Organoruthenium(II) complexes attenuate stress in Caenorhabditis elegans through regulating antioxidant machinery

Article abstract of DOI:10.1016/j.ejmech.2019.02.029

The 1:1 stoichiometric reactions of 3-methoxy salicylaldehyde-4(N)-substituted thiosemicarbazones (H₂L^1?4) with [RuCpCl(PPh₃)₂] was carried out in methanol. The obtained complexes (1–4) were characterized by ana

Full text of DOI:10.1016/j.ejmech.2019.02.029

Accepted Manuscript
Organoruthenium(II) complexes attenuate stress in Caenorhabditis elegans through
regulating antioxidant machinery
A. Mohankumar, G. Devagi, G. Shanmugam, S. Nivitha, P. Sundararaj, F. Dallemer,
P. Kalaivani, R. Prabhakaran
PII:
S0223-5234(19)30143-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.029
EJMECH 11121
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2018
Revised Date: 7 February 2019
Accepted Date: 9 February 2019
Please cite this article as: A. Mohankumar, G. Devagi, G. Shanmugam, S. Nivitha, P. Sundararaj,
F. Dallemer, P. Kalaivani, R. Prabhakaran, Organoruthenium(II) complexes attenuate stress in
Caenorhabditis elegans through regulating antioxidant machinery, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.029.
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ACCEPTED MANUSCRIPT
Organoruthenium(II) complexes attenuate stress in Caenorhabditis elegans
through regulating antioxidant machinery
A. Mohankumar, a G. Devagib, G. Shanmugama, S. Nivithac, P. Sundararaja, F. Dallemerd,
P. Kalaivanie*, R. Prabhakarana*
aDepartment of Zoology, Bharathiar University, Coimbatore 641 046, Tamilnadu, India
bDepartment of Chemistry, Bharathiar University, Coimbatore 641 046, Tamilnadu, India
cCollege of Science, Northeastern University, Boston, Massachusetts 02115, USA
dLaboratoire MADIREL CNRS UMR7246 , Université of Aix-Marseille , Centre de Saint-
Jérôme, bât. MADIREL, 13397 Marseille cedex 20, France
eDepartment of Chemistry, Nirmala College for Women, Bharathiar University, Tamilnadu,
Coimbatore 641018
Abstract
The 1:1 stoichiometric reactions of 3-methoxy salicylaldehyde-4(N)-substituted
thiosemicarbazones (H2L1–4) with [RuCpCl(PPh3)2] was carried out in methanol. The obtained
1
complexes (1-4) were characterized by analytical, IR, absorption and H-NMR spectroscopic
studies. The structures of ligand [H2-3MSal-etsc] (H2L3) and complex [RuCp(Msal-etsc)(PPh3)]
(3), were characterized by single crystal X-ray diffraction studies. The interaction of the
ruthenium(II) complexes(1–4) with calfthymus DNA (CT-DNA) has been explored by
absorption and emission titration methods. Based on the observations, an intercalative binding
mode of DNA has been proposed. The protein binding abilities of the new complexes were
monitored by quenching the tryptophan and tyrosine residues of BSA, as model protein. From
the studies, it was found that the new ruthenium metallacycles exhibited better affinity than their
precursors. The free radical scavenging assay suggests that all complexes effectively scavenged
the DPPH radicals as compared to that of standard control ascorbic acid and scavenging
activities of complexes are in the order of 4 >2 > 3 > 1. In addition, ruthenium(II) complexes (2-
4) also exhibited an excellent in vivo antioxidant activity as it was able to increase the survival of
worms exposed to lethal oxidative and thermal stresses possibly through reducing the
intracellular ROS levels. It was interesting to note that complexes 2-4 failed to increase the
lifespan of mev-1 mutant worms having shortened lifespan due to the over production of free
radicals. This data confirmed that complexes 2-4 conferred stress resistance in C. elegans, but
they also require an endogenous detoxification mechanism for doing so. The genetic and reporter
gene expression analysis revealed that complexes 2-4 maintained the intracellular redox status
and offered stress protection through transactivation of antioxidant defence machinery genes gst-
4 and sod-3 which are directly regulated by SKN-1 and DAF-16 transcription factors,
respectively. Altogether, our results suggested that complexes 2-4 might play a crucial role in
stress modulation and they perhaps exert almost similar effects in higher models, which is an
important issue to be validated in future.
Keywords: Ruthenium(II); anti oxidant; Caenorhabditis elegans; Stress resistance; ROS
Corresponding author Tel.: +91-422-2428319; Fax: +91-422-2422387.
E-mail: kalaivani19@gmail.com(P.Kalaivani); rpnchemist@gmail.com (R. Prabhakaran)
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Introduction
Organometallic compounds have effective applications in the treatments of viral, microbial and
cancer diseases and also have cardio protective effects [1]. Besides, organometallic compounds
also showed some specific characteristics like wide and diverse structural types, variation in
ligand bonding modes and redox properties which make them attractive in medicinal chemistry.
They showed properties in-between clinically verified coordination compounds and organic
molecules to some extent. Free radical damage can change the instructions coded in a strand of
DNA. It can make a circulating low-density lipoprotein molecule more likely to get trapped in an
artery wall. Or it can alter a cell’s membrane, changing the flow of what enters the cell and what
leaves it. Hence, it is important to scavenge the generated free radicals in the biological systems.
There are number of ruthenium complexes have shown better radical scavenging activity than
the conventional standard vitamin E used in the study. [2] Organometallic compounds consist of
a stable scaffold or reactive centre that can be finely tuned to enable the formulation stages of
clinical trials and subsequent drug optimization and rational drug design. Since the discovery of
cisplatin in 1965, coordination complexes were used extensively in clinical for chemotherapy
[3]. Development of cancer treatment and cancer drug research was developed by cisplatin [4].
