A comparative study of adduct formation between the anticancer ruthenium(III) compound HInd trans-[RuCl4(Ind)2] and serum proteins.

Formation of adducts between the antitumor ruthenium(III) complex [HInd]trans-[RuCl(4)(Ind)(2)] (KP1019) and the plasma proteins serum albumin and serum transferrin was investigated by UV-vis spectroscopy, for metal-to-protein ratios ranging from 1:1 to 5:1. In both cases, formation of tight metal-protein conjugates was observed. Similar spectroscopic features were observed for both albumin and transferrin derivatives implying a similar binding mode of the ruthenium species to these proteins. Surface histidines are the probable anchoring sites for the bound ruthenium(III) ions in line with previous crystallographic results. In order to assess the stability of the KP1019-protein adducts the influence of pH, reducing agents and chelators was analysed by UV-vis spectroscopy. Notably, there was no effect of addition of EDTA on the UV-vis spectra of the conjugates. The pH-stability was high in the pH range 5-8. Experiments with sodium ascorbate showed that there was just some alteration of selected bands. The implications of the present results are discussed in relation to the pharmacological behavior of this novel class of antitumor compounds.

Investigation of the effects of copper ions on protein aggregation using a model system.

Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.

Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers.

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium-NO adducts was documented by electronic absorption, FT-IR spectroscopy and (1)H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-Br-cGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Interactions of two cytotoxic organotin(IV) compounds with calf thymus DNA.

The reactions with DNA of two antitumor active organotin(IV) compounds, the dimer of bis[(di-n-butyl 3,6-dioxaheptanoato)tin] (C(52)H(108)Sn(4)O(1) x 2H(2)O), compound 1, and tri-n-butyltin 3,6,9-trioxodecanoate (C(19)H(40)SnO(5) x 1/2H(2)O), compound 2, were analysed by circular dichroism, DNA melting experiments and gel mobility shift assays. It is found that both complexes modify only slightly the B-type circular dichroism spectroscopy (CD) spectrum of calf thymus DNA. On the other hand, both complexes were found to affect significantly the parameters of the thermally induced helix-to-coil transition. Addition of 1 or 2 to calf thymus DNA samples does not favor DNA renaturation after melting ruling out formation of interstrand crosslinks. Moreover, the effects of both compounds on plasmid DNA gel mobility were investigated. From the analysis of the present results it is inferred that both organotin(IV) compounds do interact with DNA, probably at the level of the phosphate groups.

Solution chemistry and DNA binding properties of MEN 10755, a novel disaccharide analogue of doxorubicin.

The behavior under physiological conditions of MEN 10755, a novel disaccharide analogue of doxorubicin, was investigated in detail by a variety of spectroscopic techniques including spectrophotometry, fluorescence, and (1)H NMR. The pH dependent properties of MEN 10755 were also analysed by spectrophotometry and potentiometry within the pH range 5--11. It is found that MEN 10755 behaves very similarly to doxorubicin and reproduces closely its pH dependent pattern. Like doxorubicin, MEN 10755 undergoes dimerization with a significantly smaller association constant. The interaction of MEN 10755 with calf thymus DNA was studied in detail. Spectrophotometric and fluorescence titrations of MEN 10755 with calf thymus DNA show spectral patterns almost identical to those obtained with doxorubicin implying that the binding mechanism and the stability of the resulting adducts are very similar. An apparent affinity constant of 1.2 x 10(6) was determined for the interaction of MEN 10755 with calf thymus DNA to be compared with the value of 3.3 x 10(6) measured for doxorubicin, under the same conditions. The effects of both anthracyclines on the thermal denaturation profiles of calf thymus DNA were also analyzed; both compounds turned out to stabilize to a similar extent the DNA double helix and to give rise to a characteristic two-step melting profile. The implications of the present results for the pharmacological activity and the mechanism of action of this novel and promising antitumor compound are discussed.

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their metallocomplexes with duplex DNA.

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.

Interactions of selected gold(III) complexes with calf thymus DNA.

