In vitro cancer chemotherapeutic activity of 1,10-phenanthroline (phen), [Ag2(phen)3(mal)]x2H2O, [Cu(phen)2(mal)]x2H2O and [Mn(phen)2(mal)]x2H2O (malH2=malonic acid) using human cancer cells.

The chemotherapeutic potential of 1,10-phenanthroline (phen), and three of its transition metal complexes, namely [Cu(phen)(2)(mal)]x2H(2)O, [Mn(phen)(2)(mal)]x2H(2)O and [Ag(2)(phen)(3)(mal)]x2H(2)O (malH(2)=malonic acid) was determined using two human carcinoma cell lines (A-498 and Hep-G2). Phen and the three metal-phen complexes induced a concentration-dependent cytotoxic effect, with metal complexes demonstrating the greatest cytotoxic response. In comparative studies, IC(50) values show cytotoxicity of between 3 and 18 times greater than that observed for the metal-based anti-cancer agent, cisplatin. All of the phen-based complexes inhibited DNA synthesis which did not appear to be mediated through intercalation. Also, the potential cancer chemotherapeutic application of these compounds was seen to be enhanced by results obtained from Ames tests, which showed all of the test agents and their phase I metabolites were non-mutagenic. Taken together, these results suggest that phen and the three metal-phen complexes may have a therapeutic role to play in the successful treatment and management of cancer.

In vitro anti-tumour effect of 1,10-phenanthroline-5,6-dione (phendione), [Cu(phendione)3](ClO4)2.4H2O and [Ag(phendione)2]ClO4 using human epithelial cell lines.

The anti-cancer chemotherapeutic potential of 1,10-phenanthroline-5,6-dione (phendione), [Cu(phendione)(3)](ClO(4))(2).4H(2)O and [Ag(phendione)(2)]ClO(4) were determined using four human cells lines, i.e. two neoplastic (A-498 and Hep-G2) and two non-neoplastic (CHANG and HK-2). All of the phendione derivatives induced a concentration-dependant decrease in the viability of the four cell lines, with [Cu(phendione)(3)](ClO(4))(2).4H(2)O displaying greatest activity. In comparative studies, IC(50) values obtained with the two neoplastic cell lines showed a cytotoxic response which was between 3 and 35 times greater than that observed for the metal-based anti-cancer agent, cisplatin. Furthermore, metal-phendione complexes, rather than simple solvated metal ions, were responsible for the observed cytotoxicity. Despite the high level of potency associated with these compounds they did not display an apparent cyto-selective profile, as they reduced the viability of both neoplastic and non-neoplastic cells. However, selected mechanistic studies showed that phendione and its metal complexes inhibited DNA synthesis which did not appear to be mediated through intercalation. Ames testing highlighted that all three compounds and their phase I metabolites were non-mutagenic, unlike cisplatin. Taken together, these results suggest that phendione and its Cu(II) and Ag(I) complexes may be capable of acting as highly effective anti-cancer therapies, which with careful administration could provide very potent and effective alternatives to cisplatin.

Discovery of antagonist peptides against bacterial helicase-primase interaction in B. stearothermophilus by reverse yeast three-hybrid.

Developing small-molecule antagonists against protein-protein interactions will provide powerful tools for mechanistic/functional studies and the discovery of new antibacterials. We have developed a reverse yeast three-hybrid approach that allows high-throughput screening for antagonist peptides against essential protein-protein interactions. We have applied our methodology to the essential bacterial helicase-primase interaction in Bacillus stearothermophilus and isolated a unique antagonist peptide. This peptide binds to the primase, thus excluding the helicase and inhibiting an essential interaction in bacterial DNA replication. We provide proof of principle that our reverse yeast three-hybrid method is a powerful "one-step" screen tool for direct high-throughput antagonist peptide selection against any protein-protein interaction detectable by traditional yeast two-hybrid systems. Such peptides will provide useful "leads" for the development of new antibacterials.

Synthesis, X-ray crystal structure, anti-fungal and anti-cancer activity of [Ag2(NH3)2(salH)2] (salH2=salicylic acid).

[Ag(2)(NH(3))(2)(salH)(2)] (salH(2)=salicylic acid) was synthesised from salicylic acid and Ag(2)O in concentrated aqueous NH(3) and the dimeric Ag(I) complex was characterised using X-ray crystallography. The complex is centrosymmetric with each metal coordinated to a salicylate carboxylate oxygen and to an ammonia nitrogen atom in an almost linear fashion. The two [Ag(NH(3))(salH)] units in the complex are linked by an Ag-Ag bond. Whilst metal-free salH(2) did not prevent the growth of the fungal pathogen Candida albicans [Ag(2)(NH(3))(2)(salH)(2)], [Ag(2)(salH)(2)] and some simple Ag(I) salts greatly inhibited cell reproduction. SalH(2), [Ag(2)(NH(3))(2)(salH)(2)] [Ag(2)(salH)(2)] and AgClO(4) produced a dose-dependent cytotoxic response against the three human derived cancer cell lines, Cal-27, Hep-G2 and A-498, with the Ag(I)-containing reagents being the most effective.