Even though cisplatin is widely used in the clinical, its side-effects made the research for the
discovery of complexes with enhanced properties without affecting the normal cells. More than
30 years ago the antitumor activity of metallocenes was investigated [5]. Conventionally,
anticancer drug screening programs with coordination or organometallic complexes are based on
choosing complexes with a high level of genotoxicity and in vitro cytotoxicity studies. However,
numerous compounds later failed to enter the clinical trials due to their stability and general
toxicity issues. There are few complexes [trans-tetrachlorobis(1H-indazole)ruthenate(III)],
KP1019 [6,7] and trans-[tetrachloro-(S-dimethyl-sulfoxide)(1H-imidazole)ruthenate(III)],
NAMI-A [8,9] proved them as better candidates in exhibiting better activity against cancer cells.
An organometallic species, [Ru(η6-toluene)Cl2(pta)] (pta = 1,3,5- triaza-7-phosphatricyclo-
[3.3.1.1]decane), termed RAPTA-T showed similar in vitro and in vivo effects to NAMI-A,
demonstrating selectivity towards metastatic tumors. Both complexes are based on a ruthenium
metal centre and showed a low general toxicity that apparently reduces the side-effects
associated with chemotherapy.
It is extensively considered that ROS have constricted connection with aging [10,11].
Approximately sixty years ago Harman proposed the free radical theory of aging which states
that free radicals produced during metabolism can cause oxidative damages in proteins, lipids
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and DNA, and lead to cell dysfunctions and aging [12]. It was later mentioned as the oxidative
stress theory of aging because many kinds of ROS are not free radicals [13].The discovery of
SODs, catalases, glutathione S-transferase and other antioxidants suggests that organisms have
developed elegant mechanisms during evolution to tackle against the persistently generated ROS
[10]. As organism ages, oxidative damages such as protein carbonization and lipid peroxidation
accumulate [14,15]. Whereas, direct evidences are lacking whether or not these damages are
responsible for aging [11,16]. There are many methods for measuring free radical production in
cells. The most straight forward techniques use cell permeable fluorescent and chemiliminescent
probes. 2'-7'- Dichlorodihydrofluoresceindiacetate (DCFH-DA) is one of the most widely used
techniques for directly measuring the redox state of a cell. It has several advantages over other
techniques. It is very easy to use, extremely sensitive to redox state of a cell, inexpensive and
can be used to follow changes in ROS over time.
Caenorhabditis elegans (C. elegans)is an ideal organism for aging research due to its
short lifespan of only three weeks, the body transparency and easy culturing, the high efficiency
of carrying out RNAi experiments, and the availability of numerous transgenic or gene mutation
strains [17]. It was found in C. elegans that genetic or environmental perturbations that
postponed aging such as reduced insulin/IGF-1 signaling (IIS), mitochondrial dysfunctions and
dietary restriction (DR), usually activated the expression of antioxidant enzymes and enhanced
oxidative stress resistance [18,19]. The DAF-16/FoxO3a-dependent longevity signal was shown
to be initiated by antioxidants [11]. Knockout of the worm catalase gene ctl-2 and the
thioredoxin gene trx-1 shortened the lifespan and over expression of trx-1mildly prolonged
lifespan [20,21]. These studies were to some extent in accordance with the oxidative stress
theory of aging. However, there is considerable evidence conflicting with it [22]. It was shown
that deletion or knockdown of genes such as sod-2 and sod-3, or even knockout all five sod
genes in C. elegans did not shorten lifespan [23,24].
Herein, the current manuscript deals with the synthesis and structural characterization of
new cyclopentadienyl ruthenium(II) complexes and the potential biological effectiveness have
been screened by using multifaceted model organism C. elegans.
Results and Discussion
The reaction of [RuCl(PPh3)2(η5-C5H5)] with an equimolar amount of various
4(N)-substituted thiosemicarbazones (H2L1–H2L4) in methanol resulted in the formation of new
complexes (Scheme 1), the analytical data of which confirmed the stoichiometry of the
complexes (1-4). The structures of the ligand (H2L3) and complex 3 were confirmed by X-ray
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crystallographic studies. Attempts were made to grow single crystals of complex 1, 2 and 4 in
various organic solvents but were unsuccessful. The complexes are soluble in common organic
solvents such as dichloromethane, chloroform, benzene, acetonitrile, ethanol, methanol,
dimethylformamide and dimethylsulfoxide.
OCH3
Ph3P
OH
CH
Ru
N
S
S
+
Ru
Cl
R
N
Methanol, 5 h
C
N
Ph3P
PPh3
NH
NH
N
H
R
H2L1, R = H
H
O
H2L2, R = CH3
H2L3, R = C2H5
H2L4, R = C6H5
OCH3
1. R = H
2. R = CH3
3. R = C2H5
4. R = C6H5
Scheme 1 Synthesis of new ruthenium(II) complexes
Spectroscopic studies
The coordination of the ligands to the Ru(II) metal centre was confirmed by elemental
1
13
analyses, IR, UV-visible, H, C-NMR spectroscopy and ESI mass spectroscopy (Fig S1-S16).
All the spectral details have been discussed in the supporting information.
