Publications by authors named "Verena Fetz"

21 Publications

  • Page 1 of 1

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high efficacy.

Chem Sci 2019 Dec 10;11(5):1316-1334. Epub 2019 Dec 10.

Department of Chemical Biology, Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric -aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL, and the previously identified spectrum gap of could be closed compared to the natural products (MIC of 0.5 μg mL). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first proof of concept was obtained in an infection mouse model, where derivative led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
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http://dx.doi.org/10.1039/c9sc04769gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148378PMC
December 2019

As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

Environ Sci Technol 2019 Nov 7;53(21):12725-12733. Epub 2019 Oct 7.

German Federal Institute for Risk Assessment , German Centre for the Protection of Laboratory Animals (Bf3R) , 10589 Berlin , Germany.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode , combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.
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http://dx.doi.org/10.1021/acs.est.9b03266DOI Listing
November 2019

The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates.

Chem Sci 2019 May 15;10(20):5197-5210. Epub 2019 Apr 15.

Department of Chemical Biology , Helmholtz Centre for Infection Research , Inhoffenstrasse 7 , 38124 Braunschweig , Germany . Email:

The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines ( 16-aminoratjadone with IC = 260 pM against MCF-7 cells, or 19-oxoratjadone with IC = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate had an IC of 34.3 nM, and the LHRH conjugate had an IC of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.
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http://dx.doi.org/10.1039/c8sc05542dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540907PMC
May 2019

xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery.

SLAS Discov 2019 03 25;24(3):213-223. Epub 2019 Jan 25.

1 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.
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http://dx.doi.org/10.1177/2472555218819459DOI Listing
March 2019

Subcellular Quantification of Uptake in Gram-Negative Bacteria.

Anal Chem 2019 02 11;91(3):1863-1872. Epub 2018 Dec 11.

Department of Chemical Biology , Helmholtz Centre for Infection Research , Inhoffenstraße 7 , 38124 Braunschweig , Germany.

Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 10 cells), but owing to the smaller volume of the periplasmic compartment, its concentration in the cytoplasm was much lower (37 ± 3 vs 221 ± 10 pg/μL for the periplasm). For erythromycin and tetracycline, differences in MICs between WT and ΔTolC mutant strains were not reflected by equal differences in uptake, illustrating that additional experimental data are needed to predict antibiotic efficacy. We believe that our assay, providing the antibiotic concentration at the compartment in which the drug target is expressed, constitutes an essential piece of information for a more rational optimization of novel antibiotics against Gram-negative infections.
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http://dx.doi.org/10.1021/acs.analchem.8b03586DOI Listing
February 2019

In Vitro Model of the Gram-Negative Bacterial Cell Envelope for Investigation of Anti-Infective Permeation Kinetics.

ACS Infect Dis 2018 08 24;4(8):1188-1196. Epub 2018 May 24.

Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI) , Saarland University , Campus Building E8 1 , 66123 Saarbrücken , Germany.

The cell envelope of Gram-negative bacteria is a formidable biological barrier, inhibiting the action of antibiotics by impeding their permeation into the intracellular environment. In-depth understanding of permeation through this barrier remains a challenge, despite its critical role in antibiotic activity. We therefore designed a divisible in vitro permeation model of the Gram-negative bacterial cell envelope, mimicking its three essential structural elements, the inner membrane and the periplasmic space as well as the outer membrane, on a Transwell setup. The model was characterized by contemporary imaging techniques and employed to generate reproducible quantitative and time-resolved permeation data for various fluorescent probes and anti-infective molecules of different structure and physicochemical properties. For a set of three fluorescent probes, the permeation through the overall membrane model was found to correlate with in bacterio permeation. Even more interestingly, for a set of six Pseudomonas quorum sensing inhibitors, such permeability data were found to be predictive for their corresponding in bacterio activities. Further exploration of the capabilities of the overall model yielded a correlation between the permeability of porin-independent antibiotics and published in bacterio accumulation data; a promising ability to provide structure-permeability information was also demonstrated. Such a model may therefore constitute a valuable tool for the development of novel anti-infective drugs.
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http://dx.doi.org/10.1021/acsinfecdis.7b00165DOI Listing
August 2018

Translocation Biosensors-Versatile Tools to Probe Protein Functions in Living Cells.

Methods Mol Biol 2018 ;1683:195-210

Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), Mainz Screening Center UG & Co. KG, University of Duisburg-Essen, Essen, Germany.

