Switch in site of inhibition: A strategy for structure-based discovery of human topoisomerase IIα catalytic inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00040

A study of structure-based modulation of known ligands of hTopoIIα, an important enzyme involved in DNA processes, coupled with synthesis and in vitro assays led to the establishment of a strategy of rational switch in mode of inhibition of the enzyme's catalytic cycle. 6-Arylated derivatives of known imidazopyridine ligands were found to be selective inhibitors of hTopoIIα, while not showing TopoI inhibition and DNA binding. Interestingly, while the parent imidazopyridines acted as ATP-competitive inhibitors, arylated derivatives inhibited DNA cleavage similar to merbarone, indicating a switch in mode of inhibition from ATP-hydrolysis to the DNA-cleavage stage of catalytic cycle of the enzyme. The 6-aryl-imidazopyridines were relatively more cytotoxic than etoposide in cancer cells and less toxic to normal cells. Such unprecedented strategy will encourage research on choice-based change in target-specific mode of action for rapid drug discovery.

Full text of DOI:10.1021/acsmedchemlett.5b00040

Letter
pubs.acs.org/acsmedchemlett
Switch in Site of Inhibition: A Strategy for Structure-Based Discovery
of Human Topoisomerase IIα Catalytic Inhibitors
Ashish T. Baviskar,†,⊥ Suyog M. Amrutkar,†,⊥ Neha Trivedi,‡ Vikas Chaudhary,§ Anmada Nayak,∥
Sankar K. Guchhait,*,§ Uttam C. Banerjee,† Prasad V. Bharatam,‡,§ and Chanakya N. Kundu∥
‡
§
†Department of Pharmaceutical Technology (Biotechnology), Department of Pharmacoinformatics, and Department of Medicinal
Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar, Mohali, Punjab 160062,
India
∥School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India
S
* Supporting Information
ABSTRACT: A study of structure-based modulation of known ligands of hTopoIIα, an
important enzyme involved in DNA processes, coupled with synthesis and in vitro assays led
to the establishment of a strategy of rational switch in mode of inhibition of the enzyme’s
catalytic cycle. 6-Arylated derivatives of known imidazopyridine ligands were found to be
selective inhibitors of hTopoIIα, while not showing TopoI inhibition and DNA binding.
Interestingly, while the parent imidazopyridines acted as ATP-competitive inhibitors, arylated
derivatives inhibited DNA cleavage similar to merbarone, indicating a switch in mode of
inhibition from ATP-hydrolysis to the DNA-cleavage stage of catalytic cycle of the enzyme.
The 6-aryl-imidazopyridines were relatively more cytotoxic than etoposide in cancer cells and
less toxic to normal cells. Such unprecedented strategy will encourage research on “choice-
based change” in target-specific mode of action for rapid drug discovery.
KEYWORDS: Human topoisomerase IIα, anticancer agent, chemotype-modulation, drug discovery
oward rapid finding of new chemical entities (NCE), the
overcome the multidrug resistance (MDR).9 Many catalytic
Tstructural modulation of a known drug or bioactive agent inhibitors are now clinically used in combination therapy, such
as aclarubicin,10 MST-16,11 ICRF-187,12 suramin,13 and
novobiocin.14 Merbarone has a unique feature of being the
only agent that causes inhibition of TopoII-mediated cleavage
of DNA without affecting protein−DNA binding.15
has been recognized as a valuable approach.1,2 Many times,
structurally derived compounds have been found to interact
with a new molecular target, rather than an original one.
Podophyllotoxin is tubulin polymerization inhibitor,3 whereas
its synthetic derivative etoposide is a topoisomerase II-inhibitor
(TopoII). In contrast, the structural modulation leading to
identification of a new agent that interacts with the same target
at different binding site is rare in literature. The examples of
such approach are the development of allosteric modulators of
the G-protein coupled receptors4 and structural modulation of
RORc inverse agonistic tertiary sulfonamides, leading to
agonistic effects.5
DNA topoisomerase maintains the DNA topology and thus
plays a crucial role in various DNA processes.6 About 50% of
chemotherapeutic regimens use at least one drug that targets
these enzymes.7 There are several hTopoIIα-targeting anti-
cancer drugs. Etoposide, teniposide, doxorubicin, and mitox-
antrone form ternary complex with the enzyme and DNA and
are called hTopoIIα poisons. The hTopoIIα catalytic inhibitors
(Scheme 1a) hamper the enzyme−DNA cycle in the stages
other than ternary complex formation.8 Topoisomerase II
poisons were found to be responsible for triggering secondary
leukemias by causing certain chromosomal translocations.8 In
contrast TopoII catalytic inhibitors are known to modulate the
cytotoxic effects of the poisons and the alkylating agents and
In a rational structural modulation of a known hTopoIIα
catalytic inhibitor class of compounds, imidazo-pyridines/
pyrazines,16,17 our initial docking studies revealed an astonish-
ing important feature. The C2-biaryl and C6-aryl derivatizations
implied the possibility of a switch in binding with hTopoIIα
from the ATP-hydrolysis to DNA cleavage stage of the catalytic
cycle. This prompted us to consider a study of detailed
exploration/understanding of molecular interactions of the
hTopoIIα with DNA or known ligands and structure-based
rational modulations of the ligands. The present work illustrates
an unprecedented target-specific ligand-structural modulation
approach that provides “choice-based change” in the mode of
inhibition of an enzyme, hTopoIIα.
