Structural and kinetic studies on the activators of succinate dehydrogenase

1. 1. Diverse classes of compounds such as dicarboxylates, pyrophosphates, quinols and nitrophenols are known to activate mitochondrial succinate dehydrogenase (EC 1.3.99.1). Examples in each class — malonate, pyrophosphate, ubiquinol and 2,4-dinitrophenol — are selected for comparative studies on the kinetic constants and structural relationship. 2. 2. The activated forms of the enzyme obtained on preincubating mitochondria with the effectors exhibited Michaelian kinetics and gave doublereciprocal plots which are nearly parallel to that of the basal form. On activation, Km for the substrate also increased along with V. The effectors activated the enzyme at low concentrations and inhibited, in a competitive fashion, at high concentrations. The binding constant for activation was lower than that for inhibition for each effector. 3. 3. These compounds possess ionizable twin oxygens separated by a distance of Image and having fractional charges in the range of −0.26 to −0.74 e. The common twin-oxygen feature of the substrate and the effectors suggested the presence of corresponding counter charges in the binding domain. The competitive nature of effectors with the substrate for inhibition further indicated the close structural resemblance of the activation and catalytic sites.

Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting

A probe, 9-(anthrylmethyl)trimethylammonium chloride, 1, was prepared. 1 binds to calf-thymus DNA or Escherichia coli genomic DNA with high affinity, as evidenced from the absorption titration. Strong hypochromism, spectral broadening and red-shifts in the absorption spectra were observed. Half-reciprocal plot constructed from this experiment gave binding constant of 5±0.5×104 M−1 in base molarity. We employed this anthryl probe-DNA complex for studying the effects of addition of various surfactant to DNA. Surfactants of different charge types and chain lengths were used in this study and the effects of surfactant addition to such probe-DNA complex were compared with that of small organic cations or salts. Addition of either salts or cationic surfactants led to structural changes in DNA and under these conditions, the probe from the DNA-bound complex appeared to get released. However, the cationic surfactants could induce such release of the probe from the probe-DNA complex at a much lower concentration than that of the small organic cations or salts. In contrast the anionic surfactants failed to promote any destabilization of such probe-DNA complexes. The effects of additives on the probe-DNA complexes were also examined by using a different technique (fluorescence spectroscopy) using a different probe ethidium bromide. The association complexes formed between the cationic surfactants and the plasmid DNA pTZ19R, were further examined under agarose gel electrophoresis and could not be visualized by ethidium bromide staining presumably due to cationic surfactant-induced condensation of DNA. Most of the DNA from such association complexes can be recovered by extraction of surfactants with phenol-chloroform. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA melting temperatures by a few °C and at high [surfactant], the corresponding melting profiles got broadened.

Photocytotoxic Oxovanadium(IV) Complexes Showing Light-Induced DNA and Protein Cleavage Activity

Oxovanadium(IV) complexes [VO(L)(B)]Cl-2 (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline(phen),dipyrido[3,2-d:2',3'-f]quinoxaline(dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells, The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON5 coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III)couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M-1. The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor ``chemical nuclease'' activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells giving IC50 value of 17 mu M in visible light(IC50 = 175 mu M in dark).

DNA cleavage in red light promoted by copper(II) complexes of -amino acids and photoactive phenanthroline bases

Ternary copper(II) complexes [Cu(L-trp)(B)(H2O)](NO3) ( 1–3) and [Cu(L-phe)(B)(H2O)](NO3) ( 4–6) of L-tryptophan (L-trp) and L-phenylalanine (L-phe) having phenanthroline bases (B), viz. 1,10-phenanthroline (phen, 1 and 4), dipyrido[3,2-d:2,3-f]quinoxaline (dpq, 2 and 5) and dipyrido[3,2-a:2,3-c]phenazine (dppz, 3 and 6), were prepared and characterized by physico-chemical techniques. Complexes 3 and 6 were structurally characterized by X-ray crystallography and show the presence of a square pyramidal (4 + 1) CuN3O2 coordination geometry in which the N,O-donor amino acid (L-trp or L-phe) and N,N-donor phenanthroline base bind at the equatorial plane with an aqua ligand coordinated at the elongated axial site. Complex 3 shows significant distortion from the square pyramidal geometry and a strong intramolecular – stacking interaction between the pendant indole ring of L-trp and the planar dppz aromatic moiety. All the complexes display good binding propensity to the calf thymus DNA giving an order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding constant (Kb) values are in the range of 2.1 × 104–1.1 × 106 mol-1 with the binding site size (s) values of 0.17–0.63. The phen and dpq complexes are minor groove binders while the dppz analogues bind at the DNA major groove. Theoretical DNA docking studies on 2 and 3 show the close proximity of two photosensitizers, viz. the indole moiety of L-trp and the quinoxaline/phenazine of the dpq/dppz bases, to the complementary DNA strands. Complexes 2 and 3 show oxidative DNA double strand breaks (dsb) of supercoiled (SC) DNA forming a significant quantity of linear DNA along with the nicked circular (NC) form on photoexposure to UV-A light of 365 nm and red light of 647.1 nm (Ar–Kr laser). Complexes 1, 5 and 6 show only single strand breaks (ssb) forming NC DNA. The red light induced DNA cleavage involves metal-assisted photosensitization of L-trp and dpq/dppz base resulting in the formation of a reactive singlet oxygen (1O2) species.

