A series of 4H-1,2,3-thiadiazolo[5,4-b]indoles were synthesized by novel tandem of oxidative cyclization of 3-alkoxycarbonylhydrazonoindoline-2-thiones, 1,5-H-shift and elimination of tert-butoxy(ethoxy)carbonyl group. The simple method for their modifications by the reactions with electrophilic agents were elaborated and as a result of the synthetic investigation a number of N-alkyl-, N-acyl- and N-sulfonyl-4H-1,2,3-thiadiazolo[5,4-b]indoles were prepared in good yields. Preliminary biological tests for the three examples of synthesized compounds with different substituents at the nitrogen atom indole ring have shown that the biological behavior of the investigated 1,2,3-thiadiazolo[5,4-b]indoles is substantially directed by this structural fragment.

The photophysical properties of [Re(CO)3 (L-N3)]Br (L-N3 =2-azido-N,N-bis[(quinolin-2-yl)methyl]ethanamine), which could not be localized in cancer cells by fluorescence microscopy, have been revisited in order to evaluate its use as a luminescent probe in a biological environment. The Re(I) complex displays concentration-dependent residual fluorescence besides the expected phosphorescence, and the nature of the emitting excited states have been evaluated by DFT and time-dependent (TD) DFT methods. The results show that fluorescence occurs from a (1) LC/MLCT state, whereas phosphorescence mainly stems from a (3) LC state, in contrast to previous assignments. We found that our luminescent probe, [Re(CO)3 (L-N3)]Br, exhibits an interesting cytotoxic activity in the low micromolar range in various cancer cell lines. Several biochemical assays were performed to unveil the cytotoxic mechanism of the organometallic Re(I) bisquinoline complex. [Re(CO)3 (L-N3)]Br was found to be stable in human plasma indicating that [Re(CO)3 (L-N3)]Br itself and not a decomposition product is responsible for the observed cytotoxicity. Addition of [Re(CO)3 (L-N3)]Br to MCF-7 breast cancer cells grown on a biosensor chip micro-bioreactor immediately led to reduced cellular respiration and increased glycolysis, indicating a large shift in cellular metabolism and inhibition of mitochondrial activity. Further analysis of respiration of isolated mitochondria clearly showed that mitochondrial respiratory activity was a direct target of [Re(CO)3 (L-N3)]Br and involved two modes of action, namely increased respiration at lower concentrations, potentially through increased proton transport through the inner mitochondrial membrane, and efficient blocking of respiration at higher concentrations. Thus, we believe that the direct targeting of mitochondria in cells by [Re(CO)3 (L-N3)]Br is responsible for the anticancer activity.

Solid-phase peptide synthesis (SPPS) is a versatile technique for the assembly of small to medium size peptides, that can help in the delivery of bound metal complexes to certain cellular compartments, for example in cancer cells. This work shows a new route to gold-peptide bioconjugates via a non-catalyzed [ 3 + 2] cycloaddition reaction of gold azides with alkynyl peptides. Gold(I) tetrapeptide conjugates with a mitochondria-targeting sequence were synthesized and display prolonged stability in the presence of thiol-containing biological media. Their antiproliferative potency against selected cancer cells (2-50 mu M) corresponds to the lipophilicity of the conjugates. The cellular uptake of Au, determined by atomic absorption spectroscopy (AAS), shows that high initial uptake equals strong cytotoxicity. Respiration and acidification rates react immediately upon treatment with the Au-peptide conjugates, and a terminal breakdown of essential cellular functions is complete within ca. 12 h at most, as observed by online monitoring of the cancer cell metabolism in a microfluidic biosensor device (Bionas sensorchip system). The mode of action of these Au-peptide bioconjugates was elucidated by a variety of biochemical and cell biological experiments. First, a strong selective inhibition of the enzyme thioredoxin reductase (TrxR), a regulator of cellular redox processes, was found. In this context, elevated levels of reactive oxygen species (ROS) and strong effects on the respiration of isolated mouse liver mitochondria were found. These finally lead to cell death via apoptotic pathways, as indicated by flow cytometry, low mitochondrial membrane potential (MMP) and DNA fragmentation. Intriguingly, cisplatin-resistance in p53-mutant MDA-MB231 breast cancer cells could be overcome by the Au-peptide conjugates presented herein.

Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner.Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation.

The cytostatic properties and cellular effects of novel diene–ruthenium(II) complexes of the types OC-6-13-[RuCl2(pp)(cod)] 1–5 (pp=2,2′-bipyridyl (bpy), phen=1,10-phenanthroline (phen), 5,6-dimethylphenanthroline (5,6-Me2phen), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), ethylenediamine (en)) and OC-6-24-[RuCl{(Me2N)2CS}(pp)(cod)](CF3SO3) 6–8 (pp=phen, 5,6-Me2phen, dpq) have been studied for the human cancer cell lines MCF-7 and HT-29 and for Jurkat leukemia cells. CD spectra indicate that 7 causes a massive distortion of the CT DNA B double helix toward the A form. Whereas the neutral complexes 1, 2 and 5 exhibit only modest antiproliferative activity toward MCF-7 and HT-29 cells, the monocationic complexes are significantly more active, in particular the DNA-distorting complex 7 with its IC50 values of 0.73 and 0.42μM, respectively. As established by online monitoring with a cell-based sensor chip, this potent 5,6-Me2phen complex invokes dose-dependent decreases in MCF-7 cellular respiration and extracellular acidification rates and causes a time-delayed decrease in the impedance of the cell layers, that can be ascribed to cell death. Treatment of Jurkat cells with 7 leads to high concentrations of reactive oxygen species and the induction of apoptosis. The pronounced dose-dependent inhibition of oxygen consumption by isolated mice mitochondria indicates the involvement of an intrinsic mitochondrial pathway in the programmed cell death process.

Half-sandwich organorhodium(III) complexes and their trichloridorhodium(III) counterparts are potent anticancer agents that enhance the formation of reactive oxygen species and invoke a strong induction of apoptosis in leukemia cells. The antiproliferative activity towards human MCF-7 and HT-29 adenocarcinoma cells of novel nonintercalating complexes containing the 5-substituted phenanthroline ligands 5,6-dimethylphenanthroline, 5-chlorophenanthroline, and 5-nitrophenanthroline (phen*) increases dramatically in the order [(η5-C5Me5)IrCl(phen*)](CF3SO3) < [(η5-C5Me5)RhCl(phen*)](CF3SO3) < mer-[RhCl3(DMSO)(phen*)] (DMSO is dimethyl sulfoxide) . Improved activity was also achieved by attaching a cell-penetrating peptide to the dipyrido[3,2-a:2′,3′-c]phenazine (dppz) ligand of an organorhodium(III) complex. Whereas 5-substitution led to significant improvements in the activity of the organoiridium(III) and trichloridorhodium(III) compounds in comparison with the parent phenanthroline complex, the IC50 values of their organorhodium(III) counterparts remained effectively invariable. The high activities of the trichloridorhodium(III) complexes (IC50 = 0.06–0.13 μM) were accompanied by pronounced selectivity towards human cancer cells in comparison with immortalized HEK-293 cells. In contrast, [(η5-C5Me5)RhCl(5,6-Me2phen)](CF3SO3) (phen is phenanthroline) was markedly more active towards BJAB lymphoma cells than ex vivo healthy leukocytes and caused an immediate decrease in cellular adhesion possibly associated with interactions with membrane proteins. Its dppz analogue invoked an initial increase in glycolysis to compensate for reduced respiration before inducing a delayed onset of cell death. Strong antimitochondrial activity with respiration impairment and release of cytochrome c was established for both complexes.

