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Ruthenium complex as enzyme modulator Ruthenium complex as enzyme modulator Document Transcript

  • Current Enzyme Inhibition, 2007, 3, 243-253 243 Ruthenium Complex as Enzyme Modulator: Modulation of Lactate Dehydrogenase by a Novel Ruthenium(II) Complex Containing 4-Carboxy N-Ethylbenzamide as a Ligand Surendra K. Trigun1 , Raj K. Koiri1 , Lallan Mishra*,2 , Santosh K. Dubey2 , Santosh Singh1 and Pankaj Pandey3 1Biochemistry& Molecular Biology Section, Centre of Advanced Studies in Zoology, Banaras Hindu University, Varanasi-221005, India 2Department of Chemistry, Banaras Hindu University, Varanasi-221005, India 3Department of Nephrology, Vanderbilt University, MCN, South Nashville, TN 37232, USA Abstract: Ruthenium complex-protein interaction, particularly with respect to modulation of the enzymes associated to tumor development, is an evolving concept in understanding the mechanism of action of these complexes as anticancer agent. Lactate dehydrogenase (LDH; EC: 1.1.1.27) is critically implicated in n maintaining tumor growth via ‘Warburg effect’ in cancerous cells. This article presents current status of Ru- tio complexes as enzyme inhibitors in general and a state of art on a novel ruthenium(II) complex containing 4- Carboxy-N-ethylbenzamide as an inhibitor of LDH. The 4-carboxy-N-ethylbenzamide (CNEB) was synthesized and characterized by single crystal X-ray measurement and complexed with cis-Ru(bpy)2 Cl 2 .2H2 O u (bpy=2,2’bipyridine) resulting into synthesis of a [Ru(CNEB)2 (bpy) 2 ] 2PF 6 .0.5 NH4 PF6 ] complex, Ru(II)- rib CNEB. The complex showed appreciable cytotoxicity on Dalton’s lymphoma cells and a significant Ru(II)- CNEB-LDH interaction (Kc = 1.525 x 105 M -1). The later was further confirmed from luminescence quenching st and gel retardation assays. The complex also caused a significant decline in the activities of purified LDH and LDH from mice liver extract. The complex was further characterized as a non-competitive inhibitor of LDH (Ki = i 0.032 mM). Ru(II)-CNEB complex perfused mice liver also showed a significant decline in LDH activity coinciding with similar changes in the intensity of LDH bands on polyacrylamide gel electrophoresis. Thus, D Ru(II)-CNEB complex, as a non-competitive inhibitor of LDH, seems to be a candidate for potential therapeutic or applications. Keywords: CNEB, Ru(II) complex, structural characterization, lactate dehydrogenase, metallodrug-protein interaction, gel mobility shift, enzymatic modulation, glycolytic enzymes, Warburg effect. t F RUTHENIUM COMPLEX: THE POINT OF cell function would be another important strategy to o CONCERN understand therapeutic potential of Ru-complexes [5, 6]. During recent past, the Ru-complex-protein interactionsN Ruthenium complexes are of much current interest as anti-cancer non-platinum metallo-drugs due to the various have been focused mainly for an effective tool to deliver the reasons including diversity in the synthetic chemistry of Ru- complex at tumor site. Transferrin, a serum iron transport complexes to yield reliable preparations of predictable protein, is transported to the tumor sites at a much faster rate structures with the ability to tune ligand affinities and than to the normal cells due to the hypoxic importantly, their low toxicity and effective bio-distribution microenvironment and increased energy metabolism in the & reproducible bioactivities [1]. Until recent, cytotoxicity of cancerous cells. It has been demonstrated that several Ru- Ru-complexes has been correlated with their ability to bind complexes readily bind to transferrin, and thus, they are with and damage DNA [2,3]. However, the dictum that delivered preferentially to the tumor cells [7-9]. Anti tumor DNA is the primary target for anticancer metallodrugs is activity of some Ru-complexes following transferrin rapidly declining and different Ru-complexes have been mediated delivery at tumor sites in vivo has also been described to affect cellular functions in different ways. A reported [8]. Studies on the binding of Ru(III) complexes Ru(III) complex inhibits DNA replication as well as reduce with albumin, another important protein in serum, have also RNA synthesis in vivo [1]. NAMI-A, trans[Ru(IM) substantiated protein interacting potential of Ru-complexes (DMSO)Cl4], produces cytotoxicity by interacting with [5]. These findings provide basis to speculate that some of DNA as well as by inhibiting type IV collagenase activity the Ru-complexes may be exploited to modulate activities of [1]. Cytotoxicity of [Ru(η6C 6H6).DMSOCl 2] is mediated cancer related specific proteins. Cell signaling proteins, via inhibition of topoisomerase II [4]. Thus, suggesting that transporters and metabolic enzymes associated with the formulation of Ru-complexes targeted to proteins of critical induction and maintenance of tumors could be potential targets in this respect. RU-COMPLEXES AS ENZYME MODULATORS *Address correspondence to this author at the Department of Chemistry, Banaras Hindu University, Varanasi-221005, India; Tel: +91-9450871777; The complex behavior of enzymatic proteins can be E-mail: lmishrabhu@yahoo.co.in regulated by allosteric interactions and targeted for drug- 1573-4080/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.
  • 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 severalN 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
  • Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 245 nFig. (1). Molecular structure of CNEB (ORTEP diagram). tioFig. (1), whereas, major crystallographic property of CNEB respectively, which were further supported by its 1H NMR uis 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) stTable 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 -1N 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 View slide
  • 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 QuenchingN 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 View slide
  • 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 DN 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.
  • 248 Current Enzyme Inhibition, 2007, Vol. 3, No. 3 Trigun et al. tio n rib u D i st tF orN 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
  • Ruthenium Complex as Enzyme Modulator Current Enzyme Inhibition, 2007, Vol. 3, No. 3 249 tio n rib u D i st tF orN 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
  • 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 orN 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).
  • 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 orN 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).
  • 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 futureN 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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