• 2-Hydroxysorangiadenosine: Structure and Biosynthesis of a Myxobacterial Sesquiterpene-Nucleoside.

      Okoth, Dorothy A; Hug, Joachim J; Garcia, Ronald; Spröer, Cathrin; Overmann, Jörg; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-06-09)
      Myxobacteria represent an under-investigated source for biologically active natural products featuring intriguing structural moieties with potential applications, e.g., in the pharmaceutical industry. Sorangiadenosine and the here-discovered 2-hydroxysorangiadenosine are myxobacterial sesquiterpene-nucleosides with an unusual structural moiety, a bicyclic eudesmane-type sesquiterpene. As the biosynthesis of these rare terpene-nucleoside hybrid natural products remains elusive, we investigated secondary metabolomes and genomes of several 2-hydroxysorangiadenosine-producing myxobacteria. We report the isolation and full structure elucidation of 2-hydroxysorangiadenosine and its cytotoxic and antibiotic activities and propose a biosynthetic pathway in the myxobacterium Vitiosangium cumulatum MCy10943T.
    • Thioholgamide A, a New Anti-Proliferative Anti-Tumor Agent, Modulates Macrophage Polarization and Metabolism.

      Dahlem, Charlotte; Siow, Wei Xiong; Lopatniuk, Maria; Tse, William K F; Kessler, Sonja M; Kirsch, Susanne H; Hoppstädter, Jessica; Vollmar, Angelika M; Müller, Rolf; Luzhetskyy, Andriy; et al. (MDPI, 2020-05-19)
      Natural products represent powerful tools searching for novel anticancer drugs. Thioholgamide A (thioA) is a ribosomally synthesized and post-translationally modified peptide, which has been identified as a product of Streptomyces sp. MUSC 136T. In this study, we provide a comprehensive biological profile of thioA, elucidating its effects on different hallmarks of cancer in tumor cells as well as in macrophages as crucial players of the tumor microenvironment. In 2D and 3D in vitro cell culture models thioA showed potent anti-proliferative activities in cancer cells at nanomolar concentrations. Anti-proliferative actions were confirmed in vivo in zebrafish embryos. Cytotoxicity was only induced at several-fold higher concentrations, as assessed by live-cell microscopy and biochemical analyses. ThioA exhibited a potent modulation of cell metabolism by inhibiting oxidative phosphorylation, as determined in a live-cell metabolic assay platform. The metabolic modulation caused a repolarization of in vitro differentiated and polarized tumor-promoting human monocyte-derived macrophages: ThioA-treated macrophages showed an altered morphology and a modulated expression of genes and surface markers. Taken together, the metabolic regulator thioA revealed low activities in non-tumorigenic cells and an interesting anti-cancer profile by orchestrating different hallmarks of cancer, both in tumor cells as well as in macrophages as part of the tumor microenvironment.
    • Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine.

      Gampfer, Tanja M; Wagmann, Lea; Park, Yu Mi; Cannaert, Annelies; Herrmann, Jennifer; Fischmann, Svenja; Westphal, Folker; Müller, Rolf; Stove, Christophe P; Meyer, Markus R; et al. (Springer Nature, 2020-04-05)
      The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. N-Dealkylation, N-deacylation, hydroxylation, and N-oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded
    • 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.
    • 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.
    • The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

      Hoppstädter, Jessica; Valbuena Perez, Jenny Vanessa; Linnenberger, Rebecca; Dahlem, Charlotte; Legroux, Thierry M; Hecksteden, Anne; Tse, William K F; Flamini, Sara; Andreas, Anastasia; Herrmann, Jennifer; et al. (Wiley, 2020-02-06)
      Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.
    • Myxobakterielle Naturstofffabriken

      Krug, Daniel; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2020-02-01)
      Myxococcus xanthus is a prime example of soil-living myxobacteria featuring a complex lifestyle, including coordinated movement through swarming, predatory feeding on other microorganisms, and the formation of multicellular fruiting bodies. Due to its biosynthetic capabilities for secondary metabolite production and its applicability as biotechno-logical chassis organism for heterologous expression, Myxococcus stands out as a biochemical factory for bioactive molecules with future applications, not only in human therap
    • Cystobactamid 507: Concise Synthesis, Mode of Action and Optimization toward More Potent Antibiotics.

      Elgaher, Walid A M; Hamed, Mostafa M; Baumann, Sascha; Herrmann, Jennifer; Siebenbürger, Lorenz; Krull, Jana; Cirnski, Katarina; Kirschning, Andreas; Brönstrup, Mark; Müller, Rolf; et al. (Wiley-VCH, 2020-01-26)
      Lack of new antibiotics and increasing antimicrobial resistance are the main concerns of healthcare community nowadays, which necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids - a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe a concise total synthesis of cystobactamid 507, the identification of the bioactive conformation using non-covalently bonded rigid analogs, the first structure–activity relationship (SAR) study for cystobactamid 507 leading to new analogs with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor groove binding as part of the drug–target interaction without showing significant intercalation. By designing a new analog of cystobactamid 919-2 we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.
    • Discovery of Novel Latency-Associated Nuclear Antigen Inhibitors as Antiviral Agents Against Kaposi's Sarcoma-Associated Herpesvirus.