Single crystal X-ray diffraction studies
The crystals of H2L3 and 3 suitable for X- ray diffraction were obtained from
dichloromethane/ n-heptane (1:1) mixture. The crystallographic data, selected bond distances
and bond angles are listed in Tables 1 and S1. The ORTEP diagram of H2L3 and 3 are shown in
Figure 1 and 2. The ligand H2L3 showed thermal disorder on hydrogen atoms of C10 and C12
carbon atoms of ethyl group and ligand coordinated to ruthenium metal in complex 3 as NS
chelating donor by forming a stable five member ring with a bite angle N(1)-Ru(1)-S(1) of 80.79
(7)° and leaving the phenolic oxygen not involved in bonding. The Ru(1)-N(1) and Ru(1)-S(1)
bond distances are found to be 2.098 (3) and 2.3559 (9) Å respectively. The remaining sites are
occupied by phosphine atom of triphenylphosphine with Ru-P(1) distance of 2.2978 (9) Å and
five carbons of cyclopentadienyl ligand with Ru(1)-C(12), Ru(1)-C(13), Ru(1)-C(14), Ru(1)-
C(15), Ru(1)-C(16) distance of 2.208 (3), 2.212 (4), 2.184 (4), 2.174 (3), and 2.188 (3) Å
respectively. The Ru(1)-C(13-17),Ru(1)-S(1), Ru(1)-N(1) and Ru(1)-P(1) are comparable with
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the distances found in other piano stool ruthenium(II) complexes containing triphenylphosphine
and cyclopentadiene [25].
Figure 1ORTEP diagram of [H2-3MSal-etsc] (H2L3) showing thermal ellipsoids at the 50%
probability level.
Figure 2 ORTEP diagram of [RuCp(Msal-etsc)(PPh3)] (3) showing thermal ellipsoids at the
50% probability level (Solvent molecules were omitted for clarity).
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Table
1
Crystal Data and Structure refinement for [H2-3MSal-etsc] and
[RuCp(MSal-etsc)(PPh3)]
[H2-3MSal-etsc] (H2L3)
1510793
[RuCp(Msal-etsc)(PPh3)] ( 3)
1573744
Identification code
CCDC No
Empirical formula
C34H34N3O2PRuS.CHCl3.H2O
C11H15N3OS
253.32
Formula weight
818.13
Temperature
293 (2) K
0.71073 Å
Monoclinic
I 2/a
293(2) K
1.54184 Å
Monoclinic
P21/c
Wavelength
Crystal system
Space group
Unit cell dimensions
a
b
c
18.9393 (4) Å
11.3806 (3) Å
34.2693 (8) Å
13.1288(9) Å
6.2311(5) Å
15.9692(19) Å
90°
90°
α
97.572(10)°
β
112.941(10)°
90°
1203.06(19)Å3
90°
γ
Volume
7322.0(3) Å3
8
Z
4
1.399 Mg/m3
2.359 mm-1
536
Density
1.484 Mg/m3
0.786 mm-1
3344.0
Absorption coefficient,
F(000)
0.2 × 0.12 × 0.02 mm3
0.13 × 0.03 × 0.02 mm3
Crystal size
Crystal shape
Block, orange
Colourless
θ
range for data collection
3.75 to 24.57°
3.656 to 70.805°
-12≤h≤15,-5≤k≤7,-19≤l≤17
985
Limiting indices
-25≤h≤ 25, -15 ≤k≤14, -45 ≤l≤ 45
Reflections collected
Independent reflections
21093
8658
2224
Completeness to
θ
25.30° 99.6 %
64.99° 98.54 %
multi-scan
Full-matrix least-squares on F2
Absorption correction
Refinement method
multi-scan
Full-matrix least-squares on F2
Data / restraints / parameters
Goodness-of-fit on F2
8658 / 0 / 430
2224/ 0/161
1.036
1.049
R1 = 0.0831, wR2 = 0.1559
R1 = 0.0491, wR2= 0.0994
R1 = 0.0824, wR2 = 0.1128
Final R indices [I>2
R indices (all data)
σ(I)]
R1 = 0.0610, wR2 = 0.1760
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In addition, ligand H2L3 contains two intermolecular hydrogen bonds through oxygen
atom of hydroxyl group with the hydrogen atom of terminal nitrogen (N3) atom of another
molecule with a O(1)−H(2)···N(2) distance of 2.999 Å and hydrogen bond through oxygen atom
of hydroxyl group of hydrogen atom with sulphur atom (S1) atom of another molecule with a
O(1)−H(1)···S(1) distance of 3.156 Å (Figure S17-S20). However, the complex 3 contains one
intramolecular hydrogen bond through the hydrogen atom of the hydroxy group with the
hydrazinic nitrogen (N2) of thiosemicarbazone moiety with a O(1)−H(1)···N(2) distance of
2.519 Å and three intermolecular hydrogen bond through the hydrogen (H3) atom of terminal
nitrogen (N3) with the oxygen (O3) atom of water molecule, the hydrogen atom of the hydroxy
group with the hydrogen (H3B) atom of water molecule and the hydrogen atom of the another
molecule hydroxy group with the hydrogen (H3C) atom of water molecule (Table S2). In
agreement with the NMR spectra of the complexes and the results of X-ray analysis revealed
that the intramolecular hydrogen bond between N2 hydrazinic nitrogen and phenolic –OH
prevents the C-C single bond rotation and subsequent coordination of phenolic oxygen to
ruthenium metal prior to the deprotonation [26].
DNA binding studies
UV absorption titration experiments were carried out to study the DNA binding property
of the new Ru(II) complexes (1-4). The absorption spectra of the new complexes at constant
concentration (10 µM) in the presence of different concentration of CT-DNA (5-50 µM) are
given in (Figure S21). While increasing the concentration of DNA, the hyperchromism with red
shift of 8-9 nm (up to 256 nm) was observed in all the complexes (1-4). The binding constant Kb
of complexes can be determined by monitoring the changes in the absorbance of IL band at the
corresponding λmax with increasing concentration of DNA and is given by the ratio of slope to
the intercept in plots if DNA/(εa-εf) versus [DNA] (Figure S22). The observed range of Kb
values ((0.70 × 105) - (5.59 × 105) M-1) is lower than those observed for typical classical
intercalator (EthBr, Kb, 1.4 × 106 M-1 and certain partially intercalating ruthenium(II) complexes
([Ru(bipy)2(dppz)]2+, (dppz =dipyrido-[3,2-d:2′,3′-f]-phenazine), Kb > 106 M-1) [27]. The
obtained Kb values with hyperchromic red shift of 8-9 nm indicated that the complexes may
have comparatively weaker DNA binding interactions with DNA double helix which may be due
to partial intercalation.[27] This is well accordance with the reported partially DNA-intercalating
ruthenium(II)
complexes
[Ru(bpy)2(diimine)]2+,
[Ru(phen)2(diimine)]2+,
[Ru(5,6-
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dmp)2(diimine)]2+ and [Ru(NH3)4(phen)]2+ which are known to exhibit significantly higher red-
shifts. [27,28] From the binding constant values (Table 2), it is inferred that the complex 4
exhibited better binding than other complexes and following order of DNA binding with respect
to the electron donating/withdrawing ability 4>2>3>1. Further all the complexes exhibited better
binding ability as compared with their parent ligands [29].
