This document summarizes the synthesis and characterization of a novel ruthenium(II) complex containing 4-carboxy-N-ethylbenzamide (CNEB) as a ligand, and its potential as an inhibitor of lactate dehydrogenase (LDH). The CNEB ligand was synthesized and characterized using techniques such as X-ray crystallography. It was then complexed with cis-Ru(bpy)2Cl2 to form [Ru(CNEB)2(bpy)2]2PF6. The complex showed cytotoxic effects against cancer cells and interacted with LDH. In vitro and in tissue experiments demonstrated that the complex acts as a non-competitive inhibitor of LDH. Thus,
2. 244 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al.
discovery efforts [10]. In case of Ru-complexes as anticancer factors including oncogenic alterations and mitochondrial
agent, the main focus during the last 2-3 decades has been dysfunction [21]. In either case, this critical step is catalyzed
on the screening of Ru-complexes interfering with DNA by lactate dehydrogenase (LDH; EC: 1.1.1.27).
structure [1]. However, yet pharmacological targets of these During the last quarter of 19 th century, the dominancy of
complexes remain undefined. Targeting down stream molecular biology eclipsed the study of tumor bioenergetics,
abnormal cellular biochemistry associated to cancer however, a growing stream of recent papers are now making
development is a relevant alternative and therefore, enzymes the link between the cancer genes and the Warburg effect
of specific cell functions are important in this respect. [22]. Also, there are some reports on direct implications of
Although scarce, but there are some reports that describe Ru- glycolytic enzymes in cellular immortalization [23, 24]. A
complexes modulated enzymes of specific cell function. recent article appeared in Science on ‘Energy deregulation:
NADPH oxidase is involved in oxidative cell damage and it Licensing tumors to grow’ is an example of revival of
has been observed that this enzyme is activated by an anti researches on targeting the tumor bioenergetics and the
tumor PDTA (Ru-propylene-1, 2-diaminotetra acetic acid) in glycolytic pathway enzymes as an effective strategy to
human neutrophil cells [11]. A more systematic study on the control tumor growth [25]. Thus, susceptible glycolytic
effect of a [RuCl2(i-nic)4]; I-nic = isonicotinic acid, on nitric enzymes and the LDH in particular, need to be re-addressed
oxide synthase (NOS), an enzyme implicated in cell for therapeutic intervention against cancer.
survival, cell death and cell signaling, revealed that the
complex could inhibit the n-NOS (neuronal NOS) more The native LDH exists as a tetramer consisting of two
n
effectively than the inducible isoform (i-NOS), however, types of subunits, the M type (pre-dominantly expressed in
tio
with no effect on e-NOS (endothelial type), and thus, the skeletal muscle & supports anaerobic energy pathway) and
complex was suggested for a potential therapeutic the H type (pre-dominantly expressed in heart and other
applications [12]. In this respect, NAMI has been studied in aerobic tissues). Combination of these two sub-units in
u
more detail than any other Ru-complexes. NAMI-A has been different ratio gives rise five isozymic forms of LDH (M4,
rib
demonstrated to inhibit matrix proteins that are necessary for M3H, M2H2, MH3 & H4), which are expressed in a tissue
tumor growth [13]. This complex was also found to inhibit specific manner in different mammalian tissues. The
st
type IV collagenase in vitro [1]. Topoisomerae II is required differential expressions of LDH isozymes in different types
for DNA replication in growing tumor cells. A Ru-complex of tumors [26] and under the influence of a variety of patho-
i
[Ru(η6-C6H6)dmso)Cl2] was found to inhibit this enzyme in physiological conditions [27-29] have already been reported.
Thus, it may be argued that LDH is a relevant and suitable
D
a ligand dependent manner [4].
candidate to study anti-cancer potential of metallo-drugs.
