Targeting of T/Tn antigens with a plant lectin to kill human leukemia cells by photochemotherapy.

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy.

Comparative study of the phototoxicity of long-wavelength photosensitizers targeted by the MornigaG lectin.

Morniga G is a plant lectin selective for high density of tumor-associated carbohydrate T and Tn antigens on the surface of cells. The interaction of the protein with Tn induces its cell penetration. This property was used for targeting photosensitizers (consisting of the porphyrins TrMPyP and TPPS, the Al(III)-phthalocyanin AlPcS(4), and the chlorin e6) against leukemic Jurkat T cells after covalent coupling to the protein. The control of MornigaG/photosensitizer loading allowed the comparison of the toxicity of the different photosensitizer conjugates. Conjugate including a single AlPcS(4) per protein appeared promising, since it is poorly toxic when irradiated under white light, while it shows a strong phototoxicity (LD(50) = 4 nM) when irradiated in the therapeutic window, it preferentially kills cancerous lymphocytes, and the sugar binding specificity of the lectin part of the molecule remains unaltered.

Morniga G: a plant lectin as an endocytic ligand for photosensitizer molecule targeting toward tumor-associated T/Tn antigens.

Porphyrins are used as photosensitizer (PS) in photodynamic therapy in cancer treatment. Nevertheless, the development of photochemotherapy in oncology remains limited, because of the low selectivity of PSs. In order to allow PS targeting toward tumor-associated antigens, for the first time a white-light activatable porphyrin, [5-(4-(5-carboxy-1-butoxy)-phenyl)-10,15,20-tris(4-N-methyl)-pyridiniumyl)-porphyrin] (TrMPyP) was covalently linked to Morniga G (MorG), a galactose-specific binding plant lectin, known to recognize with high-affinity tumor-associated T/Tn antigen in cell-free systems. Firstly, using fluorescein-labeled MorG, the sugar-dependent binding and uptake of lectin by Tn-positive (Jurkat lymphoid leukemia) cells was demonstrated. Secondly, the TrMPyP-MorG conjugate was molecularly characterized. Cytometric and confocal microscopic analysis demonstrated that PS covalent linking to MorG preserved sugar-dependent specific binding and uptake of lectin by Jurkat leukemia lymphocytes. Thirdly, the conjugate (with a 1:1 PS:lectin ratio) that was bound and quickly (5 min) taken-up, induced greater than 90% cytotoxicity upon irradiation at 10 nm concentration, whereas the free PS was absolutely nontoxic. On the contrary, normal lymphocytes strongly resisted to the conjugate-mediated phototoxicity. Thus, owing to their binding and endocytosis capacities, plant lectins represent promising molecules for targeting of tumor glycan alteration and to enhance the efficiency of specific delivery of PSs to tumor cells.

Novel heteronuclear ruthenium-copper coordination compounds as efficient DNA-cleaving agents.

The DNA-cleavage ability of novel bifunctional heterobimetallic copper-ruthenium complexes is demonstrated by using gel electrophoresis.

Alkylating ability of artemisinin after Cu(I)-induced activation.

The reductive activation of artemisinin by copper(I)-dipyrrin or copper(I)-(2-Clip-Phen) complexes generates an artemisinin derived alkylating species leading to covalent artemisinin-copper complex adducts. The reactivity of the peroxide function of artemisinin toward Cu(I) complexes is similar to that of Fe(II) analogues, even though the reaction is more sluggish and product distribution slightly different.

DNA cleavage and binding selectivity of a heterodinuclear Pt-Cu(3-Clip-Phen) complex.

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum-copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen 1-(1, 10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts.

Platinated copper(3-clip-phen) complexes as effective DNA-cleaving and cytotoxic agents.