In this chapter, you will learn how to use translocation biosensors to investigate protein functions in living cells. We here present three classes of modular protein translocation biosensors tailored to investigate: (1) signal-mediated nucleo-cytoplasmic transport, (2) protease activity, and (3) protein-protein interactions. Besides the mapping of protein function, the biosensors are also applicable to identify chemicals and/or (nano) materials modulating the respective protein activities and can also be exploited for RNAi-mediated genetic screens.
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http://dx.doi.org/10.1007/978-1-4939-7357-6_12DOI Listing
June 2018

TFIIA transcriptional activity is controlled by a 'cleave-and-run' Exportin-1/Taspase 1-switch.

J Mol Cell Biol 2018 02;10(1):33-47

Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, 45141 Essen, Germany.

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.
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http://dx.doi.org/10.1093/jmcb/mjx025DOI Listing
February 2018

Superior Fluorogen-Activating Protein Probes Based on 3-Indole-Malachite Green.

Org Lett 2017 09 18;19(17):4496-4499. Epub 2017 Aug 18.

Department of Chemical Biology, Helmholtz-Zentrum für Infektionsforschung (HZI) , Inhoffenstrasse 7, 38124 Braunschweig, Germany.

A series of novel fluorogenic dyes based on 3-indole-Malachite Green, MGs 5-7, have been developed that are dark in solution but highly fluorescent when bound to the cognate reporter, fluorogen-activating protein (FAP). Significantly, the new MGs 5-7 probes are superior to the traditional MG 1 with high fluorescent efficiency and low toxicity to cells while maintaining the large "pseudo-Stokes" shifts (Δλ = λ - λ) and the malachite green (MG)-like fluorescence OFF-ON switching mechanism in both live mammalian cells and bacterial cells when bound to FAP.
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http://dx.doi.org/10.1021/acs.orglett.7b02055DOI Listing
September 2017

Multivalent Siderophore-DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections.

Angew Chem Int Ed Engl 2017 07 13;56(28):8272-8276. Epub 2017 Jun 13.

Department of Chemical Biology, Helmholtz Centre for Infection Research and German Centre for Infection Research (DZIF), Inhoffenstrasse 7, 38124, Braunschweig, Germany.

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein-malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.
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http://dx.doi.org/10.1002/anie.201701358DOI Listing
July 2017

Synthesis and Biological Investigation of Δ(12)-Prostaglandin J3 (Δ(12)-PGJ3) Analogues and Related Compounds.

J Am Chem Soc 2016 05 12;138(20):6550-60. Epub 2016 May 12.

Stemcentrx Inc. , 450 East Jamie Court, South San Francisco, California 94080, United States.

A series of Δ(12)-prostaglandin J3 (Δ(12)-PGJ3) analogues and derivatives were synthesized employing an array of synthetic strategies developed specifically to render them readily available for biological investigations. The synthesized compounds were evaluated for their cytotoxicity against a number of cancer cell lines, revealing nanomolar potencies for a number of them against certain cancer cell lines. Four analogues (2, 11, 21, and 27) demonstrated inhibition of nuclear export through a covalent addition at Cys528 of the export receptor Crm1. One of these compounds (i.e., 11) is currently under evaluation as a potential drug candidate for the treatment of certain types of cancer. These studies culminated in useful and path-pointing structure-activity relationships (SARs) that provide guidance for further improvements in the biological/pharmacological profiles of compounds within this class.
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http://dx.doi.org/10.1021/jacs.6b02075DOI Listing
May 2016

Arginine residues within the DNA binding domain of STAT3 promote intracellular shuttling and phosphorylation of STAT3.

Cell Signal 2014 Aug 8;26(8):1698-706. Epub 2014 Apr 8.

Department of Toxicology, University Medical Center, Obere Zahlbacher Str. 67, 55131 Mainz, Germany. Electronic address:

Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine-glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine-lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling.
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http://dx.doi.org/10.1016/j.cellsig.2014.03.033DOI Listing
August 2014

Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology.

Nat Nanotechnol 2013 Oct 22;8(10):772-81. Epub 2013 Sep 22.

1] Institute for Immunology, University Medical Center of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany [2].

In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
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http://dx.doi.org/10.1038/nnano.2013.181DOI Listing
October 2013

Functional characterization of novel mutations affecting survivin (BIRC5)-mediated therapy resistance in head and neck cancer patients.

Hum Mutat 2013 Feb 20;34(2):395-404. Epub 2012 Dec 20.

Institute for Molecular Biology, Centre for Medical Biotechnology, ZMB, University of Duisburg-Essen, Germany.

Survivin (BIRC5) is an acknowledged cancer therapy-resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation-induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin-based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.
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http://dx.doi.org/10.1002/humu.22249DOI Listing
February 2013

A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer.