Table 2 The Kb, Ksv and Kapp values for the interactions of complexes with CT-DNA
Complex
Kb/M-1
Ksv/M-1
Kapp/M-1
4.31×106
6.19×106
6.61×106
7.01×106
1
2
3
4
0.70 ×105
5.52 ×105
1.81 ×105
5.59 ×105
2.87 ×103
1.12 ×104
0.76 ×104
1.14 ×104
Ethidium Bromide displacement study
The result obtained from the above experiments suggested that all the compounds can
bind with CT-DNA. However, the exact mode of binding cannot be proposed by these studies.
Hence, Ethidium bromide (EB) displacement studies were carried out. Ethidium bromide
competitive binding studies using new ruthenium(II) complexes 1-4 as quenchers may give
further information about the binding of these complexes to DNA. When complexes 1-4 were
added to DNA pretreated with EB, the DNA induced emission intensity at 609-603 nm was
decreased (Figure S23). This indicated that the complexes could replace EB from the DNA-EB
system. From this observation, it may be concluded that all the complexes could bind to DNA
through the intercalation mode. From the quenching constant values, it is inferred that complex 4
replaced the EB more effectively than other complexes (Figure S24 and Table 2).
DNA cleavage study
The preliminary identification of DNA cleavage proficiency of the free ligands and
ruthenium(II) complexes was done by taking supercoiled (SC) pBR322 DNA was incubated
with complexes and ligands in 5 mM Tris HCl/ 50 mM NaCl buffer at pH 7.2 for 2 h without
added reductant. Upon gel electrophoresis of the reaction mixture, DNA cleavage was observed
(Figure S25). The relatively fast migration is the supercoiled form (Form I) and the slower
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migration is the open circular form (Form II), which were produced from supercoiled DNA
when scission occurred on its one strand [30]. The complexes at same concentration are able to
perform cleavage of pBR322 plasmid DNA. The intensity of supercoiled SC (Form I)
diminished and partly converted to nicked form NC (Form II) in complexes (1, 2 and 3), as the
intensity of NC (Form II) increases, the production of LC (Form III) increased. Whereas in
complex 4, the intensity of super coiled SC (Form I) increased and partly converted to nicked
form NC (Form II), whereas the production of linear form LC (Form III) of DNA decreased. The
more electrons withdrawing phenyl group in the complex 4 may have relatively better
interaction with the DNA base pair and promotes better DNA cleavage. It is obvious that the
ruthenium(II) complexes have the ability to cleave the supercoiled plasmid DNA and this
cleavage system does not require addition of any external agents.
UV absorption spectra of BSA
A common method to distinguish between static and dynamic quenching is by careful
examination of the absorption spectra of the BSA in the presence of complexes [31]. The UV
absorption spectra of BSA in the presence of complexes showed an increase in the absorption
intensity when the complexes were added (1- 4) (Figure S26). The changes in the absorbance
spectra for BSA + compounds indicate that test compounds interact with the BSA [32]. It is well
known that dynamic quenching only affects the excited state of fluorophore and does not change
the absorption spectrum. However, the formation of non-fluorescence ground-state complex
induced the change in the absorption spectrum of fluorophore. Thus, possible quenching
mechanism of BSA by compounds was found as static quenching [33].
Fluorescence quenching studies of BSA
In order to get more information on the binding of the compounds with BSA,
fluorescence spectra of BSA was studied by the addition of the test compounds. Changes in the
emission spectra of tryptophan are common in response to protein conformational transitions,
subunit associations, substrate binding, or denaturation. Hence, the interaction of BSA with our
compounds was studied by fluorescence measurement at room temperature and the binding
constants of the compounds were calculated. In a typical experiment, the fluorescence spectra
were recorded in the range of 290–500 nm upon excitation at 280 nm. On increasing the
concentration of compounds, a progressive decrease in the fluorescence intensity was observed,
accompanied with a blue shift (Figure S27). The observed blue shift may be due to the binding
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of compounds with the active site in BSA[34]. The binding constant Ksv value was obtained
from the plot of Io/Icorr versus [Q] in Stern–Volmer equation (Figure S28 and Table 3).
Io/Icorr = 1+ Ksv [Q]
The observed linearity in the plots indicated the ability of the complexes to quench the
emission intensity of BSA. From Ksv values, it is seen that the complexes exhibited strong
protein-binding ability with enhanced hydrophobicity.
Binding constants and the number of binding sites
For the static quenching interaction, if it is assumed that there are similar and
independent binding sites in the biomolecule, the binding constant (Kb) and the number of
binding sites (n) can be determined according to the Scatchard equation (5), [35] (Figure S29
and Table 3).
log [(Fo−F)/F]= log Kb+ n log [Q]
The values of 'n' at room temperature are approximately equal to 1 for the complexes 1-3, which
indicates that there is just one single binding site in BSA for the compounds. However, the
complex 4 has the value of 1.53 indicate that there may be a weaker interaction with tyrosine
base pair which weakly affect the micro environment of BSA and stronger interaction with
tryptophan base pair by affecting the micro environment to a large extent. This has been further
confirmed with synchronous fluorescence spectroscopic titration.
