• 14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall.

      Voigt, Jürgen; Frank, Ronald; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2003-06)
      The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists predominantly of Hyp-rich glycoproteins, which also occur in the extracellular matrix of multicellular green algae and higher plants. In addition to the Hyp-rich polypeptides, the insoluble glycoprotein framework of the Chlamydomonas cell wall contains minor amounts of 14-3-3 proteins, as revealed by immunochemical studies and mass spectroscopic analysis of tryptic peptides. Polypeptides immunologically related to the 14-3-3 proteins also were found in the culture medium of Chlamydomonas. The levels of two of these 14-3-3-related polypeptides were decreased in the culture medium of the wall-deficient mutant cw-15. These findings indicate that 14-3-3 proteins are involved in the cross-linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall.
    • Advances and Challenges of Biodegradable Implant Materials with a Focus on Magnesium-Alloys and Bacterial Infections

      Rahim, Muhammad; Ullah, Sami; Mueller, Peter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MPDI, 2018-07-10)
      Medical implants made of biodegradable materials could be advantageous for temporary applications, such as mechanical support during bone-healing or as vascular stents to keep blood vessels open. After completion of the healing process, the implant would disappear, avoiding long-term side effects or the need for surgical removal. Various corrodible metal alloys based on magnesium, iron or zinc have been proposed as sturdier and potentially less inflammatory alternatives to degradable organic polymers, in particular for load-bearing applications. Despite the recent introduction of magnesium-based screws, the remaining hurdles to routine clinical applications are still challenging. These include limitations such as mechanical material characteristics or unsuitable corrosion characteristics. In this article, the salient features and clinical prospects of currently-investigated biodegradable implant materials are summarized, with a main focus on magnesium alloys. A mechanism of action for the stimulation of bone growth due to the exertion of mechanical force by magnesium corrosion products is discussed. To explain divergent in vitro and in vivo effects of magnesium, a novel model for bacterial biofilm infections is proposed which predicts crucial consequences for antibacterial implant strategies.
    • Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study.

      Westermann, J; Flörcken, A; Willimsky, G; van Lessen, A; Kopp, J; Takvorian, A; Jöhrens, K; Lukowsky, A; Schönemann, C; Sawitzki, B; et al. (2011-04)
      Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.
    • Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

      Dalby, Andrew R; Emam, Ibrahim; Franke, Raimo; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2012)
      Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC) can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.
    • Anti-biofilm Agents against Pseudomonas aeruginosa: A Structure-Activity Relationship Study of C-Glycosidic LecB Inhibitors

      Sommer, Roman; Rox, Katharina; Wagner, Stefanie; Hauck, Dirk; Henrikus, Sarah S; Newsad, Shelby; Arnold, Tatjana; Ryckmans, Thomas; Brönstrup, Mark; Imberty, Anne; et al. (American Chemical