• Search for hits and early leads from soil bacteria to combat infectious diseases.

      Herrmann, Jennifer; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Open Learning on Enteric Pathogens, 2018-02-22)
    • Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase.

      Thomas, Lea; Rao, Zhigang; Gerstmeier, Jana; Raasch, Martin; Weinigel, Christina; Rummler, Silke; Menche, Dirk; Müller, Rolf; Pergola, Carlo; Mosig, Alexander; et al. (2017-02-09)
      Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1β, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.
    • Self-resistance guided genome mining uncovers new topoisomerase inhibitors from myxobacteria.

      Panter, Fabian; Krug, Daniel; Baumann, Sascha; Müller, Rolf; HIPS, Helmholtz-Institute für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2018-06-07)
      There is astounding discrepancy between the genome-inscribed production capacity and the set of known secondary metabolite classes from many microorganisms as detected under laboratory cultivation conditions. Genome-mining techniques are meant to fill this gap, but in order to favor discovery of structurally novel as well as bioactive compounds it is crucial to amend genomics-based strategies with selective filtering principles. In this study, we followed a self-resistance guided approach aiming at the discovery of inhibitors of topoisomerase, known as valid target in both cancer and antibiotic therapy. A common host self-defense mechanism against such inhibitors in bacteria is mediated by so-called pentapeptide repeat proteins (PRP). Genes encoding the biosynthetic machinery for production of an alleged topoisomerase inhibitor were found on the basis of their collocation adjacent to a predicted PRP in the genome of the myxobacterium
    • Semisynthesis and biological evaluation of amidochelocardin derivatives as broad-spectrum antibiotics.

      Grandclaudon, Charlotte; Birudukota, N V Suryanarayana; Elgaher, Walid A M; Jumde, Ravindra P; Yahiaoui, Samir; Arisetti, Nanaji; Hennessen, Fabienne; Hüttel, Stephan; Stadler, Marc; Herrmann, Jennifer; et al. (Elsevier, 2019-12-20)
      To address the global challenge of emerging antimicrobial resistance, the hitherto most successful strategy to new antibiotics has been the optimization of validated natural products; most of these efforts rely on semisynthesis. Herein, we report the semisynthetic modification of amidochelocardin, an atypical tetracycline obtained via genetic engineering of the chelocardin producer strain. We report modifications at C4, C7, C10 and C11 by the application of methylation, acylation, electrophilic substitution, and oxidative C-C coupling reactions. The antibacterial activity of the reaction products was tested against a panel of Gram-positive and Gram-negative pathogens. The emerging structure-activity relationships (SARs) revealed that positions C7 and C10 are favorable anchor points for the semisynthesis of optimized derivatives. The observed SAR was different from that known for tetracyclines, which underlines the pronounced differences between the two compound classes.
    • Setting Our Sights on Infectious Diseases.

      De Rycker, Manu; Horn, David; Aldridge, Bree; Amewu, Richard K; Barry, Clifton E; Buckner, Frederick S; Cook, Sarah; Ferguson, Michael A J; Gobeau, Nathalie; Herrmann, Jennifer; et al. (American Society for Chemistry, 2019-12-06)
      In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
    • Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

      Wang, Mingxun; Carver, Jeremy J; Phelan, Vanessa V; Sanchez, Laura M; Garg, Neha; Peng, Yao; Nguyen, Don Duy; Watrous, Jeramie; Kapono, Clifford A; Luzzatto-Knaan, Tal; et al. (2016-08-09)
      The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.
    • Sonogashira diversification of unprotected halotryptophans, halotryptophan containing tripeptides; and generation of a new to nature bromo-natural product and its diversification in water.

      Corr, M J; Sharma, S V; Pubill-Ulldemolins, C; Bown, R T; Poirot, P; Smith, D R M; Cartmell, C; Abou Fayad, A; Goss, R J M; Hel,holtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-03-01)
      The blending together of synthetic chemistry with natural product biosynthesis represents a potentially powerful approach to synthesis; to enable this, further synthetic tools and methodologies are needed. To this end, we have explored the first Sonogashira cross-coupling to halotryptophans in water. Broad reaction scope is demonstrated and we have explored the limits of the scope of the reaction. We have demonstrated this methodology to work excellently in the modification of model tripeptides. Furthermore, through precursor directed biosynthesis, we have generated for the first time a new to nature brominated natural product bromo-cystargamide, and demonstrated the applicability of our reaction conditions to modify this novel metabolite.
    • Squalenyl Hydrogen Sulfate Nanoparticles for Simultaneous Delivery of Tobramycin and an Alkylquinolone Quorum Sensing Inhibitor Enable the Eradication of P. aeruginosa Biofilm Infections.