or
Furthermore, the importance of Ru metal center, in
organizing different ligands for specific bio-activities, has Abnormally high LDH activity in cancerous tissues vis a
been demonstrated with respect to the binding of these vis decreased LDH activity with tumor regression have been
tF
ligands at ATP binding site of protein kinases resulting into demonstrated in animal models [26,30,31]. There is a report
inhibition of the enzyme in ligand dependent manner [14]. [32] on designing active site inhibitors of human LDH for
The reactivity of poly(amino carboxylate) ruthenium(III) therapeutic applications. A Ru(II) complex containing
o
complexes have also been screened to inhibit cysteinyl and flavones was synthesized in our lab and when administered
serine proteases involved in apoptotic cell death and various orally to mice, inhibited LDH activity reversibly in several
N
physiological functions respectively [15,16]. tissues [18]. In a drive to formulate Ru(II) complexes that
could interact with cellular proteins, we synthesized a Ru(II)-
Nonetheless, baring few exceptions, studies are scanty on CNEB complex, which on NMR study could show
Ru-complexes as modulators of the enzymes implicated in dimerization property in aqueous medium with a potential to
sustaining increased bioenergetics of the cancerous cells. A establish weak interactions with biomolecules. The complex
Ru(III) complex, HInd [Ru(ind)2Cl 4)], has been found to was examined in detail and was found to produce
interact with and inhibit cytochrome c, an important enzyme cytotoxicity against Dalton’s lymphoma cells (unpublished
of mitochondrial electron transport chain [17]. We have also results). This article reports that this complex acts as a non-
demonstrated that a Ru(II) complex containing flavones competitive inhibitor of LDH, both in vitro and at tissue
inhibit LDH, the key enzyme of Warburg effect in tumor level, and thus, the first Ru-complex to be described as LDH
cells [18]. inhibitor.
LDH AS A POTENTIAL TARGET SYNTHESIS AND CHARACTERIZATION OF 4-
CARBOXY N-ETHYLBENZAMIDE (CNEB)
LDH gets implicated in a critical way to maintain
immortal nature of the cancerous cells. Tumors rapidly 4-Carboxybenzaldehyde condensed with hydroxylamine
utilize oxygen and nutrients mainly for angiogenesis hydrochloride in ethanol under appropriate conditions
(neovascularization) leading to a hypoxic condition in produced 4-carboxy N-ethylbenzamide (CNEB), M.P.178ºC.
cellular milieu. Consequently, cancer cells depend more on The compound was characterized by elemental analysis (%)
glycolysis for their increased energy demand and in turn, [C, 57.8; H, 5.03; N, 6.8; calculated for C 10H11NO3.H2O;
generate an excess of lactic acid [19, 20]. Also, what was C, 56.9; H, 6.1; N, 6.6] and spectroscopic IR, 1H NMR &
described as ‘Warburg effect’ in 1920s, emphasized that the UV/vis λ max(nm) (ε dm3 mol-1 cm-1 in Methanol 10-4 M;
propensity of cancer cells to utilize glucose and convert 365(1230), mass measurements (m/z=193). Crystals were
glucose to lactic acid even in the presence of oxygen is one grown in ethanol-diethyl ether and molecular structure
of the hall marks of tumerogenesis induced by the multiple obtained by its X-ray crystallographic study is shown in
3. Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 245
n
Fig. (1). Molecular structure of CNEB (ORTEP diagram).
tio
Fig. (1), whereas, major crystallographic property of CNEB respectively, which were further supported by its 1H NMR
u
is presented in Table 1. spectrum recorded in DMSOd6 showing peaks at 13.01,
rib
8.22, 4.35 and 1.38 ppm corresponding to carboxyl, NH,
IR spectrum of the compound showed major peaks at
CH2 and CH 3 protons respectively.
1687 and 1633 cm-1 assigned to ν(C=O) and (NH)
st
Table 1. Crystal Data and Structure Refinement for CNEB
D i
Identification code CNEB
or
Empirical formula C10 H11 N O3
Formula weight 193.20
tF
Temperature 293(2) K
Wavelength 0.71073 A
o
Crystal system, space group Triclinic, P -1
N
Unit cell dimensions a = 5.0631 (9) A α= 79.563(3)o.
b = 7.1528(12)A β = 83.865(3)o
c = 13.801 (2) A γ = 78.838(3)o
Volume 480.91(14) A^3
Z, Calculated density 1, 0.667 Mg/m^3
Absorption coefficient 0.050 mm^-1
F(000) 102
θ range for data collection 1.50 to 25.00o
Index ranges -6<=h<=6, -8<=k<=8, -16<=l<=16
Reflections collected /unique 4630 / 1684 [R(int) = 0.0268]
Completeness to 2 θ 25.00 99.7%
Refinement method Full-matrix least-squares on F^2
Data / restraints / parameters 1684 / 0 / 148
Goodness-of-fit on F^2 1.126
Final R indices [I>2σ(I)] R1 = 0.1022, wR2 = 0.2998
R indices (all data) R1 = 0.1173, wR2 = 0.3176
Largest diff. peak and hole 0.424 and -0.331 e.A^-3
4. 246 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al.
