгата порфирин–имидазо[4,5-b]феназин и его Zn(II) и Mn(III) комплексов как лигандов G-квадруплексной (G4) ДНК. Методы. Метод замещения флуоресцентного интеркалятора (FID) использован для оценки аффинности соединений к модельной дуплексной ДНК и квадруплексу Tel22 при разных значениях ионной силы раствора. Молекулярное моделирование взаимодействия конъюгата с G4-ДНК проведено при помощи вычислений теории функционала плотности (DFT) с использованием функционала M06-2X и базисного набора 6-31G(d). В качестве модели G4 использован гуаниновый октет, стабилизированный ионом K+. Результаты. Определены значения DC50 и константы диссоциации комплексов трех конъюгатов с дуплексной и квадруплексной ДНК. Рассчитаны структуры и энергетические параметры комплексов G-октета с Zn-металлизированным конъюгатом. Выводы. Все соединения проявляют высокую аффинность к квадруплексу Tel22. Увеличение ионной силы приводит к возрастанию селективности к квадруплексной относительно дуплексной ДНК и снижению аффинности связывания лигандов. Структуру комплексов лиганд–G4 определяет прежде всего стекинговое взаимодействие порфиринового фрагмента с G-квартетом, а не интеркаляционное связывание лиганда. 2018 Article Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling / Yu.V. Didan, M.M. Ilchenko, V.V. Negrutska, L.V. Dubey, O.A. Ryazanova, I.Ya. Dubey // Вiopolymers and Cell. — 2018. — Т. 34, № 5. — С. 387-399. — Бібліогр.: 56 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00098B https://nasplib.isofts.kiev.ua/handle/123456789/154345 577.323+577.332 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Bioorganic Chemistry Bioorganic Chemistry |
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Bioorganic Chemistry Bioorganic Chemistry Didan, Yu.V. Ilchenko, M.M. Negrutska, V.V. Dubey, L.V. Ryazanova, O.A. Dubey, I.Ya. Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling Вiopolymers and Cell |
| description |
Aim. To study the efficiency of tricationic porphyrin–imidazo[4,5-b]phenazine conjugate and its Zn(II) and Mn(III) complexes as G-quadruplex (G4) DNA ligands. Methods. FID (Fluorescent Intercalator Displacement) assay was used to evaluate the affinity of compounds for a model duplex and Tel22 quadruplex DNA at various ionic strengths. Molecular modeling of the conjugate interaction with G4 DNA was performed using the Density Functional Theory (DFT) calculations with M06-2X functional and 6-31G(d) basis set. Guanine octet stabilized with K+ ion was used as a G4 model. Results. DC50 values and dissociation constants were determined for the complexes of three conjugates with duplex and quadruplex DNA. The structures and energetic parameters of G-octet complexes with Zn-metalated conjugate were obtained. Conclusions. All complexes have a strong affinity to the Tel22 quadruplex. The increase of ionic strength results in an increase in selectivity for quadruplex over duplex DNA and the decrease of binding affinity of the ligands. The structure of ligand–G4 complexes is determined by stacking interaction of porphyrin fragment with G-quartet, rather than an intercalative binging of the ligand. |
| format |
Article |
| author |
Didan, Yu.V. Ilchenko, M.M. Negrutska, V.V. Dubey, L.V. Ryazanova, O.A. Dubey, I.Ya. |
| author_facet |
Didan, Yu.V. Ilchenko, M.M. Negrutska, V.V. Dubey, L.V. Ryazanova, O.A. Dubey, I.Ya. |
| author_sort |
Didan, Yu.V. |
| title |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling |
| title_short |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling |
| title_full |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling |
| title_fullStr |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling |
| title_full_unstemmed |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling |
| title_sort |
interaction of cationic porphyrin-imidazophenazine conjugates with dna quadruplex: fid assay and quantum-chemical modeling |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2018 |
| topic_facet |
Bioorganic Chemistry |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154345 |
| citation_txt |
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling / Yu.V. Didan, M.M. Ilchenko, V.V. Negrutska, L.V. Dubey, O.A. Ryazanova, I.Ya. Dubey // Вiopolymers and Cell. — 2018. — Т. 34, № 5. — С. 387-399. — Бібліогр.: 56 назв. — англ. |