Photocytotoxic Lanthanum(III) and Gadolinium(III) Complexes of Phenanthroline Bases Showing Light-Induced DNA Cleavage Activity

Lanthanide complexes of formulation [La(B)(2)(NO3)(3)] (1-3) and [Gd(B)(2)(NO3)(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4),dipyrido[3,2-d2',3'-f]quinoxaline (dpq in 2,5) and dipyrido[3,2-a2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibitligand centered bands in the UV region The dppz complexes show thelowest energy band at 380 nm in N,N-dimethylformamide (DMF) The La(III)complexes are diamagnetic. The Gd(III) complexes (4-6) have magneticmoments that correspond to seven unpaired electrons The complexes are1(.)1 electrolytic in aqueous DMF The dpq and dppz complexes in DMFshow ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M-1 with a relative order. 3, 6 (dppz)> 2, 5 (dpq) > 1, 4 (phen) The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form onexposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) as the cleavage active species.The complexes show binding propensity to bovine serum albumin (BSA)protein giving K-BSA values of similar to 10(5) M-1. The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC50 values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC50 > 100 mu M in dark for both the complexes) Control experiments show significant dark and phototoxicity of the dppz base alone (IC50 = 413 nM in light with 4 h incubation in dark and 116 mu M in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observedon binding to the lanthanide ions while retaining similar phototoxicity.

A spectroscopic study of the interaction of isoflavones with human serum albumin

Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 X 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal.mol(-1), -21 cal.mol(-1)K(-1) and -6.86 kcal.mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and Forster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

Photocytotoxicity and near-IR light DNA cleavage activity of oxovanadium(IV) Schiff base complexes having phenanthroline bases

Oxovanadium(IV) complexes VO(L)(B)] (1-3), where H2L is a Schiff base ligand 2-(2-hydroxybenzylideneamino) phenol and B is 1,10-phenanthroline (phen for 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq for 2) or dipyrido3,2-a:2',3'-c]phenazine (dppz for 3), have been prepared, characterized and their DNA binding property and photo-induced DNA cleavage activity studied. Complex 3 which is structurally characterized by X-ray crystallography shows the presence of an oxovanadium(IV) moiety in a six coordinate VO3N3 coordination geometry. The complexes show a d-d band within 800-850 nm in DMF. The complexes display an oxidative response near 0.7 V versus SCE for V(V)-V(IV) and a reductive response within -1.1 to -1.3 V due to V(IV)-V(III) couple in DMF-0.1 M TBAP. The complexes are avid binders to calf thymus DNA giving binding constant values of 4.2 x 10(4) to 1.2 x 10(5) M (1). The complexes do not show any ``chemical nuclease'' activity in dark. The dpq and dppz complexes are photocleavers of plasmid DNA in UV-A light of 365 nm via O-1(2) pathway and in near-IR light (752.5 to 799.3 nm IR optics) by HO* pathway. Complex 3 exhibits significant photocytotoxicity in visible light in HeLa cells giving IC50 value of 13 mu M, while it is less toxic in dark (IC50 = 97 mu M). (C) 2010 Elsevier B.V. All rights reserved.