Table 3 Quenching constant (Ksv), binding constant (Kb) and number of binding sites (n) for the
interactions of complexes with BSA.
Complex
Ksv/M-1
Kb/M-1
n
1
2
3
4
8.26 ×103
2.96 ×105
3.63 ×104
1.95 ×106
8.37 ×103
9.21 ×103
6.64 ×103
1.34 ×104
1.00
1.36
1.18
1.53
Synchronous fluorescence spectroscopic studies of BSA
Synchronous fluorescence spectral study was used to obtain information about the
molecular environment in the vicinity of the fluorophore moieties of BSA[36]. Synchronous
fluorescence spectra showed tyrosine residues of BSA only at the wavelength interval (∆λ) of 15
nm whereas tryptophan residues of BSA at ∆λ of 60 nm. The concentration of complexes (0–50
µM) added to BSA (10 µM) is increased, a decrease in the fluorescence intensity with a blue
shift in the tryptophan emission maximum is observed for all the complexes
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(Figure S30). In contrast, the emission intensity of tyrosine residue increases without any change
in the wavelength of emission. These observations indicate that the test compounds did not
affect the microenvironment of tyrosine residues during the binding process significantly but the
tryptophan microenvironment to a larger extent.
Three-dimensional fluorescence spectroscopy
It is well known that 3D fluorescence spectra can provide more detailed information
about the conformational changes of proteins. Three-dimensional fluorescence spectra and
contour maps are shown in figure 3.The normal fluorescence peaks are usually located in the
lower right of the Rayleigh scattering regions [37,38]. Two typical fluorescence peaks could be
easily found in three-dimensional fluorescence spectra, which are marked as peaks
1 and 2. It was obvious that both fluorescence peaks of BSA have been quenched by complexes
1- 4. As described by Zhang et al. [39] peak 1 (λexc = 280.0 nm and λemi = 315.0 nm) mainly
revealed the spectral behaviour of tryptophan and tyrosine residues, while peak 2 (λexc = 230.0
nm and λemi = 326.0 nm) may mainly exhibit the fluorescence characteristic of polypeptide
backbone structures. Therefore we can conclude that the interaction of complexes with BSA
alternate the secondary structure of BSA particularly interacting with tryptophan residue.
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Figure 3 The 3D fluorescence spectra of BSA (A) and BSA+ 1 (B), BSA+ 2 (C), BSA+ 3 (D),
BSA+ 4 (E)
3.1. In vitro and in vivo antioxidant activity of new ruthenium(II) complexes
Free radical scavenging activity of newly synthesized ruthenium(II) complexes 1-4 was
investigated in a cell-free system using DPPH, a stable free radical. When tested in the DPPH
reduction assay, all complexes effectively scavenged the free radicals as compared to that of
standard control. The radical scavenging ability of 1-4 was in the range of IC50DPPH = 6.15±0.25,
2.62±0.41, 3.09±0.41, and 2.53±0.33 µg/mL respectively (Table 4). Complexes 2-4 exhibited
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better antioxidant activity than the conventional standard ascorbic acid. The IC50 values showed
that the radical scavenging activities of the complexes are in the order of 4 >2 > 3 >1. The
results of in vitro antioxidant activity was in good agreement with our previous studies [40,41].
In the in vivo assay system, we first evaluated the safety profile of 1-4 on wild-type C. elegans.
Among the tested pharmacological doses, 10, 20 and 40 µM concentration of 1-4 did not
adversely affect the survival of nematodes. In addition, 80 and 160 µM concentrations were
found to be toxic and significantly (p˂0.001) reduced the percentage survival of C. elegans when
compared to control group worms (Fig. 4). Additionally, 10, 20 and 40 µM concentrations of 1-4
did not obviously alter the development and fecundity rate of C. elegans (data not shown). From
these observations, it was apparent that the optimal concentrations of new ruthenium(II)
complexes (10, 20 and 40 µM) are relatively safe in C. elegans and these non-toxic molarities
have been used in subsequent experiments.
IC50 (µg/mL)
S. No
Test compound
p value
Mean ±SEM
6.15±0.25
2.62±0.41
3.09±0.41
2.53±0.33
5.20±0.10
1
2
3
4
5
ns
1
2
3
4
p˂0.01
p˂0.05
p˂0.01
Ascorbic acid
Table 4. Antioxidant activity of complexes 1-4 with reference to DPPH. Ascorbic acid was used
as a conventional standard. For the analysis of significance, DPPH reduction by complexes was
compared with ascorbic acid.
Fig.4. Toxicity assay with various pharmacological doses of complexes 1-4 on wild type C.
elegans. Bar graph depicts the survival percentage of wild-type C. elegans exposed to 1-4 for 24
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h at 20°C. Data were pooled from three independent biological experiments and presented as
mean ±SEM, *p<0.05, **p<0.01.
Considering the in vitro antioxidant potential of new ruthenium(II) complexes, we then
assayed the in vivo antioxidant efficacy using a nematode model C. elegans. The in vivo data
confirmed that 2-4can effectively protect C. elegans against lethal oxidative and thermal
stresses. In oxidative stress assay, the percentage survival of pretreated worms with 2-4 at20 µM
was significantly increased to 62.14 %, 65.69 % and 49.30 % respectively when compared to the
untreated control group worms (Fig. 5A). Similarly, in thermo-tolerance assay, we observed that
2-4 supplementation at 20 µM resulted in an increased survival rate of worms by 33.19 %, 30.73
% and 25.79 % (p<0.001) respectively (Fig. 5B).These results indicated that complexes 2-4
exert protective role against oxidative and thermal stress in C. elegans. On the contrary, complex
1 displayed negligible effect on the stress resistance of C. elegans.