At this stage of study it is worth to mention that followed by the recording of OD after incubating LDH
normally this type of condensation between aldehydes and (20µg) with different concentrations of Ru(II)-CNEB
hydroxylamine hydrochloric provide oxime, but it may complex (20, 40 & 60 µg/ml) for 30 min at 25oC. The
undergo Beckmann oxime-amide rearrangement as reported reading obtained for LDH-Ru(II)-CNEB mixture was
earlier [33] and isolation of CNEB is considered as a result corrected with the absorbance of the complex alone. The data
of this rearrangement. as a mean value from four observations at each point showed
a linear decline in the absorbance of LDH at 280 nm as a
function of increasing concentration of Ru(II)-CNEB
SYNTHESIS OF RU(II)-CNEB COMPLEX complex and thus, correlated well with the pattern reported
for the interaction of a Ru(III) complex with serum
The complex was synthesized by reacting the equimolar transferrin [34].
solutions of CNEB and cis- [Ru(bpy)2Cl 2].2H2O in ethanol.
After 11 h refluxing, aqueous solution of NH4PF 6 was added Absorption data of different concentration of Ru(II)-
and the resultant solid was isolated and then purified by CNEB complex (1.73 x 10-5 M -1.73 x 10-4 M) alone and in
loading it on an alumina-column using acetonitrile as eluent. the presence of fixed concentration of purified LDH (0.143 x
Major fraction thus obtained was again re-precipitated by 10-6 M) was further used to calculate association constant
saturated aqueous solution of NH4PF 6 followed by washings (Kc) between LDH and the complex applying Benesi-Hilde
with water, methanol and diethylether then dried in vacuo. brand (BH) equation [35] as given below:
n
’
[A].[B]0/d/ = [B]0/ε + 1/Kcε’ (1)
tio
Characterization
With
u
Empirical composition of Ru(II)-CNEB complex d' = d-d0A – d 0B (2)
[Ru(CNEB)2(bpy)2].2PF6.0.5 NH4PF 6, was assigned on the
rib
basis of its elemental analysis found (%) [C, 40.7; H, 3.5; Here, [A]0 and [B]0 are the initial concentrations of LDH and
N, 8.2; calculated for, RuC40H38N6O6.2PF 6.0.5NH 4PF 6 C, Ru(II)-CNEB complex respectively. d is the absorbance of
st
41.02; H, 3.4; N, 7.7]. The complex showed M.P.> 250ºC; LDH & complex mixture and d0A & d0B are those of LDH
UV/vis λ max(nm) (ε dm3 mol-1 cm-1 in Methanol 10-4 M) and Ru(II)-CNEB complex at the same wavelength.
i
501(740), 365(2630). IR (cm-1 ), 1610 NH, 839 ν PF 6- ; A linear plot according to equation 1 was obtained and
D
1H NMR (DMSOd ) (ppm), 1.3 (CH3), 4.3 (CH2), 8.8
6 association constant (Kc) between Ru(II)-CNEB complex &
(NH), 7.0-8.0 (ph+bpy), 9.4-10.0 (bpy), 13.1b (b=broad, LDH was calculated to be 1.525 x 105 M-1 . Since this data
or
COOH). Mass spectrum of this complex though showed falls in the close range reported for the interaction of a
peak at 1085 assigned to [Ru(CNEB)(bpy) 2].2PF6 unit with Ru(III) complex [trans-tetrachlorobis(1H-indazole)ruthenate
the loss of five hydrogen atoms, yet peaks obtained at still
tF
(III)] with serum albumin and transferrin [5], it is likely that
higher mass indicated that complex exists in an aggregated both Ru(III) and (II) complexes interact with the proteins in
structure, as peak at 2029 could be seen which is found a similar manner.
o
closer to dimeric composition [(M)2 -PF6]+ . However
dimerization may also occur as a consequence of Luminescence Quenching
N
fragmentation. Therefore, attempts were made to grow single
crystals for structural characterization using X-ray Several workers have used luminescence quenching of a
crystallography of this complex but crystals obtained in protein and/or of a fluorescent metal complex as one of the
acetonitrile-water mixture, after several months, did not reliable criteria to determine protein-metal interaction [7,36].
diffract well, hence limited its characterization by It has been demonstrated that luminescence property of a
spectroscopic data only. Ru(II) complex is sensitive to different solvents including
The complex was found to be soluble in common water [37]. The complex in our hand also showed similar
organic solvents, like methanol, acetonitrile and dimethyl behavior and, only at a semi-liquid state, could give best
sulfoxide. luminescence under a fluorescent microscope at 450 nm.