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Вiopolymers and Cell |
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387
Yu. V. Didan, M. M. Ilchenko, V. V. Negrutska
© 2018 Yu. V. Didan et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
UDC 577.323+577.332
Interaction of cationic porphyrin-imidazophenazine conjugates with
DNA quadruplex: FID assay and quantum-chemical modeling
Yu. V. Didan1,2, M. M. Ilchenko1, V. V. Negrutska1, L. V. Dubey1,
O. A. Ryazanova3, I. Ya. Dubey1
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
2 Delft University of Technology
9 van der Maasweg, Delft 2629HZ, Netherlands
3 B. I. Verkin Institute for Low Temperature Physics and Engineering, NAS of Ukraine
47, Prospekt Nauky, Kharkiv, Ukraine, 61103
dubey@imbg.org.ua
Aim. To study the efficiency of tricationic porphyrin–imidazo[4,5-b]phenazine conjugate and
its Zn(II) and Mn(III) complexes as G-quadruplex (G4) DNA ligands. Methods. FID
(Fluorescent Intercalator Displacement) assay was used to evaluate the affinity of compounds
for a model duplex and Tel22 quadruplex DNA at various ionic strengths. Molecular modeling
of the conjugate interaction with G4 DNA was performed using the Density Functional Theory
(DFT) calculations with M06-2X functional and 6-31G(d) basis set. Guanine octet stabilized
with K+ ion was used as a G4 model. Results. DC50 values and dissociation constants were
determined for the complexes of three conjugates with duplex and quadruplex DNA. The
structures and energetic parameters of G-octet complexes with Zn-metalated conjugate were
obtained. Conclusions. All complexes have a strong affinity to the Tel22 quadruplex. The
increase of ionic strength results in an increase in selectivity for quadruplex over duplex DNA
and the decrease of binding affinity of the ligands. The structure of ligand–G4 complexes is
determined by stacking interaction of porphyrin fragment with G-quartet, rather than an inter-
calative binging of the ligand.
K e y w o r d s: G-quadruplex, ligands, porphyrins, imidazophenazine, FID, DFT
Introduction
Quite recently established anticancer strategy
is based on targeting the telomeres and telo-
merase by small molecules. Telomeres are
guanine-rich DNA sequences located at the
ends of the chromosomes. Telomeric DNA is
able to fold into specific four-stranded struc-
tures called G-quadruplexes (G4) formed by
the stacks of guanine quartets (G-quartets)
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2018. Vol. 34. N 5. P 387–399
doi: http://dx.doi.org/10.7124/bc.00098B
388
Yu. V. Didan, M. M. Ilchenko, V. V. Negrutska et al.
linked by non-canonical systems of hydrogen
bonds and stabilized by monovalent metal
cations located inside the channel formed by
guanine residues [1, 2] (Fig. 1). Binding and
stabilization of these structures by small mol-
ecules can result in the inhibition of telomer-
ase, an enzyme responsible for telomere elon-
gation upon cellular division [3, 4]. Telomerase
is highly active in a majority (80–95 %) of
tumor cells, in contrast to normal somatic cells
[3, 5]. So telomerase system is directly associ-
ated with cancer, and both telomerase and
telomeric DNA quadruplex structures are con-
sidered as targets for the development of nov-
el anticancer drugs [6–9].
In the cell, G4 structures are also found in
promoter regions of some genes, mainly proto-
oncogenes [10]. Stable RNA quadruplexes
have been discovered as well [11]. There is
growing evidence that DNA and RNA quadru-
plexes play a crucial biological role as a class
of regulatory elements involved in processes
such as DNA replication, transcription and
translation [12, 13].
The binding of a ligand to telomeric
G-quadruplex can interfere with telomerase
interaction with telomeric DNA blocking its
elongation and thus inducing the death of can-
cer cells [2–4, 8]. Another mechanism of an-
titumor activity of G4 ligands has been shown
to be quadruplex-mediated DNA damage [14,
15].
Usually specific G4 ligands contain large
heteroaromatic systems allowing the efficient
π-π-interaction with G-quartets [2, 7–9, 16].