The synthesis and biological activity of three heteronuclear platinum-copper complexes based on 3-Clip-Phen are reported. These rigid complexes have been designed to alter the intrinsic mechanism of action of both the platinum moiety and the Cu(3-Clip-Phen) unit. The platinum centers of two of these complexes are coordinated to a 3-Clip-Phen moiety, an ammine ligand and two chlorides, which are either cis or trans to each other. The third complex comprises two 3-Clip-Phen units and two chloride ligands bound in a trans fashion to the platinum ion. DNA-cleavage experiments show that the complexes are highly efficient nuclease agents. In addition, a markedly difference in their aptitude to perform direct double-strand cleavage is observed, which appears to be strongly related to the ability of the platinum unit to coordinate to DNA. Indeed, complex 6 is unable to coordinate to DNA, which is reflected by its incapability to carry out double-strand breaks. Nonetheless, this complex exhibits efficient DNA-cleavage activity, and its cytotoxicity is high for several cell lines. Complex 6 shows better antiproliferate activity than both cisplatin and Cu(3-Clip-Phen) toward most cancer cell lines. Furthermore, the cytotoxicity observed for 1 is for most cell lines close to that of cisplatin, or even better. Cu(3-Clip-Phen) induces very low cytotoxic effects, but a marked migratory activity. Complex 6 presents DNA-cleavage properties comparable to the one of Cu(3-Clip-Phen), but it does not show any migratory activity. Interestingly, both Cu(3-Clip-Phen) and 6 induces vacuolisation processes in the cell in contrast to complex 1 and cisplatin. Thus, the four complexes cisplatin tested, Cu(3-Clip-Phen), 1 and 6 stimulate different cellular responses.

Preparation and study of new poly-8-hydroxyquinoline chelators for an anti-Alzheimer strategy.

Fourteen different ligands have been synthesized with two covalently linked 8-hydroxyquinoline motifs that favor metal complexation. These bis-chelators include different bridges at the C2 positions and different substituents to modulate their physicochemical properties. They can form metal complexes in a ratio of one ligand per metal ion with Cu II and Zn II, two metal ions involved in the formation of amyloid aggregates of the toxic Abeta-peptides in the Alzheimer disease. The apparent affinity of all bis-8-hydroxyquinoline ligands for Cu II and Zn II are similar with logK Cu II approximately 16 and logK Zn II approximately 13 and are 10,000 times more efficient than for the corresponding 8-hydroxyquinoline monomers. Their strong chelating capacities allow them to inhibit more efficiently than the corresponding monomers the precipitation of Abeta-peptides induced by Cu II and Zn II and also to inhibit the toxic formation of H2O2 due to copper complexes of Abeta. The best results were obtained with a one-atom linker between the two quinoline units. X-ray analyses of single-crystals of Cu II, Zn II or Ni II complexes of 2,2'-(2,2-propanediyl)-bis(8-hydroxyquinoline), including a one-atom linker, showed that all heteroatoms of the bis-8-hydroxyquinoline ligand chelate the same metal ion in a distorted square-planar geometry. The Cu II and Zn II complexes include a fifth axial ligand and are pentacoordinated.

Clip-phen conjugates for the specific cleavage of nucleic acids.

For the first time Clip-Phen (1) was conjugated to oligonucleotides to provide very efficient tools for the cleavage of nucleic acids at specific positions. The synthesis of the conjugates as well as the cleavage experiments are reported.

New approach for the preparation of efficient DNA cleaving agents: ditopic copper-platinum complexes based on 3-Clip-Phen and cisplatin.

The design and synthesis of new heterodinuclear DNA-targeting agents are described. The abilities of cisplatin and Cu(3-Clip-Phen) [Cu(1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine)Cl2], an artificial DNA-cleaving agent, have been combined through their "covalent coupling". This strategy has led to bifunctional complexes that are able to cleave the DNA in a double-stranded fashion in contrast to Cu(3-Clip-Phen) alone and have promising cytotoxicities compared to cisplatin in several cell lines.

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes.

beta-Amyloid peptide (Abeta) 1-42, involved in the pathogenesis of Alzheimer's disease, binds copper ions to form AbetaxCu(n) complexes that are able to generate H(2)O(2) in the presence of a reductant and O(2). The production of H(2)O(2) can be stopped with chelators. More reactive than H(2)O(2) itself, hydroxyl radicals HO() (generated when a reduced redox active metal complex interacts with H(2)O(2)) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H(2)O(2) associated to a pre-incubation step between ascorbate and AbetaxCu(n) allows to study the formation of H(2)O(2) but also, at the same time, its transformation by the copper complexes. AbetaxCu(n) peptides produce but do not efficiently degrade H(2)O(2). The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H(2)O(2) production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from AbetaxCu(n), whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with AbetaxCu(n). The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.

Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates.

Copper-phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper-phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11-54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from beta-elimination of 2-deoxyribonolactone by copper-phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.

Cytostatic activity of 1,10-phenanthroline derivatives generated by the clip-phen strategy.