Taking this as a novel parameter, fluorescent microscopic
analysis of Ru(II)-CNEB-LDH was performed using a
STUDIES ON RU(II)-CNEB-LDH INTERACTION NIKON-ECLIPSE TS 100-F model fluorescence
microscope. A drop of semi liquid mixture of complex and
UV Absorption Pattern
LDH in the ratio of 4:1 (w/v) and the complex alone were
In general, a shift in the absorption pattern of a protein at analyzed separately and images of luminescent spots were
280 nm in the presence of a ligand suggests for protein- captured using inbuilt NIKON camera. One representative
ligand interaction in vitro. Similar approach has been (randomly selected region) from four slides has been
applied to study interaction of certain Ru(III) complexes presented in Fig. 2 wherein; randomly selected spots from
with serum proteins [7,9,34]. This parameter was also the untreated and treated samples have been magnified to
studied for Ru(II)-CNEB complex. Pilot experiments highlight the difference in the luminescence intensity.
suggested that > 20 min incubation of Ru(II)-CNEB The excitation wavelength selected could be correlated to
complex with LDH in a ratio of 10-100 µg complex vs 10- that of a Ru(II) containing dipyridophenazine [38].
30 µg purified LDH (w/v) at 25-40oC provides reproducible Moreover, the results in Fig. 2 demonstrate that the
data. Using Shimadzu UV Visible spectrophotometer, fluorescent intensity of Ru(II)-CNEB complex declines
absorbance of 20µg purified LDH was measured at 280 nm significantly when it was incubated with LDH. Though this
5. Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 247
approach is first of its kind, however, provides a direct non-randomly in a fix ratio. Thus, it could be speculated
evidence to confirm Ru(II)-CNEB-LDH interaction in vitro. that Ru(II)-CNEB complex interacts at certain fixed sites on
LDH molecules, and when these sites get occupied, no more
further binding takes place even at a higher concentration of
Gel Mobility Shift Assay the complex.
Gel mobility shift assay is another direct and reliable Based on absorption and spectrofluorometric data, gold
tool to study molecular interactions, however, it has scarcely (III) complex was described to interact with serum albumin
been used in past to ascertain metal-protein interaction. LDH via establishing week interactions [36]. There are some
is a good candidate to monitor this property, as it has been reports suggesting that histidine residues are the preferred
demonstrated that mobility of M4-LDH gets retarded on gel binding sites for Ru(III) complexes on the protein surface
electrophoresis if bound to certain organic compounds [39]. [1,2,38]. These could be the putative sites on LDH also for
In order to ascertain and measure Ru(II)-CNEB-LDH the interaction of Ru(II)-CNEB complex. Though, it would
interaction, untreated LDH and the LDH treated with 50 & be interesting to ascertain amino acid residues involved in
100 µg Ru(II)-CNEB complex were subjected for mobility LDH-Ru(II)-CNEB interaction, however, with a
shift assay on 10% SDS-PAGE (Sodium dodecyl sulphate- pharmacological view point, it is more relevant to first
polyacrylamide gel electrophoresis) following the method of characterize this complex as a catalytic modulator of this
Laemmili [40] as described in our earlier report [41]. As enzyme.
n
described earlier [42,43], a plot (log MW vs. Rf) was
constructed to determine apparent difference in the mobility
tio
of untreated and Ru(II)-CNEB treated LDH. MODULATION OF LDH BY RU(II)-CNEB
According to Fig. (3A & B), as compared to the COMPLEX IN VITRO
u
untreated sample, mobility of Ru(II)-CNEB complex treated
rib
To ascertain whether Ru(II)-CNEB complex modulates
LDH gets retarded by ~ 4.5 kDa on SDS-PAGE and thus,
LDH activity, purified LDH was incubated with the
reconfirming a significant interaction between the complex
st
increasing concentration of the complex for 30 min,
and LDH molecules. In addition, a shift in the mobility of
followed by activity measurement and non-denaturing PAGE
LDH by ~ 4.5 kDa, equal with both 50 & 100 µg complex
i
analysis.