Tetracationic porphyrin TMPyP 1 (Fig. 1) is a
well established G4 ligand and telomerase
inhibitor [17, 18]. Its tricationic analog
tris(methylpyridinium)phenylporphyrin
(TMP3) was used to attach porphyrin to a
variety of other molecules [19–22]. We have
previously synthesized TMP3 conjugate with
imidazo[4,5-b]phenazine (TMP3-ImPhz) and
their Zn(II) and Mn(III) complexes [23]
(Fig. 2). ImPhz is an efficient intercalating
agent stabilizing DNA duplexes [24–27], and
its residue was expected to enhance ligand
affinity for G4-DNA.
A
B
Fig. 1. (A) The structure of guanine
quartet; (B) side view of the stack of
three G-quartets containing two met-
al cations (K+, Na+).
389
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
Conjugates 2a-c were shown to be efficient
telomerase inhibitors in vitro active at 2–10 µM
concentration [28]. They were also found to
display antiproliferative activity in vitro (mouse
Lewis lung carcinoma cell culture), with EC50
values for 2a, its Mn and Zn complex to be
21.8, 11.2 and 5.9 μM, respectively [23].
Absorption and fluorescence spectroscopy
studies on the interaction of compounds 2a-c
with monomolecular quadruplex Tel22 [29]
and tetramolecular G4 formed by poly(G) [30,
31], as well as various double- and single-
stranded nucleic acids [29–31], revealed a
number of binding modes, including electro-
static interactions, external binding, aggrega-
tion and intercalation. However, binding pa-
rameters of these compounds to G4 and duplex
DNA have not yet been determined.
In this work we have quantitatively evalu-
ated the affinity and selectivity of porphyrin-
imidazophenazine conjugates as quadruplex
DNA ligands and their possible mode of bind-
ing to G4 structure.
Materials and Methods
Sodium cacodylate and Thiazole Orange (TO)
were obtained from “Sigma” (USA). Conju-
gates of tricationic porphyrin TMP3 and its
Zn(II) and Mn(III) complexes with
imidazo[4,5-b]phenazine were synthesized as
previously reported [23]. Oligonucleotides
were from “Eurogentec” (Belgium).
DNA quadruplex was obtained by the fold-
ing of a model oligonucleotide Tel22 of the
sequence 5’-AGGG(TTAGGG)3]-3’. Tel22
solution in a 10 mM sodium cacodylate buffer
(pH 7.3) containing 100 mM KCl was heated
at 95ºC for 5 min and slowly cooled overnight
to room temperature and then equilibrated for
a day at 4ºС. DNA duplex ds17 was prepared
by standard procedure from complementary
17-mer oligonucleotides 5’-CCAGTTCGTAG
TAACCC-3’ and 5’-GGGTTACTACGAACT
GG-3’ [34, 35].
FID assay
Fluorescent Intercalator Displacement assay
was used to study the conjugate binding to
duplex [32, 33] and quadruplex [34–36] DNA
with TO as a fluorescent dye.
FID titration experiments were performed in
96-well plastic microplates in semi-automatic
mode with Synergy HT analyzer (BioTek, USA).
For both quadruplex and duplex DNA, the mo-
Fig. 2. The structure of cationic porphyrin derivatives.
390
Yu. V. Didan, M. M. Ilchenko, V. V. Negrutska et al.
lar ratio DNA/TO was 3:1. The reaction mix-
tures (total volume 50 μL) contained 100 or
200 mM KCl, 10 mM Na-cacodylate, 1 μM TO,
3 μM Tel22 or ds17, and various concentrations
of tested compounds (solution in dimethyl sulf-
oxide). The fluorescence of samples was mea-
sured at room temperature with λex = 500 nm
and λem = 530 nm. The data were processed with
Gen5 software. The percent of fluorophore dis-
placement D was obtained for each concentra-
tion as follows: D = 100 – [(F/F0) × 100], where
F and F0 — fluorescence of DNA/TO complex
in the presence and absence of a ligand, respec-
tively. The plots of D vs. ligand concentration
were built, from which DC50 values (ligand
concentration required for 50 % displacement
of TO in dye-DNA complex) were obtained.
Dissociation constants of complexes were de-
termined using the Scatchard method from the
plots of DF vs. DF/[free ligand], where DF is a
difference between the relative fluorescence of
DNA/TO complex in the presence and absence
of a ligand, and [free ligand] is a concentration
of unbound ligand in the probe [32, 33]. Three
independent experiments were performed for
each ligand.