In the preparation of target compounds (5 and 6, Scheme
1b) by arylation at C2-aryl and C6 of imidazo[1,2-a]-pyridines/
pyrazines, 2-(4-bromophenyl) substituted imidazo-pyridines/
pyrazines (3) and 6-bromo-imidazopyridines (4) as respective
Received: February 1, 2015
Accepted: February 23, 2015
© XXXX American Chemical Society
A
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cannot enter due to its large size and appears at the top;
whereas the decatenated products (nicked (Nck), relaxed (Rel),
and supercoiled (SC) DNA) move easily into the gel.16,21
Interestingly, compared to etoposide, negligible decatenated
products were observed in the presence of compounds 5f, 5g,
and 6a−e, and relatively less decatenation occurred for
compounds 5d, 5e, and 5i (Figure 1a). In the relaxation
assay, variably relaxed topoisomers of negatively supercoiled
DNA (pRYG) were observed, which migrated relatively slow as
compared to the supercoiled form in agarose gel (Figure 1b).
The hTopoIIα-relaxation inhibitory activities of the compounds
5d−g, 5i, and 6a−e were found to be in accordance with their
hTopoIIα decatenation activities. The results of decatenation
and relaxation assay indicated that C6-arylated imidazopyridine
derivatives 6a−e were potent inhibitors and that compound 6a
was most potent (IC50 was 55.37 μM; Figure S1).
Scheme 1. (a) Representative hTopoIIα Catalytic
Inhibitors;16 (b) Synthesis of Imidazo-pyridine/pyrazine
a
Derivatives (5 and 6)
Topoisomerase II poisons such as etoposide stabilizes the
enzyme−DNA complex and thus generates linear DNA (Lin).
In our study of cleavage complex assay, the Lin form of the
DNA was observed for etoposide, but not for investigated
compounds 6a−e (Figure 1c), which indicated their hTopoIIα
catalytic inhibition property not as poison. Human topoisomer-
ase I (hTopoI)-mediated relaxation assay22 for compounds 6a−
e showed their nonsignificant activity for inhibition of the
enzyme (Figure 1d). This indicated that the compounds were
selective for catalytic inhibition of hTopoIIα.
To investigate the cytotoxicity of compounds (6a−e), an 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was performed using representative human
embryonic kidney cancer (HEK 293) and normal monkey
kidney epithelial (Vero) cell lines.16,23 With increasing
concentration of the investigated compounds, the cell viability
of HEK 293 significantly decreased in comparison to Vero cells.
Compounds killed 50% HEK 293 cells (LC50) at 10, 10, 8, 15,
and 13 μM, respectively (Figure 2). Interestingly, more than
30% cells survived even after exposure to 75 μM concentration
of compounds 6a−e in Vero cells; whereas etoposide showed
LC50 at 55 μM for HEK 293 cells and at 75 μM for Vero cells.
To investigate the topoisomerase II inhibitory properties of the
compounds directly in cancer cells, we studied an electro-
phoretic assay using nuclear lysate of HEK 293 cells (Figure 3).
The untreated lysate showed the highest topoisomerase activity
(lane 2), and the increasing movement of DNA entering into
the gel was noted with increasing concentration (5−15 μM) of
compound 6a (lane 3−5), which revealed an increasing
a
Reagents and conditions: Step 1: ZrCl4 (10 mol %), n-BuOH, 50 °C,
3−4 h.18,19 Step 2: R3/R4-B(OH)2 (1.3 equiv), Pd(PPh3)4 (10 mol %),
TBAB (1 equiv), Na2CO3 (5 equiv), DMA-H2O (1:1), 105 °C, 3−4
h.20
precursors were considered. Compounds 3 were prepared by a
ZrCl4-catalyzed Ugi−Strecker-type multicomponent reaction of
2-aminopyridine/pyrazine with 4-bromobenzaldehyde and tert-
butyl isocyanide.18 Compounds 4 were synthesized using 5-
bromo-2-aminopyridine. Pd-catalyzed Suzuki coupling of
various boronic acids with compounds 3 and 4 produced
imidazo[1,2-a]pyridines/pyrazines (5a−j and 6a−k), respec-
tively, in good yields.20
The hTopoIIα-inhibitory activities of synthesized com-
pounds were investigated by in vitro ATP-dependent
decatenation and relaxation assays in agarose gel electro-
phoresis.16 In agarose gel, catenated kinetoplast DNA (kDNA)
Figure 1. (a) Decatenation assay, (b) relaxation assay, (c) cleavage complex assay, and (d) Topo I-mediated relaxation assay of imidazo-pyridines/
pyrazines.