(Fig. (3B)), suggests that Ru(II)-CNEB complex binds LDH
or D
N otF
Fig. (2). Comparison between the luminescence intensity of Ru(II)-CNEB alone and in the presence of purified LDH. (A) 40 x
magnification. (B) 400 x magnification.
6. 248 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al.
tio n
rib u
D i st
tF or
N o
Fig. (3). Gel mobility shift assay of purified LDH after 30 min incubation with 50 & 100 µg of Ru (II)-CNEB complex. (A)
CBB stained LDH bands after 10% SDS-PAGE. 20 µg LDH was loaded in each lane and gel was run along with 15 µg
standard MW marker lane. (B) MW determination of the treated and untreated LDH samples on semi log scale. Rf values were
calculated as a ratio of mobility of the bands from the origin/distance (cm) of the migrated dye front.
Measurement of LDH Activity earlier report [47]. Suitable amount of Ru(II)-CNEB
complex-LDH mixture and untreated LDH samples were
LDH activity was measured following the method of subjected for electrophoresis followed by LDH specific
Kornberg [44] and as described in our earlier report [18]. One staining of the gels as described earlier [47]. LDH bands
unit of the enzyme was defined as conversion of 1µ mol were scanned and quantified by gel spectrometry using alpha
NADH into NAD min -1 at 25ºC, and activity was expressed imager 2200 gel documentation software. Through pilot
as units/mg protein. Protein content in all the samples was experiments, LDH bands were identified as M4 isoform
measured using Folin method [45]. Statistical analysis, (predominantly expressed in liver and skeletal muscle
wherever required, was done following Bruning and Kintz tissues) of the enzyme (M4-LDH) as described earlier [18].
[46]. Student ‘t’ test was performed to find the level of
significance between the groups.
Effect of Ru(II)-CNEB on LDH Activity
Analysis of LDH Isozymes According to Fig. 4A, there was a significant decline in
LDH activity (p<0.05) in the presence of 100 µg Ru(II)-
LDH isozymes were analyzed on non-denaturing (non- CNEB complex, suggesting that this complex is able to
SDS) 10% PAGE following the method described in our modulate LDH activity in vitro. The pattern was supported
7. Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 249
tio n
rib u
D i st
tF or
N o
Fig. (4). Effect of Ru (II)-CNEB complex on the activity of purified LDH. (A) activity pattern in the presence of different
concentrations of the complex. (B) activity stained M4-LDH bands after 10% native PAGE, 20 µg protein was loaded in each
lane. (C) represents relative intensity of LDH bands (taking total of the three lanes as 100%) based upon densitometric analysis
data. * p < 0.05 (treated vs. untreated LDH samples).
by the similar changes in the intensity of M4-LDH bands tested on this parameter [17]. The present findings suggest
after non-denaturing PAGE (Fig. (4B)). Densitometric that LDH is another such example with respect to Ru(II)-
analysis of the bands (Fig. (4C)) provides quantitative CNEB.
difference for a perceptual decline seen in Fig. 4B. Gel
electrophoretic assay has also been adopted by some other
workers to describe the inhibition of gelatinase by NAMI-A KINETICS OF LDH IN THE PRESENCE OF RU (II)-
[13]. CNEB COMPLEX
In general, binding of a ligand to an enzyme is known to Conventional kinetic studies of the enzymes provide
induce subtle changes in the enzyme conformation resulting information on mechanistic aspect of the enzyme catalysis
into alterations in the enzyme activity [48,49]. Inhibition of [32,49]. Using this approach, there is a report on
cytochrome c activity by a Ru(III) complex has already been characterizing selective active site (competitive) inhibitors of
8. 250 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al.
