Targeting cytotoxicity and tubulin polymerization by metal-carbene complexes on a purine tautomer platform

Article abstract of DOI:10.1039/c4dt00529e

This communication describes the synthesis, structural investigation and tubulin binding of purine rare imino-tautomer based Ag(i) and Hg(ii)-carbene complexes. These complexes exhibit cytotoxicity through tubulin interaction by binding to a site close to

Full text of DOI:10.1039/c4dt00529e

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Targeting cytotoxicity and tubulin polymerization
by metal–carbene complexes on a purine
tautomer platform†
Cite this: Dalton Trans., 2014, 43,
9838
Received 20th February 2014,
Accepted 9th April 2014
Shruti Khanna,a Batakrishna Jana,b Abhijit Saha,b Prashant Kurkute,b Surajit Ghosh*b
and Sandeep Verma*a,c
DOI: 10.1039/c4dt00529e
www.rsc.org/dalton
This communication describes the synthesis, structural investi- membrane potential, nuclear translocation of apoptosis-indu-
gation and tubulin binding of purine rare imino-tautomer based cing factors and caspase-12 and inhibition of cysteine-depen-
Ag(I) and Hg(II)–carbene complexes. These complexes exhibit cyto- dent protein tyrosine phosphatases and thioredoxin reductase,
toxicity through tubulin interaction by binding to a site close to to name a few.2
the GTP binding site.
Imidazolium moieties carrying synthetic ligands are the
preferred scaffolds to stabilize carbenoid species, thus the
majority of reports focus on the modification of this hetero-
cycle for NHC synthesis.3a,4 Notably, despite the presence of
an imidazole-like ring in purine heterocycles, reports of
carbene generation on natural purine nucleobases are rare,
and most reports concern xanthine and caffeine.5 Recent
advances in organometallic anticancer drugs and the possi-
bility of using natural purines, such as adenine and guanine,
gave us an impetus to design adenine-based carbenes as anti-
tumor agents.6
We have investigated metalated adenine analogues for the
generation of higher order supramolecular frameworks and
surface patterning and the synthesis of polymeric adenine
templates, and their use in catalysis and DNA cleavage.7 The
present study describes a novel methoxyadenine rare tautomer
platform for stabilizing silver- and mercury-based carbenoid
species, for possible cytotoxicity and interaction with micro-
tubules. The structural tunability of the ligand and metal ion
stabilizing carbene was considered as a beneficial feature to
exploit this framework in these studies.
It was envisaged that the two benzyl rings on the imidazole
ring in ligand 1 would render the C8 carbon susceptible for
carbene generation, which in turn could react with Ag or Hg to
create the desired organometallic species. Consequently, C8–H
abstraction was achieved using Ag2O and Hg(OAc)2, where the
metal oxide or acetate acted as a base as well as a metal center,
stabilizing the formation of the N-heterocyclic carbene. The
proposed structures of the C8 centered metallodimers, 1A and
1B, are shown in Scheme 1. The syntheses of ligand 1 and
complexes 1A and 1B are described in the ESI.† The formation
of these complexes was confirmed by spectroscopic methods.
1H NMR spectra of 1A and 1B clearly revealed complete dis-
appearance of the C8–H signals present at δ 9.26 in ligand 1
The biological action of organometallic compounds has eli-
cited considerable interest in the past and it continues to
attract close scrutiny due to the diverse mechanisms of action,
especially in anticancer chemotherapy.1 In addition to the con-
ventional nucleic acid target, organometallic anticancer com-
pounds are being designed to seek alternate macromolecular
targets and pathways. Compounds built around cyclopenta-
dienyl cores, metal–arene interactions, polynuclear clusters and
N-heterocyclic carbenes (NHCs), are promising candidates
against a variety of cancer cell lines. Their promise is centered
around a predictable three dimensional structure, fine-tuning
of their properties via the choice of metal ions and transport
to desired sites using conventional drug delivery strategies.2
In particular, metal–NHCs offer a versatile platform for dis-
covering novel metallodrugs as their synthesis is relatively
simple and it can support different metal ions such as Ag, Au,
Pt, Pd, Cu, Ni, and Ru, thus allowing faster optimization of the
structural requirements and biological activity, in a divergent
fashion.3 The anticancer action of metal–NHCs is of contem-
porary interest as these complexes exert favourable biological
action through a number of mechanisms such as the acti-
vation of apoptosis, depolarization of the mitochondria inner
aDepartment of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016,
UP, India. E-mail: sverma@iitk.ac.in
bChemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick
Road, Kolkata 700 032, WB, India. E-mail: sghosh@iicb.res.in
cDST Thematic Unit of Excellence on Soft Nanofabrication, Centre for Environmental
Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016,
India
†Electronic supplementary information (ESI) available. CCDC 965785. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4dt00529e
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Scheme 1 Synthesis of silver carbene (1A) and mercury carbene (1B) complexes from ligand 1. Insets: DFT-optimized geometries of 1A and 1B.
