XAS spectroscopy applied to tumor-inhibiting Ru compounds

Ruthenium complexes are a new class of tumor-inhibiting compounds. KP1019, indazolium-trans- [tetrachlorobis(indazole)ruthenate(III)]), and the analogous sodium salt of the complex, KP1339, are such drugs. In particular, KP1339 has entered the phase 1 of clinical studies in oncology centers in the UK and the USA in late 2009. Neither the exact coordination nor the oxidation state of these compounds in biological tissues have been unambiguously clarified so far. Hence a fundamental knowledge of the anti-tumor effect is missing, which is important for the further development of new ruthenium complexes. Through the better understanding of the oxidation states and coordination in tumor tissue and the correlated mode of action new impacts on the development of these drugs are expected. This project will make a contribution to the fundamental understanding of the molecular mode of action and its impact on cellular processes. For the analysis of the Ru complexes x-ray absorption spectroscopy (XAS) will be used. This method has an essential role in the investigation of metal coordination sites. After completion of the XAS database with biomimetic ruthenium complexes, the coordination of KP1019 and KP1339 inside the cell and its compartments will be investigated. The proposed mode of action of Ru through a pathway involving the proteins albumin, transferrin and the possible influence of the tripeptide glutathione will be studied. This will be done with the aid of suitable test systems and model compounds with known composition and coordination, respectively. These model spectra will be compared subsequently with XAS spectra gathered from different cellular fractions. With the utmost probability transferrin is responsible for the transfer of Ru into the cell. For that reason apo-transferrin and KP1339 will be co-crystallized and their structure will be investigated with x-ray diffraction techniques. The proposed mechanism of the Ru complexes (activation by reduction) will be verified with near edge XAS. With the micro-XAS method the distribution pattern of Ru compounds inside the cell and subcellular structures will be clarified. At selected points of the micro-XAS images the coordination of Ru in these areas will be determined with combined near edge and extended XAS spectroscopy methods. In addition to the afore described XAS methods scanning transmission x-ray microscopy (STXM) will be used to investigate the oxidation state and the coordination of Ru in subcellular compartments. The results of this study will clarify how the Ru drug is bound inside the cell and with which cell organelles it interacts.

Ruthenium compounds belong to the most promising candidates of metal complexes for cancer therapy. The investigated chemicals show anti-tumor activity often exceeding that of other cytostatic agents (e.g. in colorectal carcinomas in vivo and a variety of primary explanted human tumors in vitro). In comparison with Pt compounds the Ru complexes cause less side effects and resistances against the drug are less likely. The overall chemical and pharmacokinetic behavior of Ru is quite different from Pt compounds, as reflected in extensive protein binding studies in vivo. The Ru-complexes KP1019 (indazolium-trans-[tetrachloridoobis(indazole)ruthenate(III)], its Na-salt analogue KP1339, have been the main target of this studies applying both XAS and XRD techniques. KP1019 and KP1339 consist of Ru(III) coordinated by 2 nitrogen and 4 chloride ligands termed as Ru(III)N2Cl4. Neither the exact coordination nor the oxidation state of the biotranformation compounds in tissues has been unambiguously clarified so far. Hence a fundamental knowledge of the anti-tumor effect is missing, which is important for the further development of new ruthenium complexes taking into account that an activation by reduction mechanism is under debate. XANES analysis on KP1019 under in vitro conditions suggests the replacement of an average of one Cl ligand through an O atom arising from the buffer solution or a N atom from the deprotonated indazolium counteraction an Ru stays in the oxidation state(+III). XANES spectra measured on liver and tumor samples, taken from mice treated with KP1019 or KP1339, revealed identical first coordination environments in vivo. The results were independent of the dosages and schedules tested. Microprobe X-ray fluorescence of tumor thin sections showed the strong penetration of ruthenium into the tumor tissue, with the highest concentrations near blood vessels and in the edge regions of the tissue samples.The proposed accumulation mechanism of Ru through a pathway involving the protein albumin has been taken into consideration. For that reason human serum albumin and KP1019 had been co-crystallized and the structure had been investigated by single crystal X-ray diffraction. The structural data showed the binding of two ruthenium atoms to histidine residues which are both located within well-known hydrophobic binding pockets of albumin. The ruthenium centers are octahedrally coordinated by solvent molecules revealing the dissociation of both indazole ligands from the ruthenium-based drug. However, our group proposed a binding mechanism that assumes the importance of the indazole ligands for binding site recognition and thus their indispensable role for the binding of KP1019.