Molecular modeling
Quantum-chemical modeling of the interaction
of ligands with G4 structures was performed
by DFT (Density Functional Theory) approach
using M06-2X functional [37] and 6-31G(d)
basic set. The structures of all porphyrin con-
jugates have been previously optimized by the
same method [23]. Guanine octet stabilized by
a potassium cation was used as a simple mo del
of G-quadruplex [38].
Complexes of (Zn)TMP3-ImPhz with
G-octet were investigated. Full geometry op-
timization was performed for the studied sys-
tems in water. The solvent effects were treated
using the CPCM (Conductor-like Polarizable
Continuum Model) method [39, 40]. Cal cu la-
tions were carried out with Gaussian 09 pa-
ckage (revision B.01) [41].
Results and Discussion
The synthesis of tricationic porphyrin-imidaz-
ophenazine conjugates containing a flexible
linker (2a-c, Fig. 2) was based on the coupling
of carboxyalkyl-modified porphyrin with ami-
noalkyl-functionalized phenazine derivative,
as described in [23].
Ligand affinity and selectivity
Ligand interaction with two forms of DNA
was studied using a Fluorescent Intercalator
Displacement (FID) assay. FID is a simple
and fast analytical method that allows to
evaluate the DNA binding affinity and selec-
tivity of low-molecular compounds. This as-
say proposed by Boger et al. in 2001 [32] is
based on the loss of fluorescence of some
dyes, usually Thiazole Orange (TO), upon
their displacement from complexes with DNA
by a ligand. TO is virtually non-fluorescent
in a free state, whereas its complexes with
DNA are highly fluorescent. Both duplex [32,
33, 42–44] and quadruplex [34–36, 44–46]
DNA, as well as RNA [47], can be used as
targets.
22-mer oligonucleotide Tel22 (sequence
5’-AGGG(TTAGGG)3]-3’, a fragment of hu-
man telomeric DNA containing three telo-
meric repeats), was folded in the presence of
K+ cations to form G-quadruplex of parallel
topology (PDB access code 1KF1). Model
double-stranded DNA ds17 was formed by two
391
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
complementary non-quadruplex-forming 17-
mer oligonucleotides.
To evaluate the effect of ionic strength on
binding affinity and selectivity, FID experi-
ments were performed in solutions containing
100 and 200 mM KCl.
DC50 values were determined from dis-
placement–concentration plots obtained in FID
titration experiments (Fig. 3). DC50 parameter,
compound concentration resulting in 50 %
fluorophore substitution in dye-DNA complex,
is a measure of relative ligand affinity to DNA
targets; the lower is DC50, the higher is binding
efficiency of the ligand.
Dissociation constants of ligand-DNA com-
plexes (Kd) were determined from the titration
data for duplex and G4 DNA using the classic
Sсatchard analysis, according to [32, 33].
Ligand affinity data are presented in Table 1.
It should be noted that DC50 values are di-
rectly obtained from the experiment and can
be used for affinity ranking of a series of li-
gands in regard to a given DNA target. At the
same time, Kd is a calculated parameter to be
taken with care, since FID method is an indi-
rect technique involving the competitive DNA
binding of two ligands with different affinities
and binding modes. FID and the direct meth-
ods provide comparable results [32, 45], but
Table 1. Binding parameters and selectivity indexes of TMP3-ImPhz and its metal complexes at various
ionic strengths *
Ligand
100 mM KCl
DC50, µM Kd, ×10–6 M Selectivity index **
G4 ds17 G4 ds17 SIDC SIK
2a 17.4 5.0 4.6 3.7 0.29 0.81
2b 36.1 59.7 7.0 6.1 1.65 0.87
2c 14.4 14.2 1.6 2.9 0.99 1.81
200 mM KCl
2a 43.8 79.9 20.1 89.5 1.82 4.45
2b 59.6 114 46.3 359 1.91 7.75
2c 28.6 72.3 3.8 42.0 2.53 11.1
* experimental errors are within 10 % (n=3)
** SIDC = DC50(ds)/DC50(G4); SIK = Kd(ds)/Kd(G4)
Fig. 3. FID titration plots obtained for non-metalated
conjugate 2a with quadruplex (Tel22 G4) and duplex
DNA (ds17) in 200 mM KCl buffer.