The DNA binding modes and cleavage properties of novel dinuclear Ir(III) polypyridyl (pp) complexes [{(η(5)-C(5)Me(5))Ir(pp)}(2)(μ-B)](CF(3)SO(3))(4) depend on the lengths of their rigid bridging dipyridinyl ligands B. Mono-intercalation and strong DNA cleavage properties were observed for the dipyrido[2,3-a:2',3'-c]phenazine (dppz) complexes 1 (B = 4-[(E)-2-(4-pyridinyl)ethenyl]pyridine) and 3 (B = 4-(2-pyridin-4-ylethynyl)pyridine), whose intracationic Ir···Ir' distances are about 13.1 and 13.3 Å, respectively. In contrast, UV/Vis and CD spectra were in accordance with a stable intertwined bis-intercalation mode for pairs of cations of 5 (B = 1,4-di(2-pyridin-4-ylethynyl)benzene), whose much longer Ir···Ir' distance of 20.6 Å allows a stack of five aromatic chromophores to be sandwiched between its effectively parallel dppz ligands. Whereas both 1 and 3 cleaved DNA in the dark, complex 5 exhibited only photoinduced nuclease activity. A significantly higher antiproliferative activity towards MCF-7 breast carcinoma cells was observed for the nucleases 1 and 3, whose IC(50) values of 0.61 and 0.49 were much lower than that of 2.2 μM for bis-intercalator 5. Values of 3.8 μM, only slightly higher than that of 5, were recorded for the 5,6-dimethylphenanthroline complexes 4 and 6, whose bridging ligands are identical to those of 3 and 5, respectively. Marked antileukemic activity (IC(50) = 6-7 μM) associated with increased levels of reactive oxygen species and apoptosis induction was recorded for both 3 and 5 towards Jurkat cells at concentrations of 5 μM and above. Online studies with a sensor chip system indicated that 5 μM solutions of these complexes invoke a rapid and massive reduction in MCF-7 cell respiration.

Regulatory Smads (R-Smads), Smad1/5/8 and Smad2/3, are the central mediators of TGFβ and BMP signaling pathways. Here, we screened indirubin derivatives, known kinase inhibitors, and observed strong interference with BMP signaling. We found that indirubin derivative E738 inhibited both TGFβ and BMP pathways through ubiquitin-proteasome-mediated depletion of total R-Smad pools, although phospho-R-Smad levels were initially stabilized by GSK3β and cyclin-dependent kinase inhibition. E738 also enhanced p38 and JNK phosphorylation, involved in Smad-independent TGFβ/BMP signaling. Additionally, using a small siRNA screen, we showed that depletion of ubiquitin proteases USP9x and USP34 significantly reduced total R-Smad levels, mimicking E738 treatment. In fact, both USP9x and USP34 levels were significantly reduced in E738-treated cells. Our findings not only describe the complex activity profile of the indirubin derivative E738, but also reveal a mechanism for controlling TGFβ/BMP signaling, the control of R-Smad protein levels through deubiquitination.

Since the discovery of cisplatin more than 40 years ago and its clinical introduction in the 1970s an enormous amount of research has gone into elucidating the mechanism of action of cisplatin on tumor cells. With a novel cell biosensor chip system allowing continuous monitoring of respiration, glycolysis, and impedance we followed cisplatin treatment of different cancer cell lines in real-time. Our measurements reveal a first effect on respiration, in all cisplatin treated cell lines, followed with a significant delay by interference with glycolysis in HT-29, HCT-116, HepG2, and MCF-7 cells but not in the cisplatin-resistant cell line MDA-MB-231. Most strikingly, cell death started in all cisplatin-sensitive cell lines within 8 to 11 h of treatment, indicating a clear time frame from exposure, first response to cisplatin lesions, to cell fate decision. The time points of most significant changes were selected for more detailed analysis of cisplatin response in the breast cancer cell line MCF-7. Phosphorylation of selected signal transduction mediators connected with cellular proliferation, as well as changes in gene expression, were analyzed in samples obtained directly from sensor chips at the time points when changes in glycolysis and impedance occurred.Our online cell biosensor measurements reveal for the first time the time scale of metabolic response until onset of cell death under cisplatin treatment, which is in good agreement with models of p53-mediated cell fate decision.