Oncotarget 2012 Jan;3(1):31-43

Molecular and Cellular Oncology/Mainz Screening Center, University Hospital of Mainz, Germany.

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p less than 0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P less than 0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292890PMC
http://dx.doi.org/10.18632/oncotarget.430DOI Listing
January 2012

Bioassays to monitor Taspase1 function for the identification of pharmacogenetic inhibitors.

PLoS One 2011 25;6(5):e18253. Epub 2011 May 25.

Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, Essen, Germany.

Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available.

Methodology/findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor.

Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018253PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102056PMC
September 2011

An update on the pathobiological relevance of nuclear receptors for cancers of the head and neck.

Histol Histopathol 2010 08;25(8):1093-104

Molecular and Cellular Oncology, University Hospital of Mainz, Mainz, Germany.

Cancers of the head and neck are among the most common neoplasms worldwide, characterized by local tumor aggressiveness, high rate of early recurrence, development of metastasis and second primary tumors. Although disease management of head and neck cancer has improved significantly, overall survival-rates remained largely unchanged over the last decades. Thus, in addition to modern chemo-radiation treatment strategies combined with sophisticated surgery, there is still a need for molecular markers and key regulatory factors exploitable for chemoprevention and targeted therapies. A critical event in carcinogenesis is the uncontrolled modulation of genetic programs, mediated by deregulated signaling cascades, together with downstream transcriptional modulators. Hence, nuclear receptors, belonging to a superfamily of transcription factors implicated in a broad spectrum of physiological and pathophysiological processes, have also been associated with HNC. Enhanced expression of several nuclear receptors has been shown in head and neck cancer cells, and strategies targeting these molecules have been developed and tested in the clinics. In particular, the effects of retinoids targeting nuclear receptors of the thyroid hormone receptor-like receptor subfamily have been vigorously examined in large clinical chemoprevention trials. This review seeks to provide a general overview of nuclear receptors' molecular functions and summarizes their prognostic/therapeutic relevance, as well as the (pre)clinical studies targeting nuclear receptors in HNC.
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http://dx.doi.org/10.14670/HH-25.1093DOI Listing
August 2010

Inducible NO synthase confers chemoresistance in head and neck cancer by modulating survivin.

Int J Cancer 2009 May;124(9):2033-41

Department of Otorhinolaryngology, Molecular and Cellular Oncology, University Hospital of Mainz, Mainz, Germany.

The dual role of the inducible NO synthase (iNOS) and NO signaling in head and neck squamous cell carcinoma (HNSCC) is a complex and can both promote or inhibit tumor progression. However, the underlying molecular mechanisms are not yet resolved in detail. We show for the first time that conditions, favoring low NO levels conferred resistance against cisplatin/taxol-induced apoptosis in HNSCC cell lines. Cytoprotection was mediated by survivin, because we observed its upregulation subsequent to low doses of the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or ectopic expression of physiologic amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked NOs anti-apoptotic effects. Induction of survivin involves activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway, which was antagonized by the PI3K-inhibitor LY294002. Importantly, application of the iNOS-specific inhibitor 1400W combined with RNAi-mediated downregulation of survivin cooperatively enhanced drug-induced cell death. The iNOS/survivin-axis appears to be also of clinical relevance since immunohistochemistry revealed that iNOS expression correlated with enhanced survivin levels in HNSCC specimens. In contrast, high NO concentrations suppressed survivin levels in HNSCC but also in non-malignant cells resulting in apoptosis. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved activation of p38MAP-kinase, which was counteracted by the p38MAP-kinase inhibitor SB202190. Here, we provide evidence for a novel molecular mechanism how NO signaling may contribute to therapy resistance in HNSCC by modulating survivin expression. Our data further suggest pursuing pharmacogenetic iNOS/survivin-targeting approaches as potential therapeutic strategies in head and neck cancer.
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http://dx.doi.org/10.1002/ijc.24182DOI Listing
May 2009

Translocation Biosensors - Cellular System Integrators to Dissect CRM1-Dependent Nuclear Export by Chemicogenomics.

Sensors (Basel) 2009 9;9(7):5423-45. Epub 2009 Jul 9.

Department of Molecular and Cellular Oncology, University of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany; E-Mail: (V.F.); (C.B.).