DNA represents the primary target for platinum antitumor metal complexes and is the probable target for newly developed cytotoxic gold(III) complexes. To test this hypothesis the reactions with calf thymus DNA of five representative gold(III) complexes--namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2) and [Au(phen)Cl(2)]Cl--were analyzed in vitro through various physicochemical techniques including circular dichroism, absorption spectroscopy, DNA melting, and ultradialysis. It is shown that all tested complexes interact with DNA and modify significantly its solution behavior. The solution conformation of DNA is affected to variable extents by the individual complexes as shown by CD titration experiments. Notably, in all cases, the gold(III) chromophore is not largely perturbed by addition of calf thymus DNA ruling out occurrence of gold(III) reduction. Ultradialysis experiments point out that the binding affinity of the various complexes for the DNA double helix is relatively low; in most cases the gold(III)/DNA interaction is electrostatic in nature and reversible. The implications of these findings for the mechanism of action of antitumor gold(III) complexes are discussed.

Gold(III) complexes as potential antitumor agents: solution chemistry and cytotoxic properties of some selected gold(III) compounds.

Gold(III) complexes generally exhibit interesting cytotoxic and antitumor properties, but until now, their development has been heavily hampered by their poor stability under physiological conditions. To enhance the stability of the gold(III) center, we prepared a number of gold(III) complexes with multidentate ligands - namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2), and [Au(phen)Cl(2)]Cl - and analyzed their behavior in solution. The solution properties of these complexes were monitored by visible absorption spectroscopy, mass spectrometry, and chloride-selective potentiometric measurements; the electrochemical properties were also studied by cyclic voltammetry and coulometry. Since all the investigated compounds exhibited sufficient stability under physiological conditions, their cytotoxic properties were tested in vitro, via the sulforhodamine B assay, on the representative human ovarian tumor cell line A2780, either sensitive or resistant to cisplatin. In most cases the investigated compounds showed relevant cell-killing properties with IC(50) values falling in the 0.2-10 microM range; noticeably most investigated gold(III) complexes were able to overcome, to a large extent, resistance to cisplatin when tested on the corresponding cisplatin-resistant cell line. The cytotoxic properties of the free ligands were also determined under the same solution conditions. Ethylenediamine, diethylenetriamine, and cyclam were virtually nontoxic (IC(50) values > 100 microM) so that the relevant cytotoxic effects observed for [Au(en)(2)]Cl(3) and [Au(dien)Cl]Cl(2) could be quite unambiguously ascribed to the presence of the gold(III) center. In contrast the phenanthroline and terpyridine ligands turned out to be even more cytotoxic than the corresponding gold(III) complexes rendering the interpretation of the cytotoxicity profiles of the latter complexes less straightforward. The implications of the present findings for the development of novel gold(III) complexes as possible cytotoxic and antitumor drugs are discussed.

Cytotoxicity and DNA binding properties of a chloro glycylhistidinate gold(III) complex (GHAu).

The chloro glycylhistidinate gold(III) complex (GHAu) is shown to be fairly cytotoxic towards the established A2780 ovarian carcinoma human cell line either sensitive or resistant to cisplatin. Remarkably, GHAu is far more cytotoxic than the corresponding zinc(II), palladium(II), platinum(II) and cobalt(II) complexes implying that cytotoxicity is essentially to be ascribed to the presence of a gold(III) center. Circular dichroism (CD) spectra, atomic absorption measurements and DNA melting profiles suggest that GHAu in vitro is able to bind DNA, the presumed target for several antitumor metal complexes, and to modify its conformation, even if the observed changes are generally small. Implications of these findings for the mechanism of action of cytotoxic gold(III) complexes are discussed.

DNA as a possible target for antitumor ruthenium(III) complexes.

The interaction of two experimental ruthenium(III)-containing antitumor complexes-Na[trans-RuCl(4)(DMSO)(Im)] (NAMI) and dichloro(1,2-propylendiaminetetraacetate)ruthenium(III) (RAP)-with DNA was investigated through a number of spectroscopic and molecular biology techniques, including spectrophotometry, circular dichroism, gel shift analysis, and restriction enzyme inhibition. It was found that both complexes slightly alter DNA conformation, modify its electrophoretic mobility, and inhibit DNA recognition and cleavage by some restriction enzymes, though they were less effective than cisplatin in producing such effects. Notably, the effects produced by NAMI on DNA were much larger than those induced by RAP. Implications of these results for the mechanism of action of ruthenium(III) antitumor complexes are discussed.

Solvation of the Carbonyl Compound as a Predominant Factor in the Diastereofacial Selectivity of Nucleophilic Addition.