Fig 5.The effects of 1-4 on (A) oxidative stress and (B) thermal stress resistance in wild-type C.
elegans. Bar diagram depicts the percentage survival of treated worms with 1-4 at different
concentrations. Data are from three biological experiments, and presented as mean ±SEM,
*p<0.05, **p<0.01.
The enhanced survival under stress condition is often associated with altered intracellular
redox status [40,42]. To test the idea, we assessed the effect of 1-4 on ROS accumulation using
cell permeant fluorescent probe H2DCF-DA. We observed that treatment with 20 µM of 2-4
significantly reduced the intracellular ROS level by 41.93 %, 33.96 % and 29.91 % respectively
when compared with untreated worms (Fig. 6). This suggested that 2-4 hold the capacity to
reduce the intracellular ROS levels due to their potent antioxidant ability. The reduction in ROS
levels can be assumed to be the chief reason behind stress resistance in wild-type C. elegans. In
all these experiments, we have observed that complexes 2-4 displayed a hormetic-like dose-
dependent biphasic effects on C. elegans and this result was in line with our previous study [40].
According to the obtained results, we found that 20 µM concentration of 2-4 had more activity in
relation to other tested concentrations; hence, further experiments were carried out with this
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concentration. At the same time, complex 1 had no such beneficial effect at all the tested
concentrations.
Fig. 6. Intracellular ROS accumulation in C. elegans. Graphs depict the quantified ROS levels in
wild-type C. elegans using cell permeant fluorescent probe H2DCF-DA. Data were acquired
from three independent biological runs and presented as mean±SEM, *p<0.05, **p<0.01.
We also performed a lifespan assay using mitochondrial mutant strain mev-1(kn1). This
strain has mutation in succinate dehydrogenase cytochrome b560 subunit, an integral membrane
protein that is a subunit in complex II of mitochondrial respiratory chain. MEV-1 is required for
oxidative phosphorylation, mutation in mev-1results in an abnormal energy metabolism and
uncontrolled production of free radicals that leads to shortened lifespan [43]. It was found that
supplementation of 2-4 failed to prolong the mean lifespan of mev-1 mutants under standard
laboratory conditions (Fig.7, Table 5). These results indicated that 2-4 conferring stress
resistance, and they also required endogenous detoxification mechanism for doing so, which is
consistent with previous research [44].
100
80
60
40
mev-1 (kn1)
Control
20
Complex 2
Complex 3
Complex 4
0
0
5
10
15
20
25
Age (days of adulthood)
Fig 7.The effect of 2-4 on the lifespan of mev-1 loss-of- function C. elegans. Survival curves of
mev-1(kn1) mutant worms raised on the NGM plates supplemented with 20 µM of 2-4 at 20°C.
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Curves were plotted using Kaplan-Meier survival method and analyzed by log-rank test
(p<0.05).
Table 5. Lifespan analysis of mev-1(kn1) mutant C. elegans culture at 20°C supplemented with
20 µM of 2-4.
Mean lifespan
(Mean±SEM)
11.593±0.454
Log rank test Maximum lifespan
Genotype Treatment
% Change
(p value)
(days)
19
Control
Complex2 11.370±0.380
Complex 3 11.238±0.419
Complex 4 11.271±0.202
(-) 1.92
(-) 3.06
(-) 2.78
0.3783
0.6253
0.2711
18
mev-1(kn1)
19
19
3.2. Ruthenium(II) complexes modulates antioxidant machinery in C. elegans
It has long been known that the enhanced stress toleranceis concomitantly associated
with the expression of some antioxidant defense genes in C. elegans [40,45,46]. Therefore, to
address this, we investigated the expression of important antioxidant genes by utilizing the
transgenic strains carrying an inducible GFP reporter transgene for gst-4::GFP (CL2166) and
sod-3::GFP(CF1553). We found that the level of gst-4::GFP and sod-3::GFP induction in 2-4
treated worms was significantly higher than untreated control worms (Fig. 8). These results
suggest that, apart from the antioxidant property, complexes 2-4 promoted stress resistance
possibly through direct regulation of antioxidant enzyme genes. In C. elegans, gst-4 and sod-3
encode a putative glutathione-requiring prostaglandin D synthase and a Fe/Mn superoxide
dismutase respectively, which offers a conserved protection against oxidative stress defense and
promotes healthy lifespan [47,48]. The gst-4 and sod-3 are the well-known transcriptional
readouts of SKN-1 and DAF-16 transcription factors, correspondingly [47,49]. With this
understanding, we tested the role of SKN-1 and DAF-16in 2-4 mediated stress resistance and
ROS generation in C. elegans, the worm loss-of-function in skn-1(zu67) and daf-16 (mgDf50)
were used. It was observed that 2-4 treatment failed to protect the worms from juglone-induced
lethal oxidative stress (Fig. 9A) and ROS generation (Fig. 9B), this signified that SKN-1 and
DAF-16 is essential for 2-4-mediated stress tolerance in C. elegans.
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Fig 8. Influence of 2-4 on the expression of antioxidant genes in C. elegans. (A) Representative
fluorescence images ofgst-4::GFP transgene in CL2166 and sod-3::GFP in CF1553 worms. (B)
Graphs depict the quantified expression rate (in arbitrary unit) of gst-4 and sod-3. Data shown
here are mean±SEM of three biological experiments, *p<0.05, **p<0.001.
Fig 9. Genetic requirement for stress resistance in C. elegans exposed to 2-4. (A) Histogram
represents the (A) ROS levels and (B) stress resistance in skn-1 and daf-16 mutant worms
exposure to lethal concentration of juglone. Complexes 2-4 fails to protect skn-1 and daf-16
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worms from juglone-induced ROS generation and oxidative stress. Data were pooled from three
independent biological experiments and presented as mean±SEM, **p<0.01, nsnot significant.