B
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◆
●
▲
Figure 2. MTT assay: The signs − −, − −, − −, and − − represent cell survival for HEK-293/investigated, Vero/investigated, HEK-293/
etoposide, and Vero/etoposide, respectively.
site in the central domain of hTopoIIα (PDB ID: 4FM9).24
However, these compounds are not hTopoIIα poisons like
etoposide.25 Hence, the interactions of these compounds are
expected to be different from that of etoposide. Literature
search reveals that merbarone (a catalytic inhibitor of
hTopoIIα) occupies an interaction domain overlapping with
that of etoposide.15 This is supported by the fact that
merbarone can competitively impede the inhibition of cleavage
Figure 3. Inhibition of topoisomerase-activity by compound 6a in
HEK 293 cells.
complex formation by etoposide.15 To the best of our
knowledge, molecular recognition interactions of merbarone
are not yet reported in the literature. The important
inhibitory activity of hTopoIIα-mediated relaxation of DNA. In
comparison with etoposide (45 μM, lane 6), the inhibitory-
activity of compound 6a was found to be significant. We then
studied to find out the stage at which these novel compounds
inhibited the catalytic cycle of hTopoIIα. The catalytic inhibitor
that binds with DNA changes its conformation while binding
and thus prevents it from interacting with topoisomerase II in
the first step of the catalytic cycle (e.g., aclarubicin, a DNA
components of the catalytic machinery of the enzyme (Mg2+,
TOPRIM, and TyrB805) are located in the central domain
(Figure S4) near the etoposide/merbarone-binding pocket.26,27
The results from molecular docking of merbarone in
hTopoIIα (emodel28 score −64.17) showed that merbarone
formed metal coordination bond (through carbonyl oxygens)
with Mg2+ (2.09 Å), hydrogen bonding interaction with Asp543
(3.13 Å), Asp545 (1.90 Å), and Lys614 (2.91 Å), and NH···π
interactions with Lys614 (distance 3.50 Å) (Figure 4). The
intercalating agent). The absorbance study in DNA binding
assay and the DNA intercalation assay revealed that there was
binding is further stabilized by interaction with polar residues
(Gln544, Arg713, His758, and His759) and hydrophobic
residues (Gly546, Ile577, Leu592, and Tyr805). This set of
interactions with catalytically important components of
hTopoIIα may be considered as the molecular recognition
interactions for merbarone. In order to understand the in vitro
competitive binding of merbarone and etoposide, a comparative
molecular docking analysis was undertaken (see Supporting
Information for details, Figure S5).
The molecular docking of compounds 6a−e revealed that
they could interact at merbarone-binding site and showed
interactions similar to that of merbarone (Figure 4), which are
with Mg2+, Asp541, Asp543, and Asp545 (of chain A) and
little or no significant binding affinity of the compound 6a with
DNA (see Supporting Information for details, Figure S2).
From quantum chemical studies, these imidazopyridine
derivatives were established to be basic in nature (see
Supporting Information, Tables S1 and S2). Therefore, their
N1-protonated forms were considered for molecular modeling
studies. A primary structural analysis supported by the in vitro
ATPase inhibition assay (see Supporting Information for
details, Figure S3) indicated that these molecules cannot
occupy any binding site in the ATPase domain (neither the
ATP binding site nor the ICRF binding site). Accordingly,
docking studies at other possible binding sites were performed,
which revealed a binding domain close to etoposide binding
Figure 4. (a) Merbarone, (b) compound 6a, and (c) alignment of compounds 6a−e in the merbarone-binding pocket of hTopoIIα. Red dashed
lines represent the π−π, cation−π , and NH−π interactions. Black dashed lines are hydrogen bonding interactions. Mg2+ is shown as a nonbonded
sphere (magenta). Residues that are involved in hydrogen bonding are shown in stick presentation. For clarity, only polar hydrogens are displayed.