New chelating resins based on polybenzimidazole. Selective sorption of copper(II) from ammoniacal media on resin with anchored l-cysteine

Microporous polybenzimidazole (PBI) of 250–500 μm bead size has been epoxidized and subsequently reacted with l-cysteine in the presence of a phase-transfer catalyst at room temperature to obtain a sorbent having anchored l-cysteine, EPBI(Cyst). The sorption of Cu(II), Ni(II), Co(II), and Zn(II) in mildly acidic and ammoniacal solutions has been measured under comparable conditions on EPBI(Cyst) and Dowex 50W-X8(H+) resins. While the latter shows no appreciable difference in sorption of the four metals in acidic and ammoniacal media and has 40–60 % selectivity for copper(II) over the other three, EPBI(Cyst) shows a threefold increase in copper sorption and more than 90% copper selectivity over the other metals in ammoniacal media, compared to mildly acidic media. The copper binding constant and saturation capacity of EPBI(Cyst) in ammoniacal media decrease only slowly beyond pH 11.6 with the result that the resin shows significant sorption of Cu(II) even in strongly ammoniacal solutions. The sorbed copper is stripped with HCl relatively easily. The copper sorption kinetics on EPBI(Cyst) is unusually fast in ammoniacal media with more than 90 % of equilibrium sorption being attained in one minute.

Photo-activated cytotoxicity of a pyrenyl-terpyridine copper(II) complex in HeLa cells

Terpyridine copper(II) complexes Cu(L)(2)](NO3)(2) where L is (4'-phenyl)-2 2' 6' 2 `'-terpyridine (ph-tpy in 1) and 4-(1 pyrenyl)]-2 2' 6' 2'-terpyridine (py-tpy in 2) are prepared characterized and their photocytotoxic activity studied The crystal structure of complex 1 shows distorted octahedral CuN6 coordination geometry The 1 2 electrolytic and one-electron paramagnetic complexes show a visible band near 650 nm in DMF-H2O The complexes show emission band at 352 nm for 1 and 425 nm for 2 when excited at 283 and 346 nm respectively The Cu(II)-Cu(I) redox couple is observed near -0 2 V versus SCE in DMF-0 1 m TBAP The complexes are avid partial-intercalative binders to calf thymus DNA giving binding constant (K-b) values of similar to 10(6) M-1 Complex 2 with its photoactive pyrenyl moiety exhibits significant photocleavage of pUC19 DNA in red light via singlet oxygen pathway Complex 2 also exhibits significant photo-activated cytotoxicity in HeLa cancer cells in visible light giving IC50 value of 11 9 mu M while being non-toxic in dark with an IC50 value of 130 5 mu M (C) 2010 Elsevier Ltd All rights reserved

Intermolecular complexes of singly linked bisporphyrins with trinitrobenzene

Several covalently linked bisporphyrin systems, free-base (H2P---H2P), hybrid bisporphyrins (Zn---H2P) and Zn(II) dimers (ZnP---ZnP) and their 1:1 molecular complexes with sym 1,3,5-trinitrobenzene have been investigated by optical absorption and emission, and magnetic resonance spectroscopic methods. In these systems, two porphyrin units are linked singly through one of the meso aryl groups via ether linkages of variable length. The bisporphyrins cooperatively bind a molecule of a ?-acceptor; 1,3,5-trinitrobenzene (TNB). The binding constant values vary with interchromophore separation. Maximum binding is observed in the bisporphyrin bearing a two-ether covalent linkage. It is found that TNB quenches the fluorescence of the two porphyrine units in a selective manner. It is suggested that a critical distance between the two porphyrin units is necessary for the observance of maximum cooperative intermolecular binding with an acceptor.

Copper(I) complexes of modified nucleobases and vitamin B3 as potential chemotherapeutic agents: In vitro and in vivo studies

Three new complexes of Cu(I) have been synthesized using ancillary ligands like thiopyrimidine (tp) a modified nucleobase, and nicotinamide (nie) or vitamin B3, and characterized by spectroscopy and X-ray crystallography. In vitro cytotoxicity studies of the complexes on various human cancer cell lines such as Colo295, H226, HOP62, K562, MCF7 and T24 show that Cu(PPh3)(2)(tp)Cl] and Cu(PPh3)(2)(tp)ClO4 (2) have in vitro cytotoxicity comparable to cisplatin. Complex Cu(nic)(3)PPh3]ClO4 (3) is non-toxic and increases the life span by about 55 % in spontaneous breast tumor model. DNA binding and cleavage studies show that complex (3) binds to calf thymus DNA with an apparent binding constant of 5.9 x 10(5)M and completely cleaves super-coiled DNA at a concentration of 400 mu M, whereas complexes (1) and (2) do not bind DNA and do not show any cleavage even at 1200 mu M. Thus, complex (3) may exhibit cytotoxicity Via DNA cleavage whereas the mechanism of cytotoxicity of (1) and (2) probably involves a different pathway.