392
Yu. V. Didan, M. M. Ilchenko, V. V. Negrutska et al.
some authors underline the difficulties in ac-
curately determining DNA-ligand affinity con-
stants, pointing out the need for comparing
data obtained with various techniques [35].
All compounds demonstrate a strong binding
to G4 DNA. Dissociation constants Kd in
100 mM KCl are in the range (1.6–7.0)×10–6 M.
The most efficient G4 binder is Mn(III) com-
plex 2c. At high ionic strength (200 mM KCl)
binding affinity of all ligands to G4 substan-
tially decreases, according to both DC50 and Kd
data, but remains high (Kd = 3.8–46.3)×10–6 M).
High affinity of the conjugates for G4 is
associated with a good ability to inhibit telo-
merase. The most efficient Zn(II) complex 2b
was shown to completely inhibit it in vitro in
TRAP assay [5] at 2.5 µM concentration being
several times more active than non-conjugated
porphyrin. Non-metalated conjugate 2a showed
inhibition effect at 5 µM, whereas Mn(III)
derivative 2c was less active [28].
Preferential quadruplex vs. duplex binding,
along with high affinity to quadruplex DNA,
is a key requirement for specific antitumor G4
ligands allowing to minimize their unfavorable
biological side effects. Selectivity index (SI)
is defined as the ratio between either DC50
values or dissociation constants of ligands for
duplex DNA and G4. At low KCl concentra-
tion (100 mM) ligands 2a and 2b preferen-
tially bind to duplex DNA, whereas Mn com-
plex 2c has no (SI determined from DC50
values) or relatively low (SI based on Kd is
1.81) preference for G4.
At 200 mM KCl all ligands bind to G4-
DNA stronger than to duplex DNA, i.e. dem-
onstrate a good selectivity for quadruplex over
duplex DNA. For example, the selectivity in-
dex determined from Kd values is 4.45 for
TMP3-ImPhz and 7.75 and 11.1 for its Zn and
Mn complexes, respectively. Thus, an increase
in ionic strength leads to an increase of G4
selectivity of ligands.
Generally, both electrostatic and stacking
interactions may contribute to DNA binding
of the studied ligands. The electrostatic bind-
ing of positively charged porphyrin fragment
to DNA phosphate backbone was experimen-
tally shown to occur upon interaction of 2a-c
with quadruplex structures [29–31]. Ionic
strength increase suppresses strong, but non-
specific electrostatic interactions, resulting in
the increase of ligand selectivity for G4, how-
ever, this effect is obviously associated with a
decrease of binding affinity (Table 1). These
data are in line with a recent finding that low-
ering the total charge on TMPyP decreases the
affinity, but increases the selectivity to G4 vs.
dsDNA [48].
DFT modeling of complexes
Computer modeling of the interaction of li-
gands with their molecular target, telomeric
G-quadruplex, is required to get insights into
their mechanism of action and for further
structure optimization to design more potent
telomerase inhibitors.
The complexity and flexibility of quadru-
plex structures is still a challenge for theo-
retical modeling of G4 and their ligand com-
plexes. In addition to the large size of the
molecular system, one of the main problems
is a broad structural polymorphism of quadru-
plex DNA [1, 2, 8, 49].
Usually molecular modeling of quadruplex
nucleic acids and their complexes is carried out
by molecular dynamics (MD) approaches [50,
51]. For example, a series of cationic porphy-
393
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
rin-anthraquinone hybrids have been investi-
gated for their interaction with G4 DNA by
employing docking and molecular dynamics
methods [52]. MD simulations provide valu-
able information on conformation, dynamics
and thermodynamic parameters of the systems
studied. MD methods, however, have a number
of limitations, e.g. force-field imperfections and
often short simulation times resulting in insuf-
ficient accuracy of modeling. On the other
hand, quantum-chemical (quantum-mechanical,
QM) approaches are much more accurate, al-
though require more computational resources,
and have been successfully used in the field of
quadruplex modeling [38, 53–55].