      Ho, Duy-Khiet; Murgia, Xabier; de Rossi, Chiara; Christmann, Rebekka; Hüfner de Mello Martins, Antonio G; Koch, Marcus; Andreas, Anastasia; Herrmann, Jennifer; Müller, Rolf; Empting, Martin; et al. (Wiley, 2020-04-03)
      Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.
    • Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

      Olatunji, Samir; Yu, Xiaoxiao; Bailey, Jonathan; Huang, Chia-Ying; Zapotoczna, Marta; Bowen, Katherine; Remškar, Maja; Müller, Rolf; Scanlan, Eoin M; Geoghegan, Joan A; et al. (Nature publishing group, 2020-01-09)
      Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.
    • Supercritical Fluid Extraction Enhances Discovery of Secondary Metabolites from Myxobacteria.

      Bader, Chantal D; Neuber, Markus; Panter, Fabian; Krug, Daniel; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2020-11-10)
      Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study, we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites, aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains and the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite, we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed the best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents such as MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622, which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as the founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as a part of future screening workflows of microbial natural products.
    • Susceptibility of Different Mouse Wild Type Strains to Develop Diet-Induced NAFLD/AFLD-Associated Liver Disease.

      Fengler, Vera H I; Macheiner, Tanja; Kessler, Sonja M; Czepukojc, Beate; Gemperlein, Katja; Müller, Rolf; Kiemer, Alexandra K; Magnes, Christoph; Haybaeck, Johannes; Lackner, Carolin; et al. (2016)
      Although non-alcoholic and alcoholic fatty liver disease have been intensively studied, concerning pathophysiological mechanisms are still incompletely understood. This may be due to the use of different animal models and resulting model-associated variation. Therefore, this study aimed to compare three frequently used wild type mouse strains in their susceptibility to develop diet-induced features of non-alcoholic/alcoholic fatty liver disease. Fatty liver disease associated clinical, biochemical, and histological features in C57BL/6, CD-1, and 129Sv WT mice were induced by (i) high-fat diet feeding, (ii) ethanol feeding only, and (iii) the combination of high-fat diet and ethanol feeding. Hepatic and subcutaneous adipose lipid profiles were compared in CD-1 and 129Sv mice. Additionally hepatic fatty acid composition was determined in 129Sv mice. In C57BL/6 mice dietary regimens resulted in heterogeneous hepatic responses, ranging from pronounced steatosis and inflammation to a lack of any features of fatty liver disease. Liver-related serum biochemistry showed high deviations within the regimen groups. CD-1 mice did not exhibit significant changes in metabolic and liver markers and developed no significant steatosis or inflammation as a response to dietary regimens. Although 129Sv mice showed no weight gain, this strain achieved most consistent features of fatty liver disease, apparent from concentration alterations of liver-related serum biochemistry as well as moderate steatosis and inflammation as a result of all dietary regimens. Furthermore, the hepatic lipid profile as well as the fatty acid composition of 129Sv mice were considerably altered, upon feeding the different dietary regimens. Accordingly, diet-induced non-alcoholic/alcoholic fatty liver disease is most consistently promoted in 129Sv mice compared to C57BL/6 and CD-1 mice. As a conclusion, this study demonstrates the importance of genetic background of used mouse strains for modeling diet-induced non-alcoholic/alcoholic fatty liver disease.
    • Synthesis and biological activities of the respiratory chain inhibitor aurachin D and new ring versus chain analogues.

      Li, Xu-Wen; Herrmann, Jennifer; Zang, Yi; Grellier, Philippe; Prado, Soizic; Müller, Rolf; Nay, Bastien; Muséum National d'Histoire Naturelle, Unité Molécules de Communication et Adaptation des Micro-organismes (UMR 7245 CNRS-MNHN), 57 rue Cuvier (CP 54), 75005 Paris, France. (Beilstein-Institut Zur Forderung der Chemischen Wissenschaften, 2013)
      Aurachins are myxobacterial 3-farnesyl-4(1H)-quinolone derived compounds initially described as respiratory chain inhibitors, more specifically as inhibitors of various cytochrome complexes. They are also known as potent antibiotic compounds. We describe herein the first synthesis of aurachin D through a key Conrad-Limpach reaction. The same strategy was used to reach some ring as opposed to chain analogues, allowing for the description of structure-activity relationships. Biological screening of the analogues showed antiparasitic, cytotoxic, antibacterial and antifungal activities, and depletion of the mitochondrial membrane potential. The strongest activity was found on Plasmodium falciparum with a selectivity index of 345, compared to Vero cells, for the natural product and its geranyl analogue. The loss of mitochondrial membrane potential induced by aurachins in human U-2 OS osteosarcoma cells was studied, showing the best activity for aurachin D and a naphthalene analogue, yet without totally explaining the observed cytotoxic activity of the compounds. Finally, a synthetic entry is given to the complete carboheterocyclic core of aurachin H through the N-oxidation/epoxidation of aurachin D and a shorter chain analogue, followed by subsequent biomimetic cyclization.
    • Synthesis and Biological Evaluation of Modified Miuraenamides