Table 2. Apparent Kinetic Constants (Km & Vmax) of LDH Determined after 30 min. Incubation of the Enzyme with the
Increasing Concentration of Ru (II)-CNEB Complex
Experimental Condition Km (µM Pyruvate) Vmax (µM NAD min-1 mg Protein-1)
LDH (Untreated) 16.66 10.00
LDH + 50 ug Ru (II)-CNEB 16.95 8.33
LDH + 100 ug Ru (II)-CNEB 17.24 4.16
Values were calculated from a Lineweaver-Burk plot (1/V vs. 1/[pyruvate]) constructed using mean values from 4 assays at each [S] for each set of experiments separately.
human LDH for therapeutic applications [32]. Similar LDH + complex in the ratio of 1: 2 and 1: 4 (w/v). The Km
approaches were used to explain mechanism of Ru(II)-CNEB values were determined from Line-weaver Burk plot (1/V vs.
complex dependent inhibition of LDH. 1/ [pyruvate]) constructed using mean of 3-4 observations at
each point.
Kinetic Studies The Ki value of LDH for Ru(II)-CNEB complex was
determined by measuring the activity of suitably diluted
The Km and Vmax of M4-LDH was determined by
n
LDH separately at different concentrations (0.02-0.08 mM)
measuring the enzyme activity at different concentrations of
tio
of Ru(II)-CNEB complex in the presence of 0.01 & 0.05
the substrate (5-100 µM) taking LDH sample alone and
rib u
D i st
tF or
N o
Fig. (5). Effect of Ru (II)-CNEB complex on LDH from mice liver extracts. Suitably diluted liver extract was incubated with 50 & 100
µg Ru (II)-CNEB separately for 30 min followed by spectrophotometric assay (A) and identification of M4-LDH by development of
enzyme specific bands after 10% PAGE (B). 30 µg protein was loaded in each lane. Panel C represents relative intensity of LDH band
(taking total of the three lanes as 100%) based on densitometric analysis data. * p < 0.001 (treated vs. untreated samples).
9. Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 251
mM substrate (pyruvate). The Ki value was determined from µg of complex remained unchanged, however, Vmax of the
Dixon plot (1/V vs. [Ru(II)-CNEB]) constructed using mean enzyme showed a concentration dependent decline in the
of 3-4 observations at each point. presence of Ru(II)-CNEB complex. Such a pattern represents
a non-competitive type of inhibition of LDH activity by
Ru(II)-CNEB complex. The Ki value determined for the
Characterization of Ru(II)-CNEB as LDH Inhibitor complex was 0.032 mM (not shown in the table).
According to Table 2, as compared to untreated sample, Since, Ru(II)-CNEB complex is structurally different
Km values of LDH for pyruvate in the presence of 50 & 100 from that of the metabolic substrates (Pyruvate and NADH)
tio n
rib u
D i st
tF or
N o
Fig. (6). Activity (A) and PAGE pattern (B) of LDH in Ru (II)-CNEB complex perfused liver extracts. 1, untreated sham operated mice;
2, mice perfused with KRB alone and 3, mice perfused with KRB containing 100 mg/ml complex. In case of panel B, 30 µg protein
was loaded in each lane of 10% PAGE. Panel C represents relative intensity of LDH band (taking total of the three lanes as 100%)
based on densitometric analysis data. *p < 0.01 (Ru (II)-CNEB perfused vs. untreated liver extracts).
10. 252 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al.
of LDH, it is less likely that the complex would compete for whereas, PBS alone was perfused for control mice. Another
active site of the enzyme. A Ru(II)-CNEB complex group of mice were operated similarly to serve as sham-
dependent decline in the Vmax of purified LDH with no operated control. 3-4 mice were treated in each group. On
change in the Km of the enzyme (Table 2) represents a spectrophotometric assay and on native PAGE (Fig. 6A &
typical pattern for non-competitive inhibition of LDH B) analysis, Ru(II)-CNEB complex perfused liver could
activity by this complex, and thus, suggesting for allosteric show significant decline in LDH activity suggesting thereby
inhibition of LDH by Ru(II)-CNEB. Allosteric modulation that this complex acts as a potent inhibitor of LDH at tissue
of the enzymes is considered as an effective mechanism for level also.
therapeutic interventions [10]. Taking together the results
from Fig. 2 & 3 and Table 2, it is evident that Ru(II)-CNEB
binds to LDH at an allosteric site and inhibits the enzyme CONCLUSION
non-competitively. In addition, since, Ki value (0.032 mM)
of the enzyme for Ru (II)-CNEB is lower (stronger EI The wealth of literature on identifying a number of cancer
affinity) than the values obtained for the known metabolic related genes and on understanding the regulation of such
inhibitors of LDH [50], the complex seems to be a potent genes is still not able to provide a uniform biological basis
allosteric inhibitor of LDH and therefore, offers its to design therapeutic intervention against cancer at gene
pharmacological applications targeted to this enzyme. level. Alternatively, targeting down stream cellular
mechanisms seems to be a logical alternative, particularly
n
with respect to depriving the tumors for their increased
tio
RU(II)-CNEB COMPLEX AS INHIBITOR OF LDH IN energy and resource demands by chemical intervention.