      Kirsch, Philine; Jakob, Valentin; Elgaher, Walid A M; Walt, Christine; Oberhausen, Kevin; Schulz, Thomas F; Empting, Martin; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.;HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Chemical Society (ACS), 2020-01-24)
      With the aim to develop novel antiviral agents against Kaposi's Sarcoma Herpesvirus (KSHV), we are targeting the latency-associated nuclear antigen (LANA). This protein plays an important role in viral genome maintenance during latent infection. LANA has the ability to tether the viral genome to the host nucleosomes and, thus, ensures latent persistence of the viral genome in the host cells. By inhibition of the LANA-DNA interaction, we seek to eliminate or reduce the load of the viral DNA in the host. To achieve this goal, we screened our in-house library using a dedicated fluorescence polarization (FP)-based competition assay, which allows for the quantification of LANA-DNA-interaction inhibition by small organic molecules. We successfully identified three different compound classes capable of disrupting this protein-nucleic acid interaction. We characterized these compounds by IC50 dose-response evaluation and confirmed the compound-LANA interaction using surface plasmon resonance (SPR) spectroscopy. Furthermore, two of the three hit scaffolds showed only marginal cytotoxicity in two human cell lines. Finally, we conducted STD-NMR competition experiments with our new hit compounds and a previously described fragment-sized inhibitor. Based on these results, future compound linking approaches could serve as a promising strategy for further optimization studies in order to generate highly potent KSHV inhibitors.
    • The Cytotoxic Natural Product Vioprolide A Targets Nucleolar Protein 14, Which Is Essential for Ribosome Biogenesis.

      Kirsch, Volker C; Orgler, Christina; Braig, Simone; Jeremias, Irmela; Auerbach, David; Müller, Rolf; Vollmar, Angelika M; Sieber, Stephan A; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2020-01-20)
      Novel targets are needed for treatment of devastating diseases such as cancer. For decades, natural products have guided innovative therapies by addressing diverse pathways. Inspired by the potent cytotoxic bioactivity of myxobacterial vioprolides A-D, we performed in-depth studies on their mode of action. Based on its prominent potency against human acute lymphoblastic leukemia (ALL) cells, we conducted thermal proteome profiling (TPP) and deciphered the target proteins of the most active derivative vioprolide A (VioA) in Jurkat cells. Nucleolar protein 14 (NOP14), which is essential in ribosome biogenesis, was confirmed as a specific target of VioA by a suite of proteomic and biological follow-up experiments. Given its activity against ALL cells compared to healthy lymphocytes, VioA exhibits unique therapeutic potential for anticancer therapy through a novel mode of action
    • Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections.

      Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K; et al. (MDPI, 2020-01-12)
      In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
    • 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.
    • 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.
    • 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.
    • 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.
    • PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus.

      Anversa Dimer, Frantiescoli; de Souza Carvalho-Wodarz, Cristiane; Goes, Adriely; Cirnski, Katarina; Herrmann, Jennifer; Schmitt, Viktoria; Pätzold, Linda; Abed, Nadia; de Rossi, Chiara; Bischoff, Markus; et al. (Elsevier, 2019-11-18)
      Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.
    • Homologous bd oxidases share the same architecture but differ in mechanism.

      Theßeling, Alexander; Rasmussen, Tim; Burschel, Sabrina; Wohlwend, Daniel; Kägi, Jan; Müller, Rolf; Böttcher, Bettina; Friedrich, Thorsten; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Publication group, 2019-11-13)
      Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
    • The mRNA-binding Protein TTP/ZFP36 in Hepatocarcinogenesis and Hepatocellular Carcinoma.

      Kröhler, Tarek; Kessler, Sonja M; Hosseini, Kevan; List, Markus; Barghash, Ahmad; Patial, Sonika; Laggai, Stephan; Gemperlein, Katja; Haybaeck, Johannes; Müller, Rolf; et al. (MDPI, 2019-11-08)
      Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression.
    • Differential regulation of AMP-activated protein kinase in healthy and cancer cells explains why V-ATPase inhibition selectively kills cancer cells.

      Bartel, Karin; Müller, Rolf; von Schwarzenberg, Karin; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Society for biochemistry and Molecular Biology, 2019-10-11)
      The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here, we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and non-tumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In non-malignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a pro-survival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase-independent AMPK induction in the tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a pro-survival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species (ROS) production. We could attribute the pro-survival effects to AMPK's ability to maintain redox homeostasis by inhibiting ROS production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.
    • Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases.

      Gemperlein, Katja; Dietrich, Demian; Kohlstedt, Michael; Zipf, Gregor; Bernauer, Hubert S; Wittmann, Christoph; Wenzel, Silke C; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature publishing Group, 2019-09-06)
      Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.