New trends for metal complexes with anticancer activity

Medicinal inorganic chemistry can exploit the unique properties of metal ions for the design of new drugs. This has, for instance, led to the clinical application of chemotherapeutic agents for cancer treatment, such as cisplatin. The use of cisplatin is, however, severely limited by its toxic side-effects. This has spurred chemists to employ different strategies in the development of new metal-based anticancer agents with different mechanisms of action. Recent trends in the field are discussed in this review. These include the more selective delivery and/or activation of cisplatin-related prodrugs and the discovery of new non-covalent interactions with the classical target, DNA. The use of the metal as scaffold rather than reactive centre and the departure from the cisplatin paradigm of activity towards a more targeted, cancer cell-specific approach, a major trend, are discussed as well. All this, together with the observation that some of the new drugs are organometallic complexes, illustrates that exciting times lie ahead for those interested in ‘metals in medicine

Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs

This review illustrates notable recent progress in the field of medicinal bioinorganic chemistry as many new approaches to the design of innovative metal-based anticancer drugs are emerging. Current research addressing the problems associated with platinum drugs has focused on other metal-based therapeutics that have different modes of action and on prodrug and targeting strategies in an effort to diminish the side-effects of cisplatin chemotherapy

The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874–2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid

A Case of Successful Selective Abortion Using Radio-frequency Ablation in Twin Pregnancy Suffering from Severe Twin to Twin Transfusion Syndrome

Twin to twin transfusion syndrome (TTTS) is one of the major complication of monochorionic twin pregnancy which is mainly understood by placental vascular anastomosis. Perinatal mortality and morbidity is high as 80-100% if untreated and even higher if the disease is developed at early stage. Variety of methods of isolating or intercepting placental vascular anastomosis are introduced, but they are only available in centers where all the required equipments are prepared. We report here a case of TTTS complicated with severe polyhydroamnios during the second trimester. The blood supply to donor twin was interrupted successfully at 19+2 weeks of gestation by minimally invasive radio-frequency cord ablation, under ultrasound guidance. The normal recipient twin was delivered successfully at 35 weeks of gestation and had no eventful neonatal course

Cisplatin and Oxaliplatin Toxicity: Importance of Cochlear Kinetics as a Determinant for Ototoxicity

Background Cisplatin is a commonly used platinum anti-cancer drug. Regrettably cisplatin has dose-limiting ototoxic side effects, e.g. the drug can induce an irreversible hearing loss. The ototoxic mechanisms of cisplatin have not been elucidated in the human ear and no clinically useful oto-protectors are yet available. Cisplatin is a necessary part of many treatment regimes. Its beneficial therapeutic effects might be reduced if cisplatin was excluded from the treatment in order to protect the hearing function. In this work the ototoxic effects of cisplatin are studied with the aim to better understand the mechanisms behind the irreversible hearing loss induced by this drug. Oxaliplatin is a second generation platinum-derivative anti-cancer drug, free from ototoxic side effects in clinical practice. The effects of oxaliplatin on the inner ear have been studied in this work and the results are compared with cisplatin treatment. The two drugs differ regarding both anti-cancer effects and side effects, which could be attributed to differences in pharmacokinetic factors, cellular uptake and apoptotic mechanisms. The thioredoxin redox system with the enzyme thioredoxin reductase (TrxR) was studied in cochleae due to a suggested DNA-independent apoptotic mechanism of the hair cells. The cochlear pharmacokinetics of cisplatin was assessed and the transport protein organic cation transporter 2 (OCT2) was studied in relation to the ototoxic effect of cisplatin. Material and methods Cultured human colon carcinoma cells and cell cultures of rat organ of Corti were used for apoptosis studies in vitro following exposure to cisplatin and oxaliplatin. Cisplatin and oxaliplatin were administered i.v. to guinea pigs, followed by in vivo sampling of blood, cerebrospinal fluid (CSF) and scala tympani (ST) perilymph. Liquid chromatography with post-column derivatization was used to determine the concentration of parent drug in the samples. Electrophysiological hearing thresholds and the loss of hair cells were assessed to evaluate their ototoxic effects. Phenformin, a potential blocker of OCT2 was administered and the ototoxic side effect of cisplatin was evaluated. For immunohistochemical studies, cochlea from rat, guinea pig and pig were used, where TrxR and OCT2 were evaluated in the cochlea. TrxR-assays were used to measure the TrxR activity in cochlear tissue, both in vivo and in vitro. Results The results from the in vitro studies showed that addition of either cisplatin or oxaliplatin to the culture medium in organ of Corti cell cultures caused a similar amount of outer hair cell loss and inhibition of TrxR activity. Cisplatin exposure to cultured human colon carcinoma cells also reduced the activity of TrxR. The results from the in vivo studies showed that a considerable concentration of cisplatin was present in ST perilymph as compared with weak concentrations of oxaliplatin after high dose oxaliplatin i.v. Ten minutes after cisplatin administration, its concentration in ST perilymph was 4-fold higher in the basal turn of the cochlea as compared to the apex. Cisplatin could be analysed in ST perilymph for up to 120 min. Phenformin i.v. did not reduce the ototoxic side-effect of cisplatin. Positive immunoreactivity to TrxR was evident in both hair cells and spiral ganglion cells. Futhermore, OCT2 was expressed in the supporting cells of organ of Corti and in the spiral ganglion cells. Conclusion The transport of cisplatin to the vulnerable cells of hearing seems to be of major importance for the ototoxic effects. An early high concentration of cisplatin in the base of the cochlea and delayed elimination of cisplatin from ST perilymph may be related to the cisplatin-induced loss of outer hair cells in the basal turn of the cochlea. Cisplatin and oxaliplatin both cause similar ototoxic effects when the organ of Corti is directly exposed in vitro. The thioredoxin redox system with the TrxR enzyme may well play a critical role in cisplatininduced ototoxicity. The presence of OCT2 in the supporting cells indicates that this transport protein is primarily not involved in the uptake of cisplatin from the systemic circulation but rather from the deeper compartments of the cochlea. The knowledge elicited in this work will hopefully suggest objectives for further studies in order to develop oto-protective treatments to preserve the hearing of cisplatin treated patients