Fluorescent protein biosensors are powerful cellular systems biology tools for dissecting the complexity of cellular processes with high spatial and temporal resolution. As regulated nucleo-cytoplasmic transport is crucial for the modulation of numerous (patho)physiological cellular responses, a detailed understanding of its molecular mechanism would open up novel options for a rational manipulation of the cell. In contrast to genetic approaches, we here established and employed high-content cellular translocation biosensors applicable for dissecting nuclear export by chemicogenomics. A431 cell lines, stably expressing a translocation biosensor composed of glutathione S-transferase, GFP and a rational combination of nuclear import and export signals, were engineered by antibiotic selection and flow cytometry sorting. Using an optimized nuclear translocation algorithm, the translocation response could be robustly quantified on the Cellomics Arrayscan(®) VTI platform. Subsequent to assay optimization, the assay was developed into a higher density 384-well format high-content assay and employed for the screening of the 17K ChemBioNet compound collection. This library was selected on the basis of a genetic algorithm used to identify maximum common chemical substructures in a database of annotated bioactive molecules and hence, is well-placed in the chemical space covered by bioactive compounds. Automated multiparameter data analysis combined with visual inspection allowed us to identify and to rationally discriminate true export inhibitors from false positives, which included fluorescent compounds or cytotoxic substances that dramatically affected the cellular morphology. A total of 120 potential hit compounds were selected for Cellomics Arrayscan(®) VTI based rescreening. The export inhibitory activity of 20 compounds effective at concentrations < 25 μM were confirmed by fluorescence microscopy in several cell lines. Interestingly, kinetic analysis allowed the identification of inhibitors capable to interfere with the export receptor CRM1-mediated nuclear export not only in an irreversible, but also in a reversible fashion. In sum, exploitation of biosensor based screening allows the identification of chemicogenomic tools applicable for dissecting nucleo-cytoplasmic transport in living cells.
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http://dx.doi.org/10.3390/s90705423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274152PMC
September 2012

NO signaling confers cytoprotectivity through the survivin network in ovarian carcinomas.

Cancer Res 2008 Jul;68(13):5159-66

Department of Pathology, University Hospital of Frankfurt, Frankfurt, Germany.

Despite considerable success in the treatment of epithelial ovarian cancer (EOC), therapy resistance counteracts improvement of long-term survival. The dual role of survivin as an apoptosis inhibitor and mitotic regulator has been associated with disease outcome. However, the molecular mechanisms involved in the deregulated expression in EOC of survivin need further investigation. Here, we show that high amounts of the nitric oxide (NO) donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or strong overexpression of the inducible nitric oxide synthase (iNOS) suppressed survivin levels via the p38MAPK pathway and triggered apoptosis in ovarian cancer cell lines (OCC). Importantly, low NO concentrations conferred resistance against carboplatin/paclitaxel-induced apoptosis. Cytoprotection was mediated by survivin because we observed its up-regulation subsequent to low SNAP/SNP doses or ectopic expression of low amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling. Induction of survivin involves activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which was antagonized by the PI3K-inhibitor, LY294002. Interestingly, application of the iNOS-inhibitor 1400W together with RNAi-mediated survivin down-regulation cooperatively enhanced drug-induced apoptosis in OCCs. The iNOS/survivin interdependencies seem to be also of clinical relevance because immunohistochemistry revealed that low iNOS levels correlate with survivin expression (P < 0.01) in carboplatin/paclitaxel-treated EOC patients with minimal postoperative residual tumor (n = 54). Also, iNOS and survivin expression were associated with increased risk for disease progression. Our study uncovers a novel molecular mechanism of how NO signaling may contribute to therapy resistance in EOC by modulating survivin expression. Pharmacogenetic iNOS/survivin-targeting strategies may hence be pursued to complement current treatment modalities in EOC.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-0406DOI Listing
July 2008

Dynamic survivin in head and neck cancer: molecular mechanism and therapeutic potential.

Int J Cancer 2007 Sep;121(6):1169-74

Department of Otorhinolaryngology, Molecular and Cellular Oncology, University of Mainz, Mainz, Germany.

Although disease management of head and neck squamous cell carcinomas (HNSCC) has improved significantly, therapy resistance leading to tumor recurrence still counteracts improvement of long-term survival. Consequently, identification of molecular markers that signal increased risk of treatment failure or, which can be exploited by targeted therapy, is urgently needed. Survivin is strongly expressed in HNSCC, and its proposed dual role as an apoptosis inhibitor and a mitotic effector positioned survivin in the front line of cancer research. Notably, survivin is detected as a cytoplasmic and as a nuclear protein in HNSCC patients, which stimulated numerous studies to investigate and to speculate on the functional and prognostic significance of its dynamic localization. This review focuses on our current understanding of the molecular mechanisms regulating survivin's intracellular localization and discusses its potential prognostic and therapeutic relevance for head and neck cancer.
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http://dx.doi.org/10.1002/ijc.22941DOI Listing
September 2007
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