Synthesis, physical-chemical characterisation and biological evaluation of novel 2-amido-3-hydroxypyridin-4(1H)-ones: Iron chelators with the potential for treating Alzheimer's disease.

A novel class of 2-amido-3-hydroxypyridin-4-one iron chelators is described. These compounds have been designed to behave as suitable molecular probes which will improve our knowledge of the role of iron in neurodegenerative conditions. Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson disease (PD), can be considered as diverse pathological conditions sharing critical metabolic processes such as protein aggregation and oxidative stress. Interestingly, both these metabolic alterations seem to be associated with the involvement of metal ions, including iron. Iron chelation is therefore a potential therapeutic approach. The physico-chemical (pK(a), pFe(3+) and logP) and biological properties (inhibition of iron-containing enzymes) of these chelators have been investigated in order to obtain a suitable profile for the treatment of neurodegenerative conditions. Studies with neuronal cell cultures confirm that the new iron chelators are neuroprotective against ß-amyloid-induced toxicity.

Haem and folate transport by proton-coupled folate transporter/haem carrier protein 1 (SLC46A1).

Haem carrier protein 1 (HCP1) was originally identified and characterised as a mammalian haem transporter. However, recent evidence has shown that it is also a proton-coupled folate transporter (PCFT) and mutations in the gene cause hereditary folate deficiency in humans. We therefore investigated haem and folate transport characteristics of PCFT/HCP1 both in vivo and in vitro in CD-1 mice and in the presence or absence of a blocking antibody for PCFT/HCP1, and also in cultured cells (which express PCFT/HCP1 endogenously) to elucidate the specificity and selectivity of PCFT/HCP1. The in vivo study showed that the addition of folic acid inhibited 59Fe-labelled haem transport in hypoxic mice but had no effect in normal mice. Using in vitro methods, the results showed increased [3H]folate uptake into everted duodenum from hypoxic mice but uptake was reduced by the addition of haem or PCFT/HCP1 antibodies to the medium. Caco-2 cells transiently transfected with small interfering RNA (siRNA) PCFT/HCP1 duplex oligos resulted in a 69 % reduction in PCFT/HCP1 mRNA when compared with the control siRNA. Both haem and folate uptake were significantly (P < 0.05) reduced in cells transfected with PCFT/HCP1 siRNA; however, the magnitude of reduction with folic acid uptake was greater (48 %) than that of haem (22.5 %). Overall the data support PCFT/HCP1 as a primary folate transporter with a lower affinity for haem. PCFT/HCP1 could therefore play a physiological role in Fe nutrition and the data highlight the potential for the interaction of folate and haem at the level of intestinal absorption.

New imidazole-coordinated chemotherapeutics with low epithelial toxicity.

The new imidazole-coordinated chemotherapeutics with low epithelial toxicity (NICE) presented in this article feature innovative drugs that combine epithelial toxicity comparable with that of carboplatin with novel carrier ligands optimized for DNA interaction. Recent identification of the pivotal role of basolateral organic cation transporters (OCTs) in cisplatin nephrotoxicity by a new model system (electrical resistance breakdown assay) facilitated the search for substances with a favorable organotoxic profile. The assay uses the high transepithelial electrical resistance (TEER) of the C7-clone of Madin-Darby canine kidney (MDCK) cells and the exclusive basolateral expression of OCT2 in these cells. TEER and caspase-3 activity of MDCK-C7-cells grown on microfilter membranes were monitored in response to exposure of either the apical or basolateral plasma membrane to platinum complexes. The impact of complexes on cancer cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide tests. Effects of substituents on pharmacological properties of NICE were systematically investigated by introducing sterically demanding groups as well as electron-donating and electron-withdrawing groups. Derivatives of NICE showed different renal epithelial toxic profiles and effects on cancer cells. NICE were significantly less toxic than cis- or oxaliplatin. The chlorine substituted NICE had no effect on epithelial integrity but markedly cytotoxic activity against amelanotic melanoma cells. Together, side effect targeted screening for new anticancer drugs with the electrical resistance breakdown assay offers an interesting approach for identifying and investigating new compounds. NICE feature the first group of platinum-based cytostatics discovered by using this system for systematic screening of new chemotherapeutics with low renal epithelial toxicity.

Reactions of PtII diamine anticancer complexes with trypanothione and octreotide.

The products formed in reactions of the square-planar platinum(II) anticancer complexes, [Pt(en)Cl(2)] and [Pt(R,R-dach)Cl(2)] where en=ethylenediamine and dach=diaminocyclohexane, with trypanothione, a glutathione analogue found in some parasites, and octreotide, a synthetic analogue of the hormone somatostatin, have been investigated. Mononuclear and binuclear platinum adducts were formed in reactions of the cyclic disulfides in their oxidised and reduced forms, and were analysed by UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC-MS). NMR and molecular modelling studies were carried out on the mononuclear adducts.

Structure-toxicity relationships for different types of dinuclear platinum complexes.

Nine structurally distinct dinuclear platinum complexes have been evaluated in a novel model system for the investigation of renal epithelial toxicity of platinum drugs. The results showed that these compounds are toxic when applied at the basolateral side of renal epithelia, whereas their toxic effects on the apical side are negligible. Such a difference in toxicity of the complexes has been found to result from their poor uptake through the apical membrane, as compared to the basolateral membrane. Toxicity of the compounds on the basolateral side varies depending on their structure. Structure-toxicity relationships for the group of complexes with rigid ligands and for the group of complexes with flexible ligands are discussed. Among the dinuclear complexes with rigid ligands, sterically hindered complexes are less toxic, due to their poor uptake and low reactivity towards glutathione. Within the group of complexes with flexible ligands, cis-configured isomers are more toxic than their trans-counterparts.