C
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Tyr805 (of chain B). Additionally, NH···π interactions with
Arg713 and His759 also contribute. Further stabilization of the
complex is contributed by the polar residues (Lys614, Ser709,
Glu712, Lys728, His758, and Asp831) and the hydrophobic
residues (Gly546, Ile549, Gly760, Ile577, and Leu592). The
molecules 6a−e showed cation···π interaction with Mg2+ via
C6-aryl-substitution. The docking scores (emodel) of the active
compounds are quite comparable to that of merbarone, and
also a good correlation with the relative hTopoIIα-inhibitory
activities was observed (Table S3). Further, a molecular
docking analysis of the neutral imidazo-pyridines/pyrazines
revealed a poor correlation with the biological activity (see
Supporting Information, Table S4). The parent imidazo-
pyridines (nonarylated, Figure S6) were known to interact
with hTopoIIα at the ATPase domain (evident by molecular
modeling and in vitro studies).16 With the interactions involved
in the merbarone-binding site as explored in this study, it was
found that the parent nonarylated imidazo-pyridines could also
have similar interactions (Table S5). Therefore, it can be
assumed that the C6 nonarylated imidazo-pyridines preferably
bind at ATP-binding domain but can also be accommodated in
the merbarone-binding site. However, their arylated analogues
selectively bind only at the merbarone-binding site (see SI for
structure−activity relationships of these series of molecules). In
support of these molecular modeling results, we performed an
in vitro assay15 for the attenuation of etoposide-enhanced
hTopoIIα-mediated DNA cleavage using compound 6a and a
representative compound of previously reported16 C6-non-
arylated imidazo-pyridines, 4-(3-(tert-butylamino)imidazo[1,2-
a]pyridin-2-yl)benzoic acid (IP-C6-H, 7a, Figure S6). It was
observed that the DNA cleavage decreased with an increase in
the concentration (100 to 400 μM) of compound 6a (Figure
5). This inhibition is dependent on the order of sequential
addition of compounds. Adding compound 6a before etoposide
significantly reduced the formation of the cleavage complex.
These observations are in accordance with the reported15
effects of merbarone toward attenuation of etoposide-enhanced
hTopoIIα-mediated DNA cleavage.
Figure 5. (a) Attenuation of etoposide-enhanced hTopoIIα-mediated
DNA cleavage15 by compound 6a. The order of addition of inhibitors
was as follows: etoposide → 6a (etoposide first followed by compound
6a), etoposide + 6a (etoposide and compound 6a simultaneously),
and 6a → etoposide (compound 6a first followed by etoposide). (b)
Quantification of the results.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of synthetic procedure, characterization, and analytical
data, HPLC study (>95% purity), assay protocols, and
molecular modeling methodology. This material is available
free of charge via the Internet at http://pubs.acs.org.
Interestingly, compound 7a showed no such effect at 100 μM
and a small effect at 200 and 400 μM concentrations (Figure
S7). Thus, compound 7a has preferential binding at the ATPase
domain of hTopoIIα, although some interaction at merbarone-
binding site also occurs at increased concentration. Taken all
together, these results provide an interesting and unique
observation, i.e., a switch in the site of inhibition from ATP-
hydrolysis to DNA cleavage stage in catalytic cycle of hTopoIIα
with a rational structural modulation, i.e., arylation of imidazo-
pyridine.
In conclusion, the exploration/understanding of molecular
interactions of the hTopoIIα with known ligands, structure-
based rational modulations of imidazo-pyridines/pyrazines,
synthesis, and relevant in vitro evaluations have led to the
establishment of a strategy of “choice-based change” in catalytic
mode of inhibition of hTopoIIα and development of the new
potent and specific inhibitors and cytotoxic agents. A set of
residues (Mg2+, TOPRIM, TyrB805, and ArgB804) important
for interaction in merbarone-binding domain has also been
explored for the first time. This work will prompt for rational
structural modulation studies of known hTopoIIα-based drugs/
agents toward “choice-based change” in mode of action for
rapid drug discovery. The concept and the results are also
useful in the bacterial (DNA-gyrase) and leishmanial
topoisomerase II enzyme-based research.
AUTHOR INFORMATION
Corresponding Author
*E-mail: skguchhait@niper.ac.in. Phone: +91 (0)172 2214683.
Fax: +91 (0)172 2214692.
■
Author Contributions
⊥These authors contributed equally to this work.
Funding
We gratefully acknowledge financial support from DBT, DST,
and CSIR, Government of India, New Delhi, for this
investigation. N.T. and V.C. are thankful to CSIR for their
fellowships.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
hTopoIIα, human topoisomerase IIα; MDR, multidrug
resistance; TBAB, tetra-n-butylammonium bromide; DMA,
N,N-dimethylacetamide; kDNA, kinetoplast DNA; Nck,
nicked; Rel, relaxed; Lin, linear; SC, supercoiled; TAE, Tris-
acetate-EDTA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nylte-trazolium bromide; MER, merbarone; ANP, phosphoa-
minophosphonic acid-adenylate ester; ICRF-187, (+)-1,2-
Bis(3,5-dioxopiperazinyl-1-yl)propane/dexrazoxane; rmsd,
D
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root-mean-square deviation; IEFPCM, integral equation
formalism polarizable continuum model
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