4. Conclusions
The reactions of [Ru(η5-C5H5)Cl(PPh3)2] and 3-methoxysalicylaidehyde-4(N)-substituted
thiosemicarbazones (H2L1–4) were examined in 1:1 stoichiometric ratio and resulted in the
formation of four new organo-ruthenium complexes. All the new complexes (1-4) were
1
13
characterized by analytical, IR, H-NMR and C spectroscopic studies. The ligand [H2-Msal-
etsc] and complex [Ru(η5-C5H5)(Msal-etsc)(PPh3)] (3), were structurally characterized by single
crystal X-ray diffraction studies. In all the complexes, ligands H2L5-8 are coordinated as mono
negative bidentate NS donor by forming a five member chelate ring by leaving the third
potential donor atom, phenolic oxygen remained intact without being participated in bonding.
Further, interaction of complexes (1-4) with Calf-Thymus DNA (CT DNA) has been explored
by absorption and emission titration methods. Based on the observations, a partial intercalation/
intercalation mode of DNA binding with complexes has been proposed. The protein binding
abilities of the new complexes along with their precursor were monitored by quenching of
tryptophan and tyrosine residues using BSA as model protein. The order of binding affinity is
proportionately followed in DNA binding, Ethidium Bromide displacement and BSA binding
studies with the order of 4>2>3>1. The more electrons withdrawing phenyl group in the
complex 4 may have relatively better intercalation with DNA helix and thus exhibiting better
DNA/protein affinity than other complexes. Complexes 1-4 exhibited an excellent in vitro and in
vivo antioxidant activity. Moreover, we have provided evidence that 2-4 offers stress resistance
in C. elegans via inhibiting the intracellular ROS levels and activation of stress responsive genes
like gst-4 and sod-3. Therefore, 2-4 might be a good candidate for development of novel drugs
against stress-related human ailments.
5. Measurements
All the reagents used were analar grade, were purified and dried according to the
standard procedure[50]. 3-methoxysalicylaldehyde, thiosemicarbazide, 4(N)-substituted
thiosemicarbazides, Calf Thymus DNA (CT DNA), Bovine Serum Albumin (BSA), Ethidium
Bromide (EB) and pBR322 plasmid DNA were obtained from Himedia. Melting points were
measured in a Lab India apparatus. Infrared spectra were measured as KBr pellets on a Jasco FT-
IR 400- 4100 cm-1 range. Elemental analyses of carbon, hydrogen, nitrogen and sulphur were
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determined by using Vario EL III CHNS at the Department of Chemistry, Bharathiar University,
Coimbatore, India. Electronic absorption spectra of the compounds were recorded in
dichloromethane using JASCO 600 spectrophotometer and emission measurements were carried
1
13
out by using a JASCO FP-6600 spectrofluorometer. H and C NMR spectra were recorded in
CDCl3 and DMSO at room temperature with a Bruker 400 MHz instrument, chemical shift were
relative to tetramethylsilane and expressed in parts per million (ppm). Single crystal data
collections and corrections for the ligand [H2-MSal-etsc](H2L3) and complex [RuCp(MSal-
etsc)(PPh3)] ( 3) were done at 293 K with nonius Kappa CCD Diffractometer using graphite
mono chromated Mo Kα (k = 0.71073 Å) radiation[51]. The structure solution were done by
using SHELXS-97[52]and refined by full matrix least square on F2using SHELXL-2014[53].
5.1 Experimental Section
The ligand [H2L1-4] and the ruthenium complex [RuCl(PPh3)2(η5-C5H5)] were synthesized
according to the standard literature procedures[53,54].
5.2. 1. Free radical scavenging assay
The free radical scavenging activity of complexes were determined by in vitro 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical quenching experiment as described previously
[40,55]. Briefly, 1 mL of various pharmacological doses of test compounds (0, 4, 8, 12, 16 and
20 µg/mL) were added to 3 mL 0.1 mM DPPH in methanol and the solution was mixed
vigorously. The tubes were then incubated for 20 mins in the dark,and decolorization of DPPH
solution was measured at 517 nm using UV-Vis spectrophotometer (UV-1800, Shimadzu,
Japan).The percentage inhibition of DPPH radicals (IC50) were calculated using the following
equation, and comparable with ascorbic acid.
DPPH scavenging effect (%) = ([A0 − A1]/A0) × 100
Where, A0representsthe absorbance of the control reaction; A1is the absorbance of treatment
reaction. All measurements were performed in triplicate and finally IC50 was represented in
µg/mL. Three independent experiments with appropriate replicates were performed at similar
conditions.
5.2.2. C. elegans:Strain maintenance and synchronization
The C. elegans strains used in this study were Bristol N2 (wild-type), TK22 (mev-1
[kn1]),GR1307 (daf-16 [mgDf50]), EU1 (skn-1 [zu67]), CF1553 (muIs84 [pAD76 (sod-
3p
∷GFP)]) and CL2166, dvIs19 (gst-4p::GFP). All the strains were maintained, propagated and
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assayed on nematode growth media (NGM; 17 g agar, 3 g NaCl, 2.5 g peptone, 0.5 mL of 1 M
CaCl2, 1 mL of 5 mg/mL cholesterol, 1 mL of 1 M MgSO4, 25 mL KH2PO4 buffer [pH 6.0] and
1 L deionized water) agar plates carrying Escherichia coli OP50 as food source at 20°C by
following the standardized protocols [56]. For age-synchronization, the gravid adult worms were
exposed to 5M sodium hydroxide and 5% household bleach and the gathered eggs were
incubated in M9 medium (6 g Na2HPO4, 3 g KH2PO4, 5 g NaCl, 0.25 g MgSO4.7H2O) for 12 h
to favor the hatching [57].
