The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalators

Fifteen platinum(II)-based metallointercalators have been synthesised that utilise substituted 1,10-phenanthroline (phen) ligands, including 5-chloro-1,10-phenanthroline (5-Cl-phen), 5-methyl-1,10-phenanthroline (5-CH3-phen), 5-amino-1,10-phenanthroline (5-NH2-phen), 5-nitro-1,10-phenanthroline (5-NO2-phen) and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), and achiral ethylenediamine (en) and the chiral ancillary ligands 1S,2S-diaminocyclohexane (S,S-dach) and 1R,2R-diaminocyclohexane (R,R-dach). Their cytotoxicity in the L1210 murine leukaemia cell line was determined using growth inhibition assays. The most cytotoxic metal complexes are those that contain S,S-dach ancillary ligands and 5-CH3-phen intercalating ligands. One metallointercalator [Pt(5-CH3-phen)(S,S-dach)]Cl2 (5MESS), displays a 5-10-fold increase in cytotoxicity compared to the clinical agent cisplatin. From DNA binding experiments there appears to be no significant difference between any of the metal complexes, indicating that neither DNA binding affinity nor the mode of binding/DNA adduct formed is the sole determinant of the cytotoxicity of this family of platinum(II)-based metallointercalators.     

The binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase. A probe for the open-enzyme conformation

We have studied the binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10-phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10-phenanthroline to Co(II)-LADH is about 60 s-1. This is about a third of the value (169 s-1) determined for native horse-liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471-480]. For cadmium(II)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10-phenanthroline increased to 590 s-1. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10-Phenanthroline is shown to bind to the Co(II)-LADH.NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.     

1,10-Phenanthroline phosphorylates (activates) MAP kinase in Xenopus oocytes

The membrane-permeable intracellular heavy metal chelator, 1,10-phenanthroline, which prevents progesterone-induced germinal vesicle breakdown (GVBD), would be expected to regulate phosphorylation (activation) of the MAP kinase (MAPK) cascade in Xenopus oocytes. Here, our experiments show that 1,10-phenanthroline itself results in the phosphorylation of MAPK in both oocytes and a cell-free system. In contrast, 1,7-phenanthroline, the nonchelating analogue, had no effect. A supplement of zinc (as a heavy metal) given to 1,10-phenanthroline-loaded oocytes suppressed the stimulatory effects of 1,10-phenanthroline, while 1,10-phenanthroline withdrawal caused dephosphorylation of activated MAPK. Further, treatment with a MEK (a MAPK kinase) inhibitor, PD 098059 or U0126, suppressed 1,10-phenanthroline-stimulated MAPK phosphorylation, indicating that 1,10-phenanthroline can phosphorylate MAPK in a MEK-dependent fashion. Our results suggest that phosphorylation of MAPK by 1,10-phenanthroline depends on the interaction of MEK. Thus, the intracellular heavy metal (zinc) regulates MAPK phosphorylation and 1,10-phenanthroline can serve as a unique tool for investigating MAPK phosphorylation mechanism.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline.

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10(-5)--10(-9) M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10(-4) M. The stimulatory effect of 1,10-phenanthroline manifests itself after 6 h incubation and increased with time up to 48 h. 2,2'-dipyridyl and 5,6-dimethyl-1,10-phenanthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect. Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells. While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyzed controls. These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Ru(II) complexes of crowded delocalized diimine ligands

The ligands 3,3′-dimethylene-2,2′-bibenzo[g]quinoline and bisbenzo[2,3:9,8]-1,10-phenanthroline have been coordinated with Ru(II) to form both tris- and mixed ligand complexes. These species are highly congested about the metal center but can be formed through the use of microwave irradiation. Shielding and deshielding effects on the chemical shifts of the aryl as well as the bridge protons reveal important conformational effects. Bathochromic shifts are observed in the electronic absorption spectra, associated with increased delocalization of the ligand and lowering of the π*-energy level. Similar effects are observed for the reduction potentials while the oxidation potentials are much less sensitive to ligand structure.     

