Critical targets of Ruthenium, Platinum and Gold complexes

KP1019 is a tumor-inhibiting ruthenium complex currently being evaluated in the clinical setting. Based on a simplified assumption of analogy with platinum drugs, DNA has been considered the critical target of this compound and a variety of other ruthenium complexes, but this hypothesis has in fact never been proven. Furthermore, non-classic platinum agents differ markedly from cisplatin in their way of DNA binding, and knowledge about cellular processing of their adducts is scarce, but nevertheless DNA is implicitly accepted as their critical target without clear experimental evidence. Gold(III) complexes are being explored as antineoplastic agents in preclinical settings, but despite close chemical similarities between gold(III) and platinum(II) the role of DNA as critical target is doubtful. As a key to clarify these questions, we intend to make use of differences in the ability of human cells to repair metal-induced DNA damage and the consequences for chemosensitivity. For this purpose, transformed cell lines originated from patients with genetic deficiencies in DNA repair systems (Xeroderma pigmentosum, Cockayne syndrome, Fanconi`s anemia) will be employed and compared to otherwise isogenic cell lines with restored DNA repair. Those of the latter cell models which are not available from other sources will be established by stable transfection in our laboratory. In a complementary approach, inhibitors of DNA repair processes will be studied in subcytotoxic concentrations for their effects on chemosensitivity to the compounds under investigation. Several of these metal compounds are known to interact with DNA in some way, but these properties are far less explored than those of classic platinum drugs. Therefore, we intend to study these interactions in cell-free assays using electrophoretic mobility shift assays in order to clarify whether the compounds are capable of inducing changes in DNA secondary structure. Furthermore, the extent of DNA binding as well as the formation of cross-links or strand breaks in human cancer cells will be studied by ICP-MS quantification and the Comet Assay, respectively, irrespective of its possible relevance as critical target. In recent years, the thioredoxinhioredoxin reductase (Trx/TrxR) system, a major cellular defense against oxidative stress, is increasingly being discussed as an alternative molecular target and/or determinant for chemosensitivity to gold and platinum compounds. Therefore, it is reasonable to study the influence of TrxR expression and activity in human cancer cells on their chemosensitivity to the compounds under investigations. For this purpose, TrxR overexpression will be induced by stable transfection with appropriate plasmid vectors. Additionally, binding of these compounds to TrxR will be studied and characterized in cell-free experiments by electrophoretic and mass spectroscopic methods.

Ruthenium and non-classic platinum complexes emerging as anticancer agents are known to be capable of interacting with DNA, and in analogy to cisplatin DNA adducts have been commonly assumed to account for their activity, although clear experimental evidence is still lacking. To examine the relevance of these DNA adducts, intensive work went into establishing a variety of cell models with defined differences in proficiency regarding the DNA repair systems NER, BER and ICLR. Remarkably, a transient siRNA-based knock-down of NER- and BER-related genes with considerable efficiency (even when applied on two repair factors simultaneously) turned out to be insufficient for enhancing the sensitivity of cancer cells to cisplatin or thiotepa, the anticancer activity of which is generally accepted to be based upon DNA adducts that are substrates for NER or BER. Efforts are continuing to produce more efficient knock-downs.DNA interactions of trans-platinum complexes containing two oxime ligands were characterized for the first time, revealing an extent of genotoxicity (DNA strand breaks detected by the Comet Assay) hitherto unprecedented for platinum compounds and no evidence for DNA cross-links (contrary to cisplatin), which fundamentally changed the view on this class of compounds. In vivo, no signs of therapeutic activity in tolerable doses were observed, suggesting that the biological effects are inappropriate for cancer therapy. Closely related complexes with guanidine ligands showed on average a stronger impact on DNA secondary structure in vitro, surprisingly even in those cases where the complex did not contain any obvious leaving groups. Their effects in vivo are yet to be examined.In a complementary approach, the distribution of cisplatin in human cancer cells could be visualized by means of high-resolution secondary ion mass spectrometry (NanoSIMS) in an unprecedented quality, enabling detailed information about the subcellular localization of the drug. In cisplatin-treated cells, an accumulation of platinum within chromatin and particularly in nucleoli was observed. The accumulation of platinum within chromatin is consistent with the common notion that DNA is the critical target of cisplatin. But overall, association of platinum is more pronounced with sulfur than with phosphorus. Particularly in the cytoplasm, frequent platinum hotspots are rich in sulfur throughout and were identified as acidic vesicles (probably mainly lysosomes) by confocal fluorescence microscopy. After application of cisplatin with 15N-labeled ammine ligands, NanoSIMS analyses suggested a decrease in the 15N-to-platinum ratio especially in nucleoli, which seems to indicate a partial loss of the (allegedly stably bound) ammine ligands.