Improving efficiency of viability-PCR for selective detection of live cells.

Viability PCR (v-PCR) as a method to selectively detect intact live cells has gained considerable interest over the last years with an increasing number of applications. The principle is based on treatment of microbiological samples with a viability dye prior to extraction of genomic DNA and its amplification. The dye is selectively taken up by membrane-compromised dead cells resulting in the degradation of their DNA upon light exposure and therefore inhibition of amplification. Although the treatment greatly helps to generate more meaningful data, one of the main drawbacks of the technique is currently that the exclusion of dead cell signals can be incomplete leading to false-positive signals. The resulting overestimation of live cell population is especially problematic for the detection of pathogens. We assessed in this study different conditions to increase the penetration of propidium monoazide (PMA) into dead cells of Salmonella Typhimurium and Listeria monocytogenes as representatives of gram-negative and gram-positive bacteria. When working with a low dye concentration of 10µM, a strong relationship of PMA treatment efficiency with temperature and incubation time was observed. Exposing cells to PMA at a temperature exceeding the growth temperature by 10°C for 30min proved greatly beneficial. Co-incubation of cells with PMA and deoxycholate on the other hand was only beneficial for Salmonella, but resulted in a strong undesired uptake of PMA by live Listeria cells. This difference is in agreement with the gram-specific effect of the bile salt during growth.

Monitoring intracellular labile iron pools: A novel fluorescent iron(III) sensor as a potential non-invasive diagnosis tool.

The physiological and pathophysiological importance of intracellular redox active "labile" iron has created a significant need for improved noninvasive diagnostic tools to reliably monitor iron metabolism in living cells. In this context, fluorescent iron-sensitive chemosensors in combination with digital fluorescence spectroscopic methods have proven to be highly sensitive and indispensable tools to determine cellular iron homeostasis. Recently, application of fluorescent iron sensors has led to the identification of a complex sub-cellular iron compartmentation. Cell organelle-specific iron sensors will significantly contribute to enhance fundamental knowledge of cellular iron trafficking, representing a crucial prerequisite for the future development of therapeutic strategies in iron dysregulatory diseases. Here we present physicochemical characterization and functional investigation of a new 3-hydroxypyridin-4-one based fluorescent iron(III) sensor, exclusively monitoring labile iron pools in the endosomal/lysosomal compartments. In vitro studies of the fluorescein labeled probe were carried out in murine bone marrow derived macrophages. Endosomal/lysosomal accumulation of the probe was revealed by confocal microscopy. Flow cytometry analyses demonstrated high sensitivity of the probe towards exogenous alterations of intracellular iron concentrations as well as in response to the chelation potency of iron chelators, clinically approved for treatment of iron-overload related diseases.

Targeting the lysosome: fluorescent iron(III) chelators to selectively monitor endosomal/lysosomal labile iron pools.

Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy have led to the identification of a distinct subcellular compartmentation of intracellular redox-active "labile" iron. To investigate the distribution of labile iron, our research has been focused on the development of fluorescent iron sensors targeting the endosomal/lysosomal system. Following the recent introduction of a series of 3-hydroxypyridin-4-one (HPO) based fluorescent probes we present here two novel HPO sensors capable of accumulating and monitoring iron exclusively in endosomal/lysosomal compartments. Flow cytometric and confocal microscopy studies in murine macrophages revealed endosomal/lysosomal sequestration of the probes and high responsiveness toward alterations of vesicular labile iron concentrations. This allowed assessment of cellular iron status with high sensitivity in response to the clinically applied medications desferrioxamine, deferiprone, and deferasirox. The probes represent a powerful class of sensors for quantitative iron detection and clinical real-time monitoring of subcellular labile iron levels in health and disease.

Platinum complex cytotoxicity tested by the electrical resistance breakdown assay.

The electrical resistance breakdown assay provides a novel approach for the quantification of cytotoxic activity of platinum based anticancer drugs. It is a functional assay system for cancer cell invasion that detects nanoscale alterations of an epithelial test barrier prior to microscopic morphometric changes. We measured changes in transepithelial electrical resistance (TEER) of a tight epithelial MDCK-C7 monolayer in response to highly invasive amelanotic melanoma cells (A7-clone) in combination with different platinum complexes (cis-, oxali- and carboplatin). The efficiency of the electrical resistance breakdown assay was compared a standard method for measurement of cytostatic activity, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT-assay utilizes mitochondrial enzymatic activity to draw conclusions from a functional cell metabolism to the number of living cells in a sample. When human melanoma cells were seeded on top of an electrically tight MDCK-C7 monolayer, electrical leakage occurred within 48 h of co-culture. Electrical resistance breakdown was effectively prevented by cisplatin and its analogs (no significant difference between 100 microM cisplatin and corresponding controls with non-invasive cells). The results of the electrical resistance breakdown and MTT-assay were linearly dependent. Significance of both tests was equivalent, but the electrical resistance breakdown assay gave additional functional information. Compared to oxali- and carboplatin, cisplatin was more effective in preventing TEER-breakdown than reducing the number of tumor cells, giving rise to the assumption that cisplatin can reduce tumor cell number as well as invasiveness. In conclusion the electrical resistance breakdown assay provides a sensitive, continuous and cell-based assay system for the quantification of cancer cell invasiveness and evaluation of chemotherapeutics under physiological conditions.