Synthesis, structure and biological activity of copper(II) complexes with oxolinic acid

The neutral mononuclear copper complexes with the quinolone antibacterial drug oxolinic acid in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline, 2,2'-bipyridine or 2,2'-dipyridylamine have been synthesized and characterized with infrared, UV-visible and electron paramagnetic resonance spectroscopies. The experimental data suggest that oxolinic acid acts as a deprotonated bidentate ligand and is coordinated to the metal ion through the pyridone and one carboxylate oxygen atoms. The crystal structure of (chloro)(1,10-phenanthroline)(oxolinato) copper(II), 2, has been determined with X-ray crystallography. For all complexes a distorted square pyramidal environment around Cu(II) is suggested. The EPR (electron paramagnetic resonance) behavior of 2 in aqueous solutions indicates mixture of dimeric and monomeric species. The investigation of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and showed that the complexes are bound to calf-thymus DNA. The antimicrobial activity of the complexes has been tested on three different microorganisms. The complexes show a decreased biological activity in comparison to the free oxolinic acid.     

Analysis of the DNA Interaction of Copper Compounds Belonging to the Casiopeínas® Antitumoral Series

Casiopeínas® are mixed-chelate copper complexes with antitumor tested potential. Their activity, both in vitro and in vivo, as antiproliferative, cytotoxic, and genotoxic drugs has been assessed. Biological results of these copper compounds have deserved some of them entering clinical trials. Significant efforts have been devoted to the in-depth identification of their mechanism of action. Using gel electrophoresis analysis, we have previously shown that the interaction of the Casiopeínas® Cas II-gly, [Cu(4,7-dimethyl-1,10-phenanthroline)(glycinate)]NO₃ with DNA, triggers the cleavage of the biomolecule by a free radical mechanism. In this work, we further study the behavior of different complexes of the same Casiopeínas® series also including glycinate as co-ligand {Cas VI-gly (5,6 dimethyl-1,10-phenanthroline glycinato copper(II) nitrate), Cas VII-gly (1,10-phenanthroline glycinato copper(II) nitrate), and Cas IX-gly (2,2′-bipyridine glycinato copper(II) nitrate)} and of a Casiopeínas® with a different co-ligand (Cas III-Cs; 4,7-dimethyl-1,10-phenanthroline salicylaldehydato-copper(II) nitrate). While all of them produce DNA degradation, the performance in the presence of a radical scavenger suggests the existence of differences in their mechanism of interaction with DNA.     

Zinc complexes of the antibacterial drug oxolinic acid: Structure and DNA-binding properties

The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2′-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2′-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.     

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.     

Chemical speciation of ternary complexes of L-dopa and 1,10-phenanthroline with Co(II), Ni(II) and Cu(II) in low dielectric media

Abstract

A computer assisted pH-metric investigation has been carried out on the speciation of complexes of Co(II), Ni(II) and Cu(II) with L-dopa and 1,10-phenanthroline. The titrations were performed in the presence of different relative concentrations (M:L:X = 1.0:2.5:2.5; 1.0:2.5:5.0; 1.0:5.0:2.5) of metal (M) to L-dopa (L) and 1,10-phenanthroline (X) with sodium hydroxide in varying concentrations (0-60% v/v) of 1,2-propanediol-water mixtures at an ionic strength of 0.16 mol L^-1 and at a temperature of 303.0 K. Stability constants of the ternary complexes were refined using MINIQUAD75. The species MLXH, MLX, ML₂X and MLX₂H for Co(II) and Cu(II) and MLXH, MLX and MLX₂H for Ni(II) were detected. The extra stability of ternary complexes compared to their binary complexes was believed to be due to electrostatic interactions of the side chains of ligands, charge neutralisation, chelate effect, stacking interactions and hydrogen bonding. The species distribution with pH at different compositions of 1, 2-propanediol-water mixtures and plausible equilibria for the formation of species were also presented. The bioavailability of the metal ions is explained based on the speciation.     