Gold(I) complexes with a 1,3-diethylbenzimidazol-2-ylidene N-heterocyclic carbene (NHC) ligand of the type NHC-Au-L (L = -Cl, -NHC, or -PPh(3)) were comparatively evaluated as thioredoxin reductase (TrxR) inhibitors and antimitochondrial anticancer agents. Different effects were noted in various biochemical assays (e.g., inhibition of TrxR, cellular and mitochondrial uptake, or effects on mitochondrial membrane potential), and this was related to properties of the complexes such as bond dissociation energies and overall charge. Remarkable antiproliferative effects, a strong induction of apoptosis, and enhancement of reactive oxygen species (ROS) formation as well as other effects on tumor cell metabolism confirmed the promising potential of the complexes as novel anticancer chemotherapeutics.

Commonly used colorimetric detection applied to protein microarrays with enzymatic signal amplification leads to non‐linear signal production upon increase in analyte concentration, thereby considerably limiting the range and accuracy of quantitative readout interpretation. To extend the detection range, we developed a kinetic colorimetric detection protocol for the analysis of ELISA microarrays designed to measure multiple phosphorylated proteins using the platforms ArrayTube™ and ArrayStrip™. With our novel quantification approach, microarrays were calibrated over a broad concentration range spanning four orders of magnitude of analyte concentration with picomolar threshold. We used this design for the simultaneous quantitative measurement of 15 phosphorylated proteins on a single chip.

The present invention relates in a first aspect a new angiogenesis inhibitor based on gold (I) -Alkinylkomplexen. More specifically, the present application relates to the use of compounds having a phosphine or carbene, Au (I), alkynyl, and an optionally connected via a linker to the alkynyl aromatics. These compounds show excellent inhibition of angiogenesis in an individual. Furthermore, cytotoxic properties, in particular for treatment of cancer could be shown for carbenhaltige compounds according to the present application. Furthermore, the present application relates to novel pharmaceutical compositions containing carbene-gold (I) alkyne complexes as well as new anti-inflammatory drugs, angiogenesis inhibitors or cytostatics.

The antiproliferative properties and biological impact of octahedral iridium(III) complexes of the type fac-[IrCl3(DMSO)(pp)] containing pp=phenanthroline (1) and its 4- and 5-methyl (2, 3) and 4,7- and 5,6-dimethyl derivatives (4, 5) were investigated for both adherent and non-adherent cells. A series of similar rhodium(III) complexes were studied for comparison purposes. The antiproliferative activity toward MCF-7 cancer cells increases eightfold from IC50 = 4.6 for 1 to IC50 = 0.60 mm for 5, and an even more pronounced 18-fold improvement was established for the analogous rhodium complexes 6 and 8, the respective IC50 values for which are 1.1 and 0.06 mm. Annexin V/propidium iodide assays demonstrated that the 5,6-dimethylphenanthro-line complexes 5 and 8 both cause significant inhibition of Jurkat leukemia cell proliferation and invoke extensive apoptosis but negligible necrosis. The percentages of Jurkat cells exhibiting high levels of reactive oxygen species correlate with the percentages of cells undergoing apoptosis. The antiproliferative activity of 5 and 8 is strongly selective toward MCF-7 and HT-29 cancer cells over normal HFF-1 and immortalized HEK-293 cells. Complex 5 also exhibits high selectivity toward BJAB lymphoma cells relative to healthy leukocytes. Both 5 and 8 invoke permanent decreases in the adhesion and respiration of MCF-7 cells.