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

The selenoprotein thioredoxin reductase : functional and structural characterization of human disulfide reductases as potential drug targets

Die im Säugerorganismus als Selenocystein-haltiges Enzym vorkommende homodimere Thioredoxinreduktase ist zusammen mit der Glutathionreduktase zentraler Bestandteil des zellulären Thiolmetabolismus, der über die antioxidative Abwehr hinaus auch an der Redoxregulation intra- und extrazellulärer Prozesse beteiligt ist. Das Thioredoxin- und das Glutathionsystem arbeiten in verschiedenen Organismen als parallele, sich ergänzende oder aber auch als sich ersetzende Systeme. In der vorliegenden Dissertation wurden verschiedene Beiträge zur biochemischen und kristallographischen Charakterisierung der Flavoproteine Thioredoxinreduktase und Gluta-thionreduktase im Hinblick auf ihr Potential als Zielmoleküle neu entwickelter Disulfid-reduktaseinhibitoren geleistet. Die Inhibitorstudien wurden durch Studien an verschiedenen TrxR-Mutanten, weiterführenden differentiellen Genom- und Proteomanalysen sowie Studien am Tiermodell komplementiert. Für die TrxR-Studien wurden rekombinante und native TrxR aus humaner Plazenta verwendet. Zur Gewinnung der nativen TrxR aus Plazenta wurde ein etabliertes Aufreinigungsverfahren angewendet und weiter optimiert. Im heterologen System konnten TrxR-Mutanten ohne Selenocystein in ausreichenden Mengen exprimiert werden. Diese bestanden in einer am C-terminalen Selenocystein mutierten TrxR-Mutante sowie in anderen, C-terminal-verkürzten TrxR-Mutanten und wurden auf Substratspezifität und Katalyse-eigenschaften untersucht. Trotz ihrer homologen Bereiche in den Substratbindungsregionen sind die TrxR und GR nicht ineinander zu überführen. Die Cysteinmutante der hTrxR wurde vergleichend zum Selenocystein-Wildtyp charakterisiert. Trotz einer sonst geringen Aktivität mit den üblichen Substraten Trx und DTNB erwies sie sich als potentes Enzym in der Menadionreduktion (kcat/KM = 7.4 ± 1.5 · 106 M-1· min-1) in vitro und im Zusammenhang mit einem induzierten Lipid-Transfer in die Zelle als apoptoseauslösend. Die Kristallisation der humanen TrxR stand ebenfalls im Mittelpunkt dieser Dissertation. Es gelang, hochwertige Einkristalle der Cysteinmutante der TrxR zu produzieren, von der erste Röntgenstrukturdaten bis 2.9 Å gewonnen werden konnten. Die erste im Rahmen der vorliegenden Arbeit getestete Inhibitorklasse setzte sich aus an Nitrofurancarbonhydrazid gekoppelten Cis-Diamindichlorplatin-Komplexen (CDDP-Kom-plexe) zusammen. Die CDDP-Komplexe erwiesen sich in enzymatischen in vitro-Unter-suchungen als irreversible und selektive Inhibitoren der NADPH-reduzierten TrxR im nanomolaren Konzentrationsbereich, mit maximalen Geschwindigkeitskonstanten ki um 1 min-1 in Kinetiken pseudo-erster Ordnung. Zellkulturexperimente und DNA-Interaktions-studien bestätigten, dass die TrxR-Hemmung der CDDP-Komplexe einen bedeutenden Beitrag zur Toxizität in Tumorzellen leisten kann (Millet et al., 2005). Die bekannten in vitro-Eigenschaften der Inhibitorklasse der 2,2\u27:6\u27,2\u27\u27-Terpyridinplatin(II)-Komplexe sollten in dieser Dissertation in vivo in einem Glioblastom-Rattenmodell untersucht werden. Die in Zusammenarbeit mit dem Universitätsklinikum Heidelberg durchgeführten Tierversuchsreihen mit verschiedenen Therapiemodellen und MRI-Aus-wertungen wurden durch Messungen verschiedener biochemischer, insbesondere redox-chemischer Parameter in den Geweben der Ratten ergänzt. Es ergaben sich gewebe-spezifische Veränderungen in der Redoxaktivität mit einer spezifischen TrxR-Hemmung im Tumorgewebe, die aber nicht mit der Auslösung der apoptotischen Kaskade einhergingen. Genom- sowie Proteomanalysen von behandelten Glioblastomzellen weisen auf einen chemotherapeutisch induzierten Zellzyklusarrest hin. (Urig et al., 2005; Ahmadi et al., 2005). Das Inhibitorpotential zweier Platin- und zweier Goldphosphol-Komplexe konnte in intensiven Kinetikstudien an der TrxR und der GR bestätigt werden, mit einer deutlichen Präferenz der Platin-Phospholkomplexe für die TrxR. Gold-Phospholkomplexe sind sehr effiziente Inhibitoren beider Disulfidreduktasen im unteren nanomolaren Bereich und gehen nach einer ersten kompetitiven Kinetik sehr schnell in die irreversible Phase zur kovalenten Modifikation der Enzyme über. Es gelang im Rahmen