To find a possible mode of binding, we have
performed a QM study of the interaction of
metalated TMP3-ImPhz conjugate 2b with a
simple model of G-quadruplex, so called
G-octet previously developed by us [38]. This
system consists of two guanine quartets con-
taining a central K+ cation. Molecular model-
ing was carried out to construct the geometries
of complexes in aqueous solution. Calculations
were performed by DFT approach using M06-
2X functional [37] that was proposed for the
correct description of non-covalent interac-
tions, including the representation of geometry
and energetic parameters of p-stacking com-
plexes [56]. Taking into account the comp-
lexity of the studied system, we have used
6-31G(d) basic set.
A number of possible structures of the com-
plexes have been identified (Fig. 4, Table 2).
In all cases, the stacking interaction of the
porphyrin ring with G-quartet is observed.
ImPhz fragment can bind in several ways: it
can freely move (structure A), interact with
G-quartet (B) or porphyrin (C), or intercalate
between the pair of quartets (D). The most
energetically favorable structures are those
where ImPhz residue interacts with G-quartet
(B) or with a porphyrin (C), i.e. complexes in
which both fragments of the conjugate par-
ticipate in binding interactions. Of course,
model structure B formed upon the conjugate
binding to G-octet cannot be realized with a
real Tel22 quadruplex containing 3 stacked
G-quartets, due to the insufficient linker length.
At the same time, the formation of intercalative
complex D where imidazophenazine chromo-
phore is inserted between two G-quartets is
associated with strong deformation of G-octet
that results in significant increase of system
energy and its destabilization. So the intercala-
tive mode of binding of ImPhz moiety is hard-
ly possible. The same is true for the porphyrin
fragment. It should be noted that metal cation
located between G-quartets prevents ligands
from efficient intercalation.
Table 2. Energetic parameters of (Zn)TMP3-ImPhz conjugate complexes with G-octet obtained with
M06-2X/6-31G(d)/CPCM method.
Energy,
kCal/mol
Structure
A B C D
Total -6523385.80 -6523413.28 -6523412.74 -6523380.59
Relative 27.48 0.0 0.54 32.59
394
Yu. V. Didan, M. M. Ilchenko, V. V. Negrutska et al.
Fig. 4. DFT calculated structures of porphyrin-imidazo-
phenazine 2b complexes with G-octet (side view). N–Zn
coordination bond in complex C is shown as a dashed
line.
Importantly, both spectral-fluorescent data
[29] and DFT calculations [23] suggest that
free conjugate 2a and its Zn(II) complex 2b in
water exist mainly in the form of rather stable
intramolecular head-to-tail heterodimers. Their
formation results from the strong electronic
A
B
C
D
395
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
interactions between porphyrin and ImPhz
chromophores, via either p-p-stacking (2a) or
metal coordination (2b). Moreover, it was
shown by fluorescence spectroscopy techniques
including the fluorescence polarization data that
these conjugates can bind to G4 structures in
the form of internal heterodimers [29–31], just
as in case of the calculated complex C.
Non-intercalative binding mode of the con-
jugates 2a-c to Tel22 quadruplex of antiparal-
lel topology was proposed; non-metalated
conjugate 2a was shown to bind to G4 via the
interaction of porphyrin moiety with a terminal
G-quartet, whereas the intercalative binding of
phenazine moiety to Tel22 was not observed
[29]. Phenazine intercalation between the gua-
nine bases was observed only for the interac-
tion of (Zn)TMP3-ImPhz conjugate 2b with a
long tetramolecular quadruplex formed by
poly(G) [31]. TMPyP itself was shown to bind
to the parallel Tel22 via the external end-stack-
ing rather than intercalation [18].
Thus, DFT modeling results are in good
agreement with experimental data on the con-
jugate binding to Tel22 G4.
Conclusion
All studied compounds have a strong binding
affinity for Tel22 quadruplex, with Kd values
in micromolar range. Both binding affinity
and selectivity of ligands depend on ionic
strength of the solution. Its increase results in
the decrease of ligand affinity for both duplex
and G4 DNA. Interestingly, this decrease is
associated with a significant increase of selec-
tivity of compounds for G4 resulting presum-
ably from the suppression of non-specific
electrostatic interactions. Theoretical model-
ing has revealed that stacking interaction of
G-quartet with a porphyrin fragment, but not
the intercalative binding of any conjugate
component, determines the structure of li-
gand–G4 complexes.