Metal complexes of 1,10-phenanthroline-5,6-dione alter the susceptibility of the yeast Candida albicans to amphotericin B and miconazole.

Growth of the pathogenic yeast Candida albicans in sub-MIC (minimum inhibitory concentration) levels of Cu(ClO4)2 6H2O and [Cu(phendio)3](ClO4)2 4H2O (phendio = 1,10-phenanthroline-5,6-dione) increased the concentration of miconazole and amphotericin B required to achieve the MIC90 whereas pre-growth in AgClO4 and [Ag(phendio)2]ClO4 resulted in a small decrease in the relevant MIC90 values. The copper complexes reduce the oxygen consumption of C. albicans while the silver complexes increase oxygen consumption. In addition, pre-growth of cells in the copper complexes resulted in a lower ergosterol content while the silver complexes induced an elevation in ergosterol synthesis. The ability of copper and silver complexes to alter the susceptibility of C. albicans to miconazole and amphotericin B may be influenced by their action on respiration, since reduced respiration rates correlate with reduced cellular ergosterol which is the target for amphotericin B. Lower levels of ergosterol have previously been associated with elevated tolerance to this drug. In the case of reduced sensitivity to miconazole, tolerance may be mediated by lower ergosterol synthesis giving rise to fewer toxic side products once biosynthesis is inhibited by miconazole.

Synthesis and X-ray crystal structure of [Ag(phendio)2]ClO4 (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells.

The Cu(II) and Ag(I) complexes, [Cu(phendio)3](ClO4)2 x 4H2O and [Ag(phendio)2]ClO4 (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. 'Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH3CO2, AgNO3 and AgClO4 x H2O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to 'metal-free' phendio, simple Ag(I) salts and [Ag(phendio)2]ClO4 causes extensive, non-specific DNA cleavage. 'Metal-free' phendio and [Ag(phendio)2]ClO4 induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.

Induction of apoptosis in yeast and mammalian cells by exposure to 1,10-phenanthroline metal complexes.

1,10-Phenanthroline (phen) and metal-phen complexes display fungicidal and fungiststic activity, disrupt mitochondrial function and induce oxidative stress. We have examined the effect of these drugs on the structure of yeast and mammalian cell organelles and the integrity of cellular DNA. Exposure of Candida albicans to [Mn(phen)2(mal)].2H2O or [Ag2(phen)3(mal)].2H2O (mal H2 = malonic acid) resulted in DNA degradation whereas exposure to phen or [Cu(phen)2(mal)].2H2O did not. All drugs induced extensive changes to the internal structure of yeast cells including retraction of the cytoplasm, nuclear fragmentation and disruption of the mitochondrion. In the case of cultured mammalian cells [Cu(phen)2(mal)].2H2O induced apoptosis as evidenced by the ladder pattern of DNA fragments following gel electrophoresis and also the blebbing of the cell membrane. The other drugs produced non-specific DNA degradation in mammalian cells. In conclusion, phen and metal-phen complexes have the potential to induce apoptosis in fungal and mammalian cells. Given their distinct mode of action compared to conventional anti-fungal drugs, phen and metal-phen complexes may represent a novel group of anti-fungal agents for use either in combination with existing drugs or in cases where resistance to conventional drugs has emerged.

Mode of anti-fungal activity of 1,10-phenanthroline and its Cu(II), Mn(II) and Ag(I) complexes.

The mode of action of the anti-fungal compounds, 1,10-phenanthroline (phen), [Cu(phen)2(mal)] x 2H2O, [Mn(phen)2(mal)] x 2H2O and [Ag2(phen)3(mal)] x 2H2O (malH2 = malonic acid), was examined using the pathogenic yeast Candida albicans. The compounds have minimum inhibitory concentrations (MIC's) in the range 1.25-5.0 microg cm(-3) and at a concentration of 10 microg cm(-3) display some fungicidal activity. Yeast cells exposed to these drugs show a diminished ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC), indicating a reduction in respiratory function. Treating exponential and stationary phase yeast cells with phen and the Cu(II) and Mn(II) complexes causes a dramatic increase in oxygen consumption. All of the drugs promote reductions in the levels of cytochromes b and c in the cells, whilst the Ag(I) complex also lowers the amount of cytochrome aa3. Cells treated with phen and the Cu(II) and Ag(I) species show reduced levels of ergosterol whilst the Mn(II) complex induces an increase in the sterol concentration. The general conclusion is that the drugs damage mitochondrial function and uncouple respiration. That the drugs are not uniformly active suggests their bioactivity has a degree of metal-ion dependency. Phen and metal-phen complexes represent a novel set of highly active anti-fungal agents whose mode of action is significantly different to that of the polyene and azole prescription drugs.