Temperature-dependent selectivity in nucleophilic additions is affected by the solvent. The inversion temperature (marked with arrows in the graph) that appears in the nonlinear Eyring plots of ln (anti/syn) versus temperature for the addition of butyllithium to an O-protected alpha-hydroxy aldehyde 1 does not depend on nucleophiles (nBuLi (black triangle), tBuLi (*)), but on the solvent. Its value can be obtained from a plot of the (13)C NMR chemical shift of C=O versus temperature. TBDMS=tBuMe(2)Si.

A spectroscopic study of the reaction of NAMI, a novel ruthenium(III)anti-neoplastic complex, with bovine serum albumin.

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti-neoplastic ruthenium(III) complex, with BSA, was studied in detail by various physico-chemical techniques. It is shown that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm. Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case of transferrins it is proposed that the BSA-bound ruthenium ions are ligated to surface histidines of the protein; results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar mechanism of interaction in all cases. It is suggested that the described NAMI-BSA adducts may form in vivo and may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of action of NAMI and of related ruthenium(III) complexes are discussed.

Cytotoxicity, DNA damage, and cell cycle perturbations induced by two representative gold(III) complexes in human leukemic cells with different cisplatin sensitivity.

The gold(III) complexes [Au(phen)Cl2]Cl and [Au(dien)Cl]Cl2 were recently shown to exert important cytotoxic effects in vitro on human tumor cell lines. To elucidate the biochemical mechanisms leading to cell death, the effects produced by these gold(III) complexes on the leukemic CCRF-CEM cell line--either sensitive (CCRF-CEM) or resistant to cisplatin (CCRF-CEM/CDDP)--were analyzed in detail by various techniques. For comparison purposes the effects produced by equitoxic concentrations of cisplatin were also analyzed. First, the dependence of the IC50 values of either complex on the incubation time was investigated. Cytotoxicity experiments confirmed that both gold(III) compounds retain their efficacy against the cisplatin-resistant line: only minimal cross-resistance with cisplatin was detected. Notably, [Au(phen)Cl2]Cl is more cytotoxic than [Au(dien)Cl]Cl2, with IC50 values of 7.4 and 6.0 M at 24 and 72 h, respectively, on the resistant line. Results of the COMET assay point out that both gold(III) complexes directly damage nuclear DNA. Remarkably, DNA damage inferred by either gold(III) complex in the two cell lines is larger than that produced by equitoxic cisplatin concentrations. Finally, the effects that either gold(III) complex produces on the cell cycle were investigated by flow cytometry. It was found that both complexes cause only moderate and transient cell cycle perturbations. Larger cell cycle perturbations are induced by equitoxic concentrations of cisplatin. The implications of the present results for the mechanism of action of cytotoxic gold(III) complexes are discussed.

Biological Properties of IRIM, the Iridium(III) Analogue of (Imidazolium (Bisimidazole) Tetrachlororuthenate) (ICR).

Some biological aspects of the new complex imidazolium bisimidazole tetrachloro iridate(III)-IRIM- the iridium(III) analogue of ICR, were considered. More in detail the conformational effects produced by IRIM on DNA and the cytotoxic properties of IRIM on some selected human cell lines were measured. Dialysis experiments and DNA thermal denaturation studies are suggestive of poor binding of IRIM to DNA; formation of interstrand crosslinks is not observed. In any case CD measurements suggest that addition of increasing amounts of IRIM to calf thymus DNA results into significant spectral changes, that are diagnostic of a direct interaction with DNA. A number of experiments carried out on the A2780 human ovarian carcinoma, B16 murine melanoma, MCF7 and TS mammary adenocarcinoma tumor cell lines strongly point out that IRIM does not exhibit significant growth inhibition effects within the concentration range 10(-4)-10(-6) M. It is suggested that the lower biological effects of IRIM compared to ICR are a consequence of the larger kinetic inertness of the iridium(III) center with respect to ruthenium(III).

Comparative Analysis of [Au(en)(2)] and [Pt(en)(2)] non Covalent Binding to Calf Thymus DNA.

Reactions of the complexes bisethylendiammine gold(III) and bisethylendiammine platinum(II) with calfthymus DNA were comparatively analysed. Both complexes bind DNA non-covalently most probably on the basis of electrostatic interactions. Binding of either complex at low ratios results into modest modifications of B-type DNA conformations, as detected by CD. Far larger CD alterations are observed at high ratios. The gold(III) chromophore is scarcely perturbed by DNA addition Binding of [Au(en)(2)]Cl(3) to calf thymus DNA is reversed by sodium cyanide. By analogy with the case of [Pt(en)(2)]Cl(2) it is suggested that Auen acts as a minor groove binder.