MICE LIVER LDH, a critical enzyme of cell bioenergetics, would be a
logical target, as it is found to be implicated in tumor
u
In order to ascertain pharmacological application of a development. There is much interest in ruthenium
rib
compound, it is important to verify its action with bio- complexes as anti cancer agents, however, these complexes
components at cellular/tissue level. need to be investigated to define their pharmacological
st
targets at cellular level. This article is an attempt to draw the
attention of chemists and biologists towards formulating
Inhibition of LDH in Cell Free Extracts
i
Ru-complexes as modulators of LDH and other susceptible
enzymes with a viewpoint of therapeutic intervention against
D
To confirm whether Ru(II)-CNEB complex modulates
cancer. In an attempt of this kind, we have provided some
LDH in cell free extracts also, mice liver extracts were
or
experimental data which suggest that a novel Ru(II)-CNEB
incubated with the increasing concentration of the complex
complex interacts with LDH molecules in a specific manner
followed by activity measurement and PAGE analysis of
and acts as a potent non-competitive inhibitor of the enzyme
LDH. According to Fig. 5A, as compared to the untreated
tF
in vitro and at tissue level as well. Since, elevated level of
sample, Ru(II)-CNEB complex treated samples showed a
LDH is implicated in catering the additional energy need of
concentration dependent significant decline (p < 0.001) in
tumor cells, Ru(II)-CNEB complex seems to be a promising
o
the activity of LDH. The inhibitory pattern of LDH was
candidate to inhibit this pathway via inhibiting LDH.
further supported from a similar decline in the intensity of
Additionally, these findings may open a new area of future
N
M4-LDH bands on non-denaturing PAGE (Fig. 5B & C).
research for formulating and screening a number of anticancer
Ru-complexes targeted to LDH in different tumor models.
Inhibition of LDH at Tissue Level
To confirm these effects at tissue level, mice were ACKNOWLEDGEMENTS
perfused with the buffered Ru(II)-CNEB complex to ensure
that the complex reaches at tissue level. Liver was perfused This work was financially supported in part by
intra-cardially with the aqueous solution of the complex. Department of Biotechnology, New Delhi (joint project to
Ru(II)-CNEB complex (25 mg) diluted in phosphate LM and SKT, BT/PR5910/BRB/10/406/2005), Govt. of
buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM India, and CAS Programme, Department of Zoology, BHU.
Na2HPO4, 2 mM KH2PO4, 0.5 mM MgCl2, 1 mM CaCl2). Special thanks are due to Prof. (Mrs.) V.G. Puranik, NCL,
Through pilot experiments, it was ascertained that even up Pune, India, for assistance in providing single crystal X-ray
to 250 µg /ml (~10 µg/g body weight) of the complex data and to Prof. S.N Singh, ex-Professor of Physiology &
prepared in PBS did not produce toxic symptoms in adult Biochemistry, Department of Zoology, BHU, Varanasi,
mice. India, for correction of the manuscript.
Liver perfusion was done according to Clémence et al.
[51] with some modifications. Briefly, mice were SUPPLEMENTARY MATERIALS
anesthetized by thiopentone injection and a canula was
implanted between ventricle and atrium in the heart so that it Deposition of Crystallographic Data
feeds the ventral aorta and perfusate reaches to the tissues
Crystallographic data for the structural analysis of CNEB
directly through systemic circulation. Perfusion was done at
have been deposited with the Cambridge Crystallographic
a rate of 1 ml/min and at constant pressure by keeping the
Data Center, CCDC No. 618507. Copies of this information
solution at 1meter height above the heart for 30 min.
may be obtained free of charge from the Director, 12 Union
Experimental group mice were perfused with PBS
Road, Cambridge, CB2 1EZ, UK (fax: +44 1223 336 033;
containing 100 µg/ml (4-5 µg/g body weight) complex
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Received: November 03, 2006 Revised: December 25, 2006 Accepted: January 17, 2007