Acknowledgement
Computational facilities were provided by the
joint computer cluster of SSI Institute for
Single Crystals and Institute for Scintillation
Materials of the National Academy of Sciences
of Ukraine [57].
Funding
This research was in part supported by the
NAS of Ukraine program “Molecular and
Cellular Biotechnologies for Medicine,
Industry and Agriculture” (grant No. 43/18).
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Взаємодія кон’югатів катіонного порфірину
з імідазофеназином із ДНК-квадруплексом:
метод FID та квантово-хімічне моделювання
Ю. В. Дідан, М. М. Ільченко, В. В. Негруцька,
Л. В. Дубей, О. О. Рязанова, І. Я. Дубей
Мета. Дослідити ефективність трикатіонного
кон’югату порфірин–імідазо[4,5-b]феназин та його
Zn(II) і Mn(III) комплексів як лігандів G-квадру-
плексної (G4) ДНК. Методи. Метод заміщення флу-
оресцентного інтеркалятора (FID) використано для
оцінки афінності сполук до модельної дуплексної
ДНК та квадруплексу Tel22 за різних значень іонної
сили розчину. Молекулярне моделювання взаємодії
кон’югату з G4-ДНК проведено за допомогою об-
числень теорії функціоналу густини (DFT) з викорис-
танням функціоналу M06-2X та базисного набору
6-31G(d). Як модель G4 використано гуаніновий
октет, стабілізований іоном K+. Результати.
Визначено значення DC50 та константи дисоціації
комплексів трьох кон’югатів із дуплексною та ква-
друплексною ДНК. Розраховано структури та енер-
гетичні параметри комплексів G-октету з Zn-мета лі-
зованим кон’югатом. Висновки. Всі сполуки виявля-
ють високу афінність до квадруплексу Tel22.
Збільшення іонної сили веде до зростання селектив-
ності до квадруплексної відносно дуплексної ДНК та
зниження афінності зв’язування лігандів. Структуру
комплексів ліганд–G4 визначає передусім стекінгова
взаємодія порфіринового фрагмента з G-квартетом,
а не інтеркаляційне зв’язування ліганда.
К л юч ов і с л ов а: G-квадруплекс, ліганди, порфі-
рини, імідазофеназин, FID, DFT
Взаимодействие конъюгатов катионного
порфирина с имидазофеназином с ДНК-
квадруплексом: метод FID и квантово-
химическое моделирование
Ю. В. Дидан, Н. Н. Ильченко, В. В. Негруцкая,
Л. В. Дубей, О. А. Рязанова, И. Я. Дубей
Цель. Изучить эффективность трикатионного конъ-
югата порфирин–имидазо[4,5-b]феназин и его Zn(II)
и Mn(III) комплексов как лигандов G-квадруплексной
(G4) ДНК. Методы. Метод замещения флуоресцент-
399
Interaction of cationic porphyrin-imidazophenazine conjugates with DNA quadruplex: FID assay and quantum-chemical modeling
ного интеркалятора (FID) использован для оценки
аффинности соединений к модельной дуплексной
ДНК и квадруплексу Tel22 при разных значениях
ионной силы раствора. Молекулярное моделирование
взаимодействия конъюгата с G4-ДНК проведено при
помощи вычислений теории функционала плотности
(DFT) с использованием функционала M06-2X и
базисного набора 6-31G(d). В качестве модели G4
использован гуаниновый октет, стабилизированный
ионом K+. Результаты. Определены значения DC50
и константы диссоциации комплексов трех конъю-
гатов с дуплексной и квадруплексной ДНК.
Рассчитаны структуры и энергетические параметры
комплексов G-октета с Zn-металлизированным конъ-
югатом. Выводы. Все соединения проявляют высо-
кую аффинность к квадруплексу Tel22. Увеличение
ионной силы приводит к возрастанию селективности
к квадруплексной относительно дуплексной ДНК и
снижению аффинности связывания лигандов.
Структуру комплексов лиганд–G4 определяет прежде
всего стекинговое взаимодействие порфиринового
фрагмента с G-квартетом, а не интеркаляционное
связывание лиганда.
К л юч е в ы е с л ов а: G-квадруплекс, лиганды, пор-
фирины, имидазофеназин, FID, DFT
Received 11.08.2018
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