The cytostatic properties of novel rhodium(III) thiacrown ether complexes [RhCl(LL)([9]aneS(3))](n+) with either aromatic κ(2)N ligands (n = 2) or anionic chelate ligands (n = 1) have been investigated for the human cancer cell lines HT-29 and MCF-7 and for immortalized HEK-293 cells. Taken together with literature IC(50) values for analogous complexes with polypyridyl ligands or 1,4-dithiane, the in vitro assays indicate that dicationic complexes with soft κ(2)N (imino) or κ(2)S (thiaether) ligands exhibit significantly higher antiproliferative effects than those with hard κ(2)N (amino) ligands. Dicationic complexes are more active than monocationic complexes with similar ligands. Pronounced apoptosis-inducing properties towards Jurkat cells were established for complexes with LL = bpm, dpq, and 1,4-dithiane. The order of activity (bpm > 1,4-dithiane > dpq > bpy) contrasts to that observed for adhesive cancer cells (bpm > bpy, 1,4-dithiane > dpq). Necrosis is insignificant in all cases. The percentage of Jurkat cells exhibiting apoptosis after 24 or 48 h incubation periods is directly correlated to the percentage of cells exhibiting high levels of reactive oxygen species. As established by online monitoring with a sensor chip system, treatment of MCF-7 cells with the bpm and 1,4-dithiane complexes leads to a significant and permanent concentration-dependent decrease in oxygen consumption and cellular adhesion.

We demonstrate the cytotoxic potential of the Schiff base iron complex [FeIII(salophene)Cl] in vitro and ex vivo and illustrate its ability to overcome multiple drug resistance in vincristine and daunorubicine resistant leukemic cells (Nalm-6). Treatment of lymphoma cells (BJAB) with [FeIII(salophene)Cl] led to the exclusion of unspecific necrosis, a concentration-dependent inhibition of proliferation and a specific apoptotic cell death. We further detected a significant loss of the mitochondrial membrane potential in lymphoma cells and an up- and downregulation of various apoptosis relevant genes, respectively, indicating the involvement of the intrinsic mitochondrial pathway.

The antiproliferative properties and cellular impact of novel substitutionally inert rhodium(III) complexes of the types [Rh{(CH₃)₂ NCS₂}₂(pp)]Cl 3-5 (pp=5,6-Me₂phen, dpq, dppz) and OC-6-23-[Rh(2-S-py)₂(pp)]Cl 6 and 7 (2-S-py=pyridine-2-thiolate; pp=dpq, dppz) have been investigated for the adherent human cancer cell lines MCF-7 and HT-29 and for non-adherent Jurkat cells. Whereas CD and viscosity measurements indicate that the polypyridyl ligands of 4 and 5 intercalate into CT DNA, this is not the case for the analogous pyridine-2-thiolate complexes 6 and 7. Complexes 3-7 all exhibit a high antiproliferative activity towards MCF-7 and HT-29 cells, with IC(50) values in the range 0.055-0.285 μM. As established by online monitoring with a cell-based sensor chip, the highly cytostatic complex 6 (IC(50)=0.059 and 0.078 μM) invokes an immediate concentration-dependent reduction of MCF-7 cell respiration and a time-delayed decrease in cellular impedance, which can be ascribed to the induction of cell death. Annexin V/PI assays demonstrated that 6 also has a pronounced antiproliferative activity towards Jurkat cells and that it invokes extensive apoptosis and high concentrations of reactive oxygen species in these leukemia cells. The observation of a dose-dependent inhibition of the oxygen consumption of isolated mice mitochondria indicates the involvement of an intrinsic mitochondrial pathway in this process.

Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands represent a promising class of gold coordination compounds with a good stability against the thiol glutathione. TrxR was selectively inhibited by in comparison to the closely related enzyme glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor cells. More detailed studies on a selected complex revealed a distinct pharmacodynamic profile including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of mitochondrial respiration and activity against resistant cell lines.

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant'') that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.