dieser Arbeit, einen hGR-Phosphol-Komplex zu kristallisieren und seine Röntgenstruktur bei 2.6 Å zu lösen. Die lineare S-Au-S-Geometrie zwischen den Cysteinen im aktiven Zentrum der hGR konnte zum ersten Mal in einer Proteinstruktur beobachtet werden (Urig et al., in press; Koncarevic et al., 2005; Deponte et al., 2005).The mammalian selenocystein containing homodimeric enzyme thioredoxin reductase (TrxR) is, together with glutathione reductase (GR), one of the two major proteins of the cellular thiol metabolism. It is involved in the antioxidant defence and is responsible for the redox regulation of intracellular and extracellular processes. The thioredoxin and the glutathione system can act as parallel, complementary or even substituting systems, depending on the organism. The present thesis describes the biochemical as well as crystallographic characterization of the two flavoproteines thioredoxin reductase und glutathione reductase as potential targets for novel inhibitors of disulfide reductases. The inhibitory studies were complemented by studies with different mutants of TrxR and additional differential genome and proteome analyses. Therefore, recombinant as well as native TrxR were used. The native enzyme was prepared from human placenta, using an established purification protocol that was further optimized. The hTrxR mutants without selenocystein were heterologously expressed in sufficient amounts. A cystein mutant as well as a C-terminally truncated mutant of hTrxR were examined for their substrate specificities and catalytic properties. Despite sequenz and structure homology, particularly in the substrate binding regions, it is not possible to transfer the TrxR into a GR by C-terminal protein engineering. The cystein mutant of hTrxR was much less active than the wildtype enzyme in reaction with the two common substrates thioredoxin and DTNB. For the first time, it could be shown that this mutant is very active with menadione in vitro (kcat/KM = 7.4 ± 1.5 · 106 M-1· min-1) and is capable to induce apoptosis when introduced in the cell by a lipid-mediated delivery system. The crystallization of human TrxR was another focus in this thesis. High quality monocrystals of the hTrxR cystein mutant were obtained which diffracted to 2.9 Å (X-ray struture analysis). In the first inhibitor study different cis-diaminedichlorplatinum(II) complexes from nitro-furancarbohydrazide (CDDP complexes) were examined. It was shown in in vitro assays that the CDDP complexes are irreversible and selective inhibitors of the NADPH-reduced TrxR in the nanomolar range with maximal rate constants ki of around 1 min-1 in pseudo-first order kinetics. Cell culture experiments and DNA interaction studies confirmed that the inhibition of TrxR by the CDDP complexes contributes to the toxicity of tumor cells (Millet et al., 2005). 2,2\u27:6\u27,2\u27\u27-terpyridineplatininum(II) complexes, known for their good inhibitory potential in in vitro assays, were tested for their in vivo activity in a glioblastoma rat model. In cooperation with the university hospital in Heidelberg a set of animal experiments including different therapy models was realised and analysed by MRI. In addition, several biochemical and redox parameters were determined in the rat tissues. Tissue-specific variations in redox activity were observed with a specific inhibition of TrxR in the tumor tissue, not inducing the apoptotic cascade. The results from genome and proteome analysis of treated glioblastoma cells furthermore indicate a chemotherapeutically induced cell cycle arrest. (Urig et al., 2005; Ahmadi et al., 2005). The inhibitory potential of two platinum and two gold phosphole complexes was proved in extensive kinetic analyses using TrxR and GR. The platinum complexes showed a strong preference for TrxR while the gold phosphol complexes are very efficient inhibitors of both enzymes in the lower nanomolar range. After a first competitive kinetic a fast and irreversible phase is followed to yield the covalent modification of the enzyme. We succeded in crystallisation of hGR in complex with a gold phosphol inhibitor and the X-ray structure was solved at 2.6 Å. The linear S-Au-S geometry between the cysteines in the actice site of hGR is the first to be observed in a protein structure (Urig et al., in press; Koncarevic et al., 2005; Deponte et al., 2005)