5.2.3. Toxicity evaluation, brood size and body length
The test compounds were dissolved in dimethyl sulfoxide (DMSO), and the final
concentration of DMSO was maintained <0.5 % at all experiments. Toxicity assay was
performed to eliminate the toxic condition to the worms according to the method described
previously[41]. In brief, age synchronized young adult worms (n=50-60 individuals/experiment)
were exposed to 1.0 mL of test solution containing various pharmacological doses of complexes
1-4(10, 20, 40, 80 and 160 µM)or 0.1 % DMSO (vehicle control)and 6 mg/mL of heat killed E.
coli OP50 for 24 h in a sterile microtiter plates. Following exposure, the treated worms were
examined for inactivity under a dissecting microscope. The worms fail to respond mild physical
contact with metalwire was considered as dead. For brood size assay, wild-type worms (n=10
individuals/experiment) were treated with various concentrations of 1-4, at L4 stage they were
then individually shifted to fresh plates every day during the reproduction period. The eggs laid
by each worm was allowed to develop at 20°C and counted at L2-L3 stage in order to verify the
hatchability of the eggs. Growth of the nematode was assessed by body length, which was
determined by measuring the flat surface area of control and treated worms (n=40
worms/experiment)using OptikaIsView image analyzing system (Optika, Italy)[51]. At least
three biological trails were performed with four replicates.
5.2.4. Assessment of oxidative and thermal stress resistance
Wild-type worms were raised on the NGM plates in the absence or presence of 1-4 from
L1 stage at 20°C. Subsequently, adult worms of control and treated groups were subjected to
oxidative and thermal stresses. For oxidative stress, complexes 1-4 treated and untreatedday-2
worms were transferred to new NGM plates containing 240 µM of 5-hydroxy-1,4-
napthoquinone (Juglone, Sigma-Aldrich) for 4 h and the survival of worms were scored. To
assess the thermal stress tolerance, 1-4 treated and control worms were shifted to 35°C for 3 h on
NGM plates and then scored for viability after 2 h substantial recovery time at 20°C. The
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survival rate of the worms was confirmed by pharyngeal pumping and touch-provoked
movement.
5.2.5. Quantification of endogenous ROS accumulation in C. elegans
To examine the intracellular ROS accumulation, wild-type worms were treated with 1-4
for 72 h from L4 stage as said above. After treatment, the worms were transferred to 1 mL of M9
buffercontaining 50 µM of 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) in 24 well sterile
microtiter tissue culture plates and incubated for 3 h at 20°C in the dark. The worms were
washed twice with M9 buffer to remove the adhering bacteria, anesthetized with25mM of
sodium azide (NaN3), and mounted on 3 % agarose paddedmicroscopic slides. About 30
randomly selected immobilized worms were captured under fluorescencemicroscope (BX-41,
Olympus, Japan) and analyzed for fluorescence intensity by determining the mean pixel intensity
using imageJ software (NIH, Bethesda, MD).
5.2.6. Reporter geneexpression assay
We followedMohankumaret al[58]method to determine the effects of 1-4on gst-4and
sod-3expressions in C. elegans.Age synchronized late L4 stage C. elegans strains (n=20-25
worms/experiment) carrying inducible green fluorescence protein (GFP) reporter transgene for
gst-4::GFP (CL2166) and sod-3::GFP (CF1553) were treated with respective concentrations of
1-4for 24 h at 20°C.Photomicrographsof control and treated worms were collected and analyzed
for fluorescence intensity as described in the quantification of endogenous ROS accumulation.
5.2.7. Lifespan assays
The synchronized populations of wild-type and mitochondrial mutant strain TK22 (mev-1
[kn1]) were raised from L1 stage on the NGM plates supplemented with respective concentration
of 1-4at 20°C.At L4 stage, 50µM of 5-fluorouracil (5-FU, HiMedia) was added to each platein
order to avoid the progeny development.To avoid contamination and starvation, the worms were
transferred to fresh NGM plates for every other day until their death. Worms that fail to respond
touch-provoked movement, loss of pharyngeal pumping, and lysed body were considered as
dead, whereas the worms exhibiting internal hatching, death during handling and crawling out
the NGM plates was marked as censored. This was terminated until the last worm become
marked as censored or death. Three independent trails were performed for each concentration
with appropriate replicates.
5.2.8. Data analysis
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Means were compared with control group usingone way analysis of variance (ANOVA)
followed by Bonferronipost hoc test in IBM SPSS statistical software for Windows v.16 (IBM
Corporation, Armonk, NY, USA).For lifespan assay, survival curves was plotted using Kaplan-
Meier survival method, and log-rank test was applied to analyze the survival differences between
control and treated groups in MedCalc v.14 statistical tool (MedCalc Software, Ostend,
Belgium).The probability levels of p<0.05 was considered as statistically significant.
Supporting information
Experimental procedures, Spectroscopic discussion, Figures S1-S27, Tables S1 and S2
were given in this section. Crystallographic data for the complexes [H2-3MSal-etsc] and
[RuCp(Msal-etsc)(PPh3)] have been deposited at the Cambridge Crystallographic centre as
supplementary publication (CCDC No. 1510793 and CCDC No.1573744). The data can be
obtained free of charge at w.w.w.ccdc.cam.ac.uk/conts/retrieving.html/
Acknowledgments
The author P.K. gratefully acknowledges Department of Science and Technology (DST-
SERB), New Delhi, India (No. SB/FT/CS-056-/2014 dated 12.08.2015) for the financial
support. The authors gratefully acknowledge Caenorhabditis Genetic Centre (CGC, University
of Minnesota, MN, USA) which is funded by NIH Office of Research Infrastructure Programs
(P40 OD010440), for providing all C. elegans strains used in this study.
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Highlights
‹ New organoruthenium complexes were prepared and characterized by spectral and
analytical techniques
‹ The complexes exhibited an intercalative binding with CT-DNA
‹ Complexes exhibited better free radical scavenging activity than vitamin C
‹ Complexes 2,3 and 4 conferred stress resistance in C. elegans