DNA targeting polyaza macrobicyclic dizinc(II) complexes promoting high in vitro caspase dependent anti-proliferative activity against human carcinoma cancer cells

A series of macrobicyclic dizinc(II) complexes Zn2L1-2B](ClO4)(4) (1-6) have been synthesized and characterized (L1-2 are polyaza macrobicyclic binucleating ligands, and B is the N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)). The DNA and protein binding, DNA hydrolysis and anticancer activity of these complexes were investigated. The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence and CD spectroscopy. The DNA binding constant values of the complexes were found to range from 2.80 x 10(5) to 5.25 x 10(5) M-1, and the binding affinities are in the following order: 3 > 6 > 2 > 5 > 1 > 4. All the dizinc(II) complexes 1-6 are found to effectively promote the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 3 and 6 under physiological conditions give observed rate constants (k(obs)) of 5.56 +/- 0.1 and 5.12 +/- 0.2 h(-1), respectively, showing a 10(7)-fold rate acceleration over the uncatalyzed reaction of dsDNA. Remarkably, the macrobicyclic dizinc(II) complexes 1-6 bind and cleave bovine serum albumin (BSA), and effectively promote the caspase-3 and caspase-9 dependent deaths of HeLa and BeWo cancer cells. The cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme levels in cancer cell lysate and content media.

Cobalt(II), Nickel(II) and Copper(II) complexes of a tetradentate Schiff base as photosensitizers: Quantum yield of O-1(2) generation and its promising role in anti-tumor activity

In the present investigation, a Schiff base N'(1),N'(3)-bis(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzene-1,3-d icarbohydrazide and its metal complexes have been synthesized and characterized. The DNA-binding studies were performed using absorption spectroscopy, emission spectra, viscosity measurements and thermal denatuaration studies. The experimental evidence indicated that, the Co(II), Ni(II) and Cu(II) complexes interact with calf thymus DNA through intercalation with an intrinsic binding constant K-b of 2.6 x 10(4) M-1, 5.7 x 10(4) M-1 and 4.5 x 10(4) M-1, respectively and they exhibited potent photo-damage abilities on pUC19 DNA, through singlet oxygen generation with quantum yields of 0.32, 0.27 and 0.30 respectively. The cytotoxic activity of the complexes resulted that they act as a potent photosensitizers for photochemical reactions. (C) 2012 Elsevier B.V. All rights reserved.

DNA and protein targeting 1,2,4-triazole based water soluble dinickel(II) complexes enhances antiproliferation and lactate dehydrogenase inhibition

Water soluble dinickel(II) complexes Ni-2(L)(2)(1-2)](NO3)(4) (1-2), where L1-2 are triazole based dinucleating ligands, were synthesized and characterized. The DNA binding, protein binding, DNA hydrolysis and anticancer properties were investigated. The interactions of complexes 1 and 2 with calf thymus DNA were studied by spectroscopic techniques, including absorption and fluorescence spectroscopy. The DNA binding constant values of the complexes 1 and 2 were found to be 2.36 x 10(5) and 4.87 x 10(5) M-1 and the binding affinities are in the following order: 2 > 1. Both the dinickel(II) complexes 1 and 2, promoted the hydrolytic cleavage of plasmid pBR322 DNA under both anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 1 and 2 under physiological conditions give the observed rate constants (k(obs)) of 5.05 +/- 0.2 and 5.65 +/- 0.1 h(-1), respectively, which shows 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. Meanwhile, the interactions of the complex with BSA have also been studied by spectroscopy. Both the complexes 1 and 2 display strong binding propensity and the binding constant (K-b), number of binding sites (n) were obtained are 0.71 x 10(6) 1.47] and 5.62 x 10(6) 1.98] M-1, respectively. The complexes 1 and 2 also promoted the apoptosis against human carcinoma (HeLa, and BeWo) cancer cells. Cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme level in cancer cell lysate and content media. (c) 2013 Elsevier Ltd. All rights reserved.