Clay-nucleic acid complexes: characteristics and implications for the preservation of genetic material in primeval habitats.

The equilibrium adsorption of three nucleic acids: chromosomal DNA, supercoiled plasmid DNA, and 25S rRNA, on the clay minerals, montmorillonite (M) and kaolinite (K), were studied. Adsorption of the nucleic acid on the clays was rapid and maximal after 90 min of contact time. Chromosomal DNA was adsorbed to a greater extent than plasmid DNA and RNA, and the adsorption was also greater on M than on K. Adsorption isotherms were of the L type, and a plateau was reached with all the complexes, with the exception of chromosomal DNA adsorbed on M. To deterine where nucleic acids are adsorbed on clay minerals and the nature of the interaction, complexes were studied by X-ray diffraction (X-RD), electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. X-RD showed that nucleic acids did not penetrate the clay, indicating that the adsorption occurred primarily on the external surfaces of clay particles, as also suggested by electron microscopy observations. FT-IR spectra of clay-tightly bound nucleic acid complexes showed adsorption bands that indicate a variation of the nucleic acids status as a consequence of their adsorption on clay. Data obtained suggested that the formation of clay-nucleic acid complex could have an important role in the preservation of genetic material in primeval habitats.

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes.

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti2Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti2Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and 13C NMR spectroscopies, and shown to be stable within the pH range 5.5-9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti2Tf, even after long incubation times, although some weak interactions of Ti(IV)-NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution.

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.

Cytotoxic effects of gold(III) complexes on established human tumor cell lines sensitive and resistant to cisplatin.

Gold(III) complexes, isostructural and isoelectronic with platinum(II) complexes, are potentially attractive as anticancer agents. We have synthesized a group of square planar gold(III) complexes, all containing at least two gold-chloride bonds in cis-position, and tested their in vitro cytotoxicity on a panel of established human tumor cell lines. Remarkably, all these compounds showed significant cytotoxic effects. In particular, the complexes containing the salycilaldiminate ligand induced tumor cell growth inhibitory effects comparable to or even greater than cisplatin. All gold(III) complexes substantially retained their antitumor potency against two cisplatin-resistant tumor cell lines (CCRF-CEM/R leukemia and A2780/R ovarian carcinoma); only minimal cross-resistance with cisplatin was observed. When considering the mechanism of action, it is reasonable to assume that the cytotoxicity of these gold(III) complexes derives from DNA binding. Preliminary spectroscopic results are consistent with this hypothesis; indeed, circular dichroism experiments show that both the salycilaldiminate- and the pyridine-containing gold(III) complexes bind calf thymus DNA in vitro and alter reversibly its B-type solution conformation. These results, however, must be treated with caution; solution studies indicate that gold(III) compounds are poorly stable under physiological conditions, possibly implying that, when injected, only a small amount will reach, unchanged, the DNA target. The results of our investigations are discussed in the perspective of future work on the cytotoxic and antitumor properties of gold(III) compounds.

Carbonic anhydrase activators: X-ray crystallographic and spectroscopic investigations for the interaction of isozymes I and II with histamine.

The interaction of native and Co(II)-substituted isozymes I and II of carbonic anhydrase (CA) with histamine, a well-known activator, was investigated kinetically, spectroscopically, and X-ray crystallographically. This activator is of the noncompetitive type with 4-nitrophenyl acetate and CO2 as substrates for both HCA I and HCA II. The electronic spectrum of the adduct of Co(II)-HCA II with histamine is similar to the spectrum of the Co(II)-HCA II-phenol adduct, being only slightly different from that of the uncomplexed enzyme. This is the first spectroscopic evidence that the activator molecule binds within the active site, but not directly to the metal ion. X-ray crystallographic data for the adduct of HCA II with histamine showed that the activator molecule is bound at the entrance of the active site cavity in a position where it may actively participate in shuttling protons between the active site and the bulk solvent. The role of the activators and the reported X-ray crystal structure of the HCA II-histamine adduct has prompted us to reexamine the X-ray structures of the different CA isozymes in order to find a structural basis accounting for their large differences in catalytic rate. A tentative explanation is proposed on the basis of possible pathways of proton transfer, which constitute the rate-limiting step in the catalytic reaction.