Cellular studies on novel critical targets of anti-tumor metal chelators and complexes

Neben Platinverbindungen sind auch Nicht-Platin-Metallverbindungen mit antitumoraler Wirkung eine vielversprechende Klasse potentieller Wirkstoffe für die Krebstherapie. Da sich die biologischen Eigenschaften neuartiger Metallverbindungen wesentlich von den etablierten Platinverbindungen unterscheiden, sind Wirkmechanismusstudien von großer Bedeutung. Die in dieser Doktorarbeit vorgelegten Forschungsartikel beschäftigen sich mit zellbiologischen Untersuchungen zu neuartigen molekularen Wirkmechanismen von α-N-heterozyklischen Thiosemicarbazonen und Ruthenium-Komplexen. Triapin (3-Aminopyridin-2-carboxaldehyd-thiosemicarbazon) is derzeit der am intensivsten untersuchten Vertreter der Thiosemicarbazone und durchlief bereits zahlreiche klinische Phase I- und Phase II-Studien. Triapin geht stabile Komplexe mit den biologisch relevanten Metallen Eisen, Zink und Kupfer ein und ist ein Inhibitor der Ribonukleotid-Reduktase. Struktur-Aktivitäts-Beziehungen wurden von mehreren Triapinderivaten und dessen Eisen-, Gallium-, Zink- und Kupfer-Komplexen in humanen Tumorzellinien untersucht. Weiters wurde durch die intrinsischen Fluoreszenzeigenschaften von Triapin und Derivaten die intrazelluläre Verteilung mittels Fluoreszenzmikroskopie studiert. Die Aktivitätssteigerung durch Dimethylierung der terminalen Aminogruppe sowie die zelluläre Akkumulation im Endoplasmatischen Retikulum veranlasste uns, den Einfluss von Thiosemicarbazonen auf die Unfolded Protein Response (UPR) zu studieren. Die mit der Dimethylierung einhergehende erhöhte Zytotoxizität korreliert mit der Hochregulation von Schlüsselfaktoren der UPR auf Protein- als auch mRNA-Ebene. Zudem weisen die Aktivierung des proapoptotischen Transkriptionsfaktors CHOP im Zuge der UPR und die Depolarisierung der mitochondrialen Membranen auf einen zusätzlichen molekularen Wirkmechanismus für Thiosemicarbazone hin. Neben experimentellen Studien beinhaltet diese Doktorarbeit auch einen synoptischen Artikel über die Ruthenium-Verbindung NKP-1339, der den aktuellen Wissensstand zu dieser Verbindung und deren Wirkmechanismus zusammenfasst und diskutiert. Diese wurde kürzlich in einer klinische Phase I Studie evaluiert und zeigte in dieser sehr geringe Nebenwirkungen sowie Wirksamkeit in mehreren Fällen verschiedener fortgeschrittenen Krebserkrankungen.Non-platinum metal compounds with anticancer activity represent a promising class of investigational drugs for cancer treatment. Since biological properties of novel metal compounds can differ from those of the well established platinum drugs, investigations on their mode of actions are essential. The research work presented in this PhD thesis is focused on cell biological studies on novel molecular targets of α-N-heterocyclic thiosemicarbazones and ruthenium complexes. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), which entered several clinical phase I and II trials is currently the most extensively studied thiosemicarbazone. Triapine forms complexes with the biological relevant metals iron, zinc and copper in a highly effective manner and is a potent inhibitor of ribonucleotide reductase. Structure-activity relationships of Triapine derivatives and corresponding iron, gallium, zinc and copper complexes were studied on human cancer cell lines. Furthermore, intrinsic fluorescence properties of several of these compounds enabled us to study their intracellular distribution by fluorescence microscopy. Enhancement of cytotoxic properties by dimethylation of the terminal amino group and cellular accumulation in the endoplasmic reticulum (ER) inspired us to study the influence of thiosemicarbazones on the unfolded protein response (UPR), an adaptive rescue pathway of the ER. Investigations of key factors of the UPR on protein and mRNA levels revealed that enhanced cytotoxic properties resulting from dimethylation correlates with elevated UPR signaling. Moreover, proapoptotic signaling via the ER stress-related transcription factor CHOP and loss of mitochondrial membrane potential suggest that ER stress induction is an additional mechanism for the biological activity of α-N-heterocyclic thiosemicarbazones. Beside the experimental studies a synoptic article about the investigational ruthenium drug NKP-1339 summarizing and discussing the current knowledge about this compound and its mode of action is included in this thesis. NKP-1339 was evaluated recently in a clinical phase I trial with the result of very limited side effects and several responding cases with various refractory forms of cancer