(Fig. S2†). This was further confirmed by 13C NMR where a pathway.12 Mitochondrial targeting and the lack of genotoxi-
peak corresponding to C8 at δ 136.37 shifted to δ 178.52 for 1A city further augmented the scope of their biological action.
and 175.80 for 1B, characteristic of the corresponding
metal carbene signal (Fig. S3†). HRMS data also confirmed the metal complexes (1A and 1B) by MTT assay, which uses cytoso-
formation of carbene complexes 1A and 1B (Fig. S4†). lic redox enzymes for the reduction of tetrazolium dye as an
We assessed the anticancer properties of ligand 1 and its
Complexes 1A and 1B did not crystallize despite trying mul- indicator of cell viability, in A549 lung cancer and MCF7 breast
tiple crystallization conditions. Thus, we resorted to Density cancer cell lines, for 24 h. Curcumin, a known anticancer com-
Functional Theory (DFT) calculations to gain insight into the pound, was used as a control.13 1, 1A and 1B (40 μM) exhibited
structural features of these carbene complexes. DFT calcu- similar cytotoxicity (loss of viable cells) against the A549
lations for both complexes were performed by employing adenocarcinoma cell line, which was comparable to the curcu-
B3LYP exchange-correlation functional,8 with a 6-31G(d,p) min control (40 μM) (Fig. 1a). However, for the MCF7 cell line,
basis set for all non-metallic atoms and a LANL2DZ basis set 1, 1A and 1B (40 μM) show ∼20%, ∼60% and ∼90% cyto-
for both Ag and Hg ions9 (see the ESI† for geometry optimi- toxicity, respectively, when compared to ∼70% cytotoxicity exhibi-
zations). Normal mode analysis of the vibrational frequencies ted by curcumin (40 μM) i.e., the MCF7 cell line is 80%, 40%,
was performed and the absence of any negative frequency 10% and 30% viable after treatment with 1, 1A, 1B and curcu-
confirmed the structure to be at minima (at least local). These min, respectively, (Fig. 1b). These results suggest that while 1
calculations considered basis set superposition errors (BSSE) is least cytotoxic to the MCF7 cell line, the cytotoxicity of 1A is
in order to calculate accurate energies in the counterpoise comparable to that of curcumin with 1B, exhibiting excellent
approximation.10 Geometry optimization of 1A and 1B cytotoxicity. At this time, it can be proposed that this differ-
afforded nearly linear carbene complexes, with a C8–Ag–C8′ ence in cytotoxicity could be attributed to specificity for a
bond angle of 178.63° and a C8–Hg–C8′ bond angle of 170.62° particular cell line. After determining the cytotoxicity of these
(Scheme 1). A slight elongation in the C8–N bond was compounds, changes in the morphology of the cancer cell
observed, while the N7–C8–N9 bond angle decreased by 3.13° lines were studied after 24 h conjugate uptake and the ensuing
in 1A and by 1.9° in 1B (Table S2†). All of the DFT calculations changes in cellular morphology were compared, as evident
were performed using Gaussian 09 (see ESI†).
from the differential interference contrast images (Fig. 2, S5
Silver and other metal complexes have been studied for and S6†). Cell deformation alludes to the potential anticancer
multiple biological effects such as antiseptic activity, inhi- activity of 1A and 1B.
bition of inflammation and antibacterial and antitumor
We decided to probe the possible mechanism for morpho-
action.11 We became interested in assessing the possible anti- logical changes by these conjugates. It is well known that
cancer action of 1A and 1B, as recent studies reported the microtubules, an important constituent of the cytoskeleton,
effect of silver complexes on caspase-independent cancer cell are key targets for anticancer drug action. Moreover, micro-
apoptosis through a mitochondrial apoptosis inducing factor tubules are also one of the vital cytoskeleton filaments, which
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pounds is perhaps a consequence of disruption of the micro-
tubule networks leading to deformation of cellular morphology.
The effect of these conjugates on microtubule dynamics
was further investigated by following in vitro microtubule
assembly/polymerization, via a turbidity assay, in the presence
of 1, 1A, 1B and curcumin, at 37 °C for 40 min. Curcumin is
known to induce significant depolymerization of micro-
tubules.14 A significant increase in turbidity, when measured
at 350 nm, is observed during in vitro microtubule polymeriz-
ation.15 Interference with this process was inferred from the
turbidity data (Fig. 4a), where 1A offered the greatest inhibition
and relative inhibition was determined to be 1A > curcumin >
1 > 1B. Thus, the anticancer activity of 1A and 1B could be
ascribed to their ability to target microtubule polymerization.