      Kappler, Sarah; Karmann, Lisa; Prudel, Cynthia; Herrmann, Jennifer; Caddeu, Giulia; Müller, Rolf; Vollmar, Angelika M.; Zahler, Stefan; Kazmaier, Uli; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
    • Synthesis and Biological Evaluation of Novel 2-Substituted ­Analogues of (-)-Pentenomycin i

      Zisopoulou, Stavroula A.; Bousis, Spyridon; Haupenthal, Jörg; Herrmann, Jennifer; Müller, Rolf; Hirsch, Anna K.H.; Komiotis, Dimitri; Gallos, John K.; Stathakis, Christos I.; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Thieme, 2020-03-17)
      A library of novel 2-substituted derivatives of the antibiotic natural product pentenomycin I is presented. The new collection of analogues is divided in two main classes, 2-alkynyl- and 2-aryl- derivatives, which are accessed by the appropriate type of palladium-catalyzed cross-coupling reaction of the 2-iodo-protected pentenomycin I with suitable nucleophiles. The new derivatives were tested for their activity against certain types of bacteria and one of them, compound 8h, was found to exhibit significant inhibitory activity against several Gram-positive bacteria but also displayed cytotoxic activity against eukaryotic cell lines.
    • Synthetic and Biologic Studies on New Urea and Triazole Containing Cystobactamid Derivatives.

      Kirschning, Andreas; Planke, Therese; Cirnski, Katarina; Herrmann, Jennifer; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-Blackwell, 2019-12-13)
      he cystobactamids belong to the group of arene-based oligoamides that effectively inhibit bacterial type IIa topoisomerases. Cystobactamid 861-2 is the most active member of these antibiotics. Most amide bonds present in the cystobactamids link benzoic acids with anilines and it was found that some of these amide bonds undergo chemical and enzymatic hydrolysis, especially the one linking ring C with ring D. This work reports on the chemical synthesis and biological evaluation of thirteen new cystobactamids that still contain the methoxyaspartate hinge unit. However, we exchanged selected amide bonds either by the urea or the triazole groups and modified ring A in the latter case. While hydrolytic stability could be improved with these structural substitutes, the high antibacterial potency of cystobactamid 861-2 could only be preserved in selected cases. This includes derivatives, in which the urea group is positioned between rings A and B and where the triazole is found between rings C and D.
    • Synthetic biology approaches and combinatorial biosynthesis towards heterologous lipopeptide production.

      Yan, Fu; Burgard, Christian; Popoff, Alexander; Zaburannyi, Nestor; Zipf, Gregor; Maier, Josef; Bernauer, Hubert S; Wenzel, Silke C; Müller, Rolf; HIPS, Helmholtz-Institut füt Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2018-10-14)
      Synthetic biology techniques coupled with heterologous secondary metabolite production offer opportunities for the discovery and optimisation of natural products. Here we developed a new assembly strategy based on type IIS endonucleases and elaborate synthetic DNA platforms, which could be used to seamlessly assemble and engineer biosynthetic gene clusters (BGCs). By applying this versatile tool, we designed and assembled more than thirty different artificial myxochromide BGCs, each around 30 kb in size, and established heterologous expression platforms using a derivative of Myxococcus xanthus DK1622 as a host. In addition to the five native types of myxochromides (A, B, C, D and S), novel lipopeptide structures were produced by combinatorial exchange of nonribosomal peptide synthetase (NRPS) encoding genes from different myxochromide BGCs. Inspired by the evolutionary diversification of the native myxochromide megasynthetases, the ancestral A-type NRPS was engineered by inactivation, deletion, or duplication of catalytic domains and successfully converted into functional B-, C- and D-type megasynthetases. The constructional design approach applied in this study enables combinatorial engineering of complex synthetic BGCs and has great potential for the exploitation of other natural product biosynthetic pathways.
    • Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high: In vivo efficacy

      Testolin, Giambattista; Cirnski, Katarina; Rox, Katharina; Prochnow, Hans; Fetz, Verena; Grandclaudon, Charlotte; Mollner, Tim; Baiyoumy, Alain; Ritter, Antje; Leitner, Christian; et al. (RSC, 2020-01-01)
      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 para-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 Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL−1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL−1). 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 in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 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.
    • Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

      Pfitzer, Lisa; Moser, Christina; Gegenfurtner, Florian; Arner, Anja; Foerster, Florian; Atzberger, Carina; Zisis, Themistoklis; Kubisch-Dohmen, Rebekka; Busse, Johanna; Smith, Rebecca; et al. (Springer-Nature, 2019-04-03)
      Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.
    • Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

      Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; et al. (2014)
      Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCɛ signaling shown in a xenograft breast cancer model.
    • Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum.

      Scherer, Olga; Steinmetz, Heinrich; Kaether, Christoph; Weinigel, Christina; Barz, Dagmar; Kleinert, Hartmut; Menche, Dirk; Müller, Rolf; Pergola, Carlo; Werz, Oliver; et al. (2014-10-15)
      The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.