The Interaction of Antitumor Metal Complexes with Proteins

Despite the recognized therapeutic success of platinum-based antitumor agents, it still remains unclear in what mode, in which metabolic state and how much being inactivated they enter tumors. Since all of the platinum drugs, which are currently in clinical use, are administered intravenously, binding to serum proteins is of primary consideration, as these biological macromolecules - being the first druggable targets after administration - play the most important role in drug transport and delivery. Characterization of interactions of tumor-inhibiting platinum group compounds with serum proteins in terms of binding constants, stoichiometry and the nature of metal moiety and protein active sites involved in adduct formation, rate constants, the reactivity of adducts in the presence of extracellular complexing and reductive components, the reversibility of protein binding with respect to relevant intracellular targets (like DNA), etc. is the major research goal of the proposed project. This study will encompass a representative series of accepted and novel platinum(II) and ruthenium(III) complexes, both structurally related and different in the type of ligand part, and the principal serum transport proteins, albumin and transferrin, as well as real serum proteins. To acquire the above binding information, capillary electrophoresis (CE) and high- performance liquid chromatography (HPLC), their couplings to mass spectrometry (MS) detection techniques, operating in different ionization modes, as well as off-line MS will be employed as major research tools. Strong emphasis will be placed on modification of existing CE/HPLC methodology to measure the protein-metallodrug binding parameters, with due account for complex solution chemistry of platinum metal species and modelling physiological conditions (including natural protein concentrations and actual drug contents in the bloodstream). As a result of the analysis of the whole complex of binding data, different proteins, platinum metal complexes and other biomolecules under scrutiny will be assessed quantitatively and compared regarding their mutual and competitive binding affinity. Such information would provide insights into the mechanism of drug`s action and their targeted delivery to tumor tissue and thus present a decent step forward on the way to evaluating new anticancer drug candidates in a faster and cost-efficient manner. The multidimensional separation and detection assays to be developed will be put into appraisal with regard to routine usage by anticancer research community.

The platinum complexes cisplatin, oxaliplatin and carboplatin are used today in about 50% of the cancer chemotherapy schemes. The major disadvantages of platinum-based chemotherapy (and chemotherapy in general) comprise the occurrence of resistance, adverse effects and a limited number of treatable tumors. Currently several platinum and non-platinum compounds are evaluated in clinical trials as anticancer agents and many promising approaches are at the preclinical stage of development. A major limitation in the drug development process is the absence of early selection criteria of drug candidates for further studies, and in order to increase the efficiency of compound design, it is of enormous interest to clarify the mode of action of pharmacologically active metal complexes. The aim of this project was to deliver important information on the first biological metabolization steps after intravenous administration. From our point of view the most important research results obtained within this project resulted from experiments on the coordination behavior of the drug candidate KP1019 and analogous compounds to serum proteins. It was shown by separation methods coupled to an element selective detector that human serum albumin is the preferred binding partner in the bloodstream, whereas transferrin is loaded to a low extent. However, estimation of transferrin loading shows us that the amount bound to transferrin just meets nicely the requirements of a high uptake of the Ru(III) compound into the tumor cell. For these studies, several innovative methods were developed and ultimately the methods were shown to be applicable for studies with human blood serum samples, obtained from cancer patients included into a phase I clinical trial. The methods were validated against each other and high correlation between the obtained results was found. Furthermore, it was shown that the counter ion of the ruthenium coordination compound does not influence the protein binding behavior and therefore the sodium compound KP1339 was chosen for further clinical trials, due to its higher solubility but similar chemical behavior. Therefore, in the context of this project an important contribution was made to the drug development process and the new methods can and will be applied for studying next generation drug candidates. In future the research in our group will continue to investigate the behavior of metallodrugs in the blood of animals and of patients (accompanying a new phase I/II study) and also the targets in the cell will be of major interest.