Copper(II) Complexes with Highly Water-Soluble l- and d‑Proline–Thiosemicarbazone Conjugates as Potential Inhibitors of Topoisomerase IIα

Two proline–thiosemicarbazone bioconjugates with excellent aqueous solubility, namely, 3-methyl-(<i>S</i>)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [l-Pro-FTSC or (<i>S</i>)-H₂L] and 3-methyl-(<i>R</i>)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [d-Pro-FTSC or (<i>R</i>)-H₂L], have been synthesized and characterized by elemental analysis, one- and two-dimensional ¹H and ¹³C NMR spectroscopy, and electrospray ionization mass spectrometry. The complexation behavior of l-Pro-FTSC with copper­(II) in an aqueous solution and in a 30% (w/w) dimethyl sulfoxide/water mixture has been studied via pH potentiometry, UV–vis spectrophotometry, electron paramagnetic resonance, ¹H NMR spectroscopy, and spectrofluorimetry. By the reaction of copper­(II) acetate with (<i>S</i>)-H₂L and (<i>R</i>)-H₂L in water, the complexes [Cu­(<i>S</i>,<i>R</i>)-L] and [Cu­(<i>R</i>,<i>S</i>)-L] have been synthesized and comprehensively characterized. An X-ray diffraction study of [Cu­(<i>S</i>,<i>R</i>)-L] showed the formation of a square-pyramidal complex, with the bioconjugate acting as a pentadentate ligand. Both copper­(II) complexes displayed antiproliferative activity in CH1 ovarian carcinoma cells and inhibited Topoisomerase IIα activity in a DNA plasmid relaxation assay

Molecular mode of action of NKP-1339 – a clinically investigated ruthenium-based drug – involves ER- and ROS-related effects in colon carcinoma cell lines

Sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339) is a clinically investigated ruthenium-based metal complex, which shows promising results in solid tumors, such as non-small cell lung cancer, colorectal carcinoma, and most distinctively in gastrointestinal neuroendocrine tumors. In previous studies, fast binding to albumin as well as transferrin could be shown. The enhanced permeability and retention (EPR) effect, which is diversely being exploited for tumor targeting, could therefore be applicable for NKP-1339. Here we studied the serum dependence of its biological activity in various methods, influencing its cellular accumulation, cytotoxicity as well as the generation of reactive oxygen species (ROS). ROS lead to Nrf2 activation, which is known to activate antioxidant response gene transcription. GRP78 down-regulation on the protein level suggests ER associated protein degradation (ERAD) as a mode of action, as RNA levels are only mildly affected. Another important part for the mode of action is endoplasmic reticulum (ER) stress, as different factors are highly upregulated on the protein level. For example PERK, a transmembrane receptor which is released by GRP78 when the ER is disturbed, is upregulated and phosphorylated. EIF2α is phosphorylated, which leads to an inhibition of CAP-dependent translation and other stress responses. The transcription factor CHOP (DDIT3), which promotes ER stress dependent apoptosis, is time and concentration dependently upregulated. Finally cytotoxicity tests could prove that inhibition of ER stress and ER stress-mediated apoptosis leads to decreased cytotoxic effects of NKP-1339, which highlights the involvement of this mechanism in the mode of action