Study on nucleic acid (CT-DNA and yeast tRNA) binding behaviors and cytotoxic properties of a heterodinuclear Ru(II)-Co(III) polypyridyl complex

A heterodinuclear (Ru(II), Co(III)) metal polypyridyl complex [(phen)₂Ru(bpibH₂)Co(phen)₂]⁵⁺ {phen = 1,10-phenanthroline, bpibH₂ = 1,4-bis([1,10]phebanthroline-[5,6-d]imidazol-2-yl)-benzene} has been designed and synthesized. The comparative study on the interactions of the Ru(II)-Co(III) complex with calf thymus DNA (CT-DNA) and yeast tRNA has been investigated by UV-visible spectroscopy, fluorescence spectroscopy, viscosity, as well as equilibrium dialysis and circular dichroism (CD). The antitumor activities of the complex have been evaluated by MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} method and Giemsa staining experiment. These results indicate that the structures of nucleic acids have significant effects on the binding behaviors of metal complexes. Furthermore, the complex demonstrates different antitumor activity against selected tumor cell lines in vitro, and can make the cell apoptosis.     

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.     

Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals.

The metal chelators 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical formation from hydrogen peroxide and Cu+ or F2(2+) (Fenton reaction) as determined by electron spin resonance. o-Phenanthroline prevented both Fe- and Cu-mediated Fenton reactions whereas neocuproine only prevented the Cu-mediated Fenton reaction. Because only 1,10-phenanthroline but not neocuproine prevented DNA strand-break formation in hydrogen peroxide-treated mammalian fibroblasts it appears that the Fe-mediated, as compared to the Cu-mediated, intranuclear Fenton reaction is responsible for DNA damage.     

Synthesis, spectroscopic and structural characterization of the reaction products of quaternary cationic 2,2′-bipyridylium ligand bromide salts with metal halides

The synthesis and spectroscopic characterization of quaternary salts containing 2,2,-bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline is reported. The coordinating behaviour of the bipyridylium salts toward palladium, copper and dimethyltin(IV)halides is investigated. The reaction of N-R,2,2′-bipyridylium (= L¹) bromide (R = 4-Cl-C₆H₄ · CO · CH₂) with palladium(II) chloride in acetonitrile solution yields a crystalline product, shown by a single crystal X-ray study to be L¹PdX₃, where X is a Cl/Br (1:1) composite.     

Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases

Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively. The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes. The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases. Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II). Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II). The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases. Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Human telomeric G-quadruplex DNA interactions of N-phenanthroline glycosylamine copper(II) complexes

We report in this article the interactions of five N-(1,10-phenanthrolin-5-yl)-β-glycopyranosylamine copper(II) complexes with G-quadruplex DNA. Specifically, the interactions of these compounds with a human telomeric oligonucleotide have been assessed by fluorescence-based assays (FRET melting and G4-FID), circular dichroism and competitive equilibrium dialysis experiments. The metal complexes bind and stabilize G-quadruplex DNA structures with apparent association constants in the order of 10⁴–10⁵ M⁻¹ and the affinity observed is dependent on the ionic conditions utilized and the specific nature of the carbohydrate moiety tethered to the 1,10-phenanthroline system. The compounds showed only a slight preference to bind G-quadruplex DNA over duplex DNA when the quadruplex DNA was folded in sodium ionic conditions. However, the binding affinity and selectivity, although modest, were notably increased when the G-quadruplex DNA was folded in the presence of potassium metal ions. Moreover, the study points towards a significant contribution of groove and/or loop binding in the recognition mode of quadruplex structures by these non-classical quadruplex ligands. The results reported herein highlight the potential and the versatility of carbohydrate bis-phenanthroline metal-complex conjugates to recognize G-quadruplex DNA structures.     

Partial Purification and Characterization of Aminopeptidase II from Chara australis

Aminopeptidase II, one of the two major aminopeptidases in the giant alga Chara australis, was partially purified. Its molecular weight was estimated to be about 80,000 by gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that it is composed of a single polypeptide with a molecular weight of about 85,000. Aminopeptidase II hydrolyzed alanine-2-naphthylamide more efficiently than the naphthylamides of lysine and proline, and only weakly hydrolyzed the naphthylamides of arginine, phenylalanine, valine, and leucine. The optimal pH for the hydrolysis of alanine-2-naphthylamide was near 7.0. The activity of aminopeptidase II was inhibited by the SH-reagents p-chloromercuribenzoic acid and N-ethylmaleimide and by the metal chelator 1,10-phenanthroline.