Anti-cancer activities of our conjugates prompted us to
qualitatively interrogate their interaction with tubulin focusing
on the probable binding site, nature of binding and the amino
acids supporting this interaction. As the adenine imino tauto-
mer exhibits structural similarity to guanine, we decided to
investigate the possibility of our tautomer occupying the GTP
binding site in β-tubulin through docking studies.16 It is
known that GTP binds to both α and β-tubulin monomers,
where β-tubulin bound GTP is hydrolyzed during microtubule
assembly.17 Docking studies performed to assess the binding
of 1 in proximity to the GTP binding site suggested that 1
Fig. 1 The percentage viability of the cancer cell lines after treatment
with 1, its metal complexes (1A and 1B) and curcumin (as a control) was
assessed by MTT assay in: (a) A549 and (b) MCF7 cell lines for 24 h.
maintain cell structure. Therefore, the ability of these conju- probably binds close to the Asn117 residue of β-tubulin
gates to perturb microtubule networks inside cancer cells was (Fig. 4b and c), similar to GTP/GDP binding. Incidentally, this
assessed. As imagined, the microtubule networks in A549 and site is proximal to the GTP/GDP binding pocket of β-tubulin
MCF7 cell lines were dramatically affected after treatment with (Fig. 4d).17 Such an interaction could possibly be ascribed
the conjugates for 24 h, which resulted in drastic morphologi- to the structural similarity between GTP and rare purine
cal changes, suggesting this as a possible mechanism in elicit- tautomer, 1. Thus, it could be proposed that 1 exerts its action
ing an anticancer response (Fig. 3, S7†). Based on these through hydrophobic and hydrogen bonding interactions with
results, we surmise that the anticancer activity of our com- β-tubulin.
Fig. 2 Changes in cell morphology after treatment with 1 and its complexes for 24 h. Upper panel: (a) untreated A549 cells; (b) with 1; (c) with 1A;
(d) with 1B. Lower panel: (e) untreated MCF7 cells; (f) with 1; (g) with 1A; (h) with 1B [scale bar = 20 μm].
9840 | Dalton Trans., 2014, 43, 9838–9842
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Fig. 3 Changes in the cellular microtubule network on treatment with 1 and its complexes for 24 h. Upper panel: (a) untreated A549 cells; (b) with
1; (c) with 1A; (d) with 1B. Lower panel: (e) untreated MCF7 cells; (f) with 1; (g) with 1A; (h) with 1B. [Scale bar = 20 μm; tubulin labelled by antibody
(red) and nucleus stained by DAPI (blue)].
discovery of microtubule polymerization inhibitors. Further
investigations in this direction are currently in process.
Acknowledgements
The Department of Science and Technology, Government of
India, is acknowledged for the J C Bose National Fellowship to
SV and Ramanujan Fellowship to SG. SG also thanks CSIR-
IICB, Kolkata for financial support through network project
(BSC0113). SK, BJ, AS, and PK acknowledge CSIR, India, for
research fellowship. We thank NCCS Pune for the cell lines,
Bikramjit Sharma and Dr Shubhra Awasthi, Department
of Chemistry, IIT Kanpur, for help with DFT calculations
and Prof. K. Bhattacharya (IACS, Kolkata) and Dr N. C. Maity
(CSIR-IICB, Kolkata) for access to instrumentation. This
Fig. 4 (a) Turbidity assay indicating the inhibition of tubulin polymeriz-
ation by curcumin, 1, 1A and 1B. (b) Docked image depicting the inter-
action of 1 with Asn117 in β-tubulin. (c) Binding of 1 with the Asn117
residue of β-tubulin through H-bonding. (d) Docking studies illustrate a
work was inspired by the international and interdisciplinary
environments of the JSPS Asian CORE Program, “Asian Chemi-
cal Biology Initiative”.
similar binding pocket for 1 and guanine nucleotide, in the β-tubulin
subunit.
Notes and references
In conclusion, we have presented carbene generation on
a rare purine tautomer, its characterization by analytical
methods, its structure via DFT calculations, and its potential
anticancer activity against lung carcinoma and breast cancer
cell lines, A549 and MCF7, respectively. 1, 1A and 1B altered
the cellular morphology by inhibiting tubulin polymerization
via noncovalent interactions in the tubulin binding site. Given
their structural similarity to guanine, 1 and its metal com-
plexes are implicated in blocking the GTP binding site. We
surmise that this approach may present a new avenue for the
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9842 | Dalton Trans., 2014, 43, 9838–9842
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