The cytostatic activities of a series of twelve 1,10-phenanthroline (Phen) derivatives and of their copper complexes were studied on L1210 murine leukemia cells. Large increases in the biological activity were observed for compounds of the 3-Clip-Phen series, in which two Phen moieties were bridged at their C3 positions by an alkoxy linker, the 3-pentyl-Clip-Phen derivative showing an IC(50) value of 130 nM while Phen shows an IC(50) value of 2500 nM under the same conditions. IC(50) values seemed to be modulated not only by the position, the nature, and the length of the linker of Clip-Phen but also by hydrophobicity. Since copper complexes of Phen are chemical nucleases and nucleic acids are thus a potential target for these compounds, the corresponding copper complexes were also studied. Copper complexation of the 3-Clip-Phen ligands did not increase their biological activities. Attempts to vectorize 3-Clip-Phen derivatives with a DNA binder such as spermine or with a cell-penetration peptide failed to increase their biological activity relative to the original 3-Clip-Phen series.

DNA cleavage studies of mononuclear and dinuclear copper(II) complexes with benzothiazolesulfonamide ligands.

Copper-based transition metal complexes performing single- and double-strand scission of DNA have been studied. The dinuclear complexes [Cu(2)(L)(2)(OCH(3))(2)(NH(3))(2)] and [Cu(2)(L)(2)(OCH(3))(2)(DMSO)(2)] are more active than the corresponding mononuclear [Cu(L)(2)(py)(2)] (where HL= N-(4-methylbenzothiazol-2-yl)benzenesulfonamide), suggesting that the dinuclearity is an important factor in the oxidative cleavage of DNA. The cleavage efficiency of the complexes depends on the reducing agent used in the process, the tandem ascorbate/H(2)O(2) being the most efficient. PAGE analyses have shown that these complexes cleave DNA without sequence selectivity. The DNA degradation process takes place mainly by C1' oxidation, but C4' and C5' oxidations cannot be ruled out as minor pathways. These copper complexes preferably oxidize guanine under mild conditions, but under more drastic conditions the oxidation reactivity appears to be T>G>C>A, suggesting the intervention of hydroxyl radicals as active species.

Acridine conjugates of 3-clip-phen: influence of the linker on the synthesis and the DNA cleavage activity of their copper complexes.

To increase the DNA cleavage activity and the cell delivery of the bis(phenanthroline) DNA cleaver "3-Clip-Phen", conjugates between 3-Clip-Phen and the intercalators acridine and 6-chloro-2-methoxyacridine, through amino acid linkers of various length, were prepared. After complexation with CuCl(2), the ability of these conjugates to cleave phiX 174 DNA in the presence of a reductant and air was compared. The results indicated that (i) the coupling of 3-Clip-Phen to an acridine derivative increased the DNA cleavage efficiency of the copper complexes, (ii) the acridine derivatives were more active than 6-chloro-2-methoxyacridine derivatives, (iii) the linker length influenced cleavage efficiency, the highest DNA cleavage activity being obtained for an aminocaproic spacer.

A minor groove binding copper-phenanthroline conjugate produces direct strand breaks via beta-elimination of 2-deoxyribonolactone.

Copper-phenanthroline complexes and their conjugates are useful reagents for studying nucleic acid interactions. Although DNA cleavage by such complexes was discovered more than 20 years ago, significant questions remain unanswered regarding the chemical mechanism(s) by which DNA is damaged. Kinetic evidence is provided, which demonstrates that the major pathway for DNA damage by a minor groove binding molecule conjugated to copper phenanthroline (6) involves C1'-oxidation. Additional experiments using 6 and a DNA substrate containing 2-deoxyribonolactone (1) show that direct strand breaks are produced via beta-elimination from 1. These studies support the original mechanism for DNA damage by copper phenanthroline put forth by Sigman and a more recent proposal concerning the mechanism for direct strand break formation.

Preparation of the New Bis(phenanthroline) Ligand "Clip-Phen" and Evaluation of the Nuclease Activity of the Corresponding Copper Complex.

The best nuclease activities of 1,10-phenanthroline-copper complexes have been observed for a chelate/metal stoichiometry of 2/1. To favor this stoichiometry, the "Clip-Phen" ligand was synthesized with two 1,10-phenanthroline units linked via their C2-carbons by a short flexible arm. An exogenous amine function was present to allow future vectorization of this new chelating ligand. X-ray analysis of monocrystals of Clip-Phen confirmed its structure. An EPR study of (Clip-Phen)CuCl(2) showed that the cupric ion was pentacoordinated with a water molecule as the possible fifth ligand. The Cu(I/II) redox couple was found to be near 85 mV vs SCE, close to the redox couple of (Phen)(2)CuCl(2). A comparison of the nuclease activity of copper complexes of Clip-Phen and 1,10-phenanthroline indicated that (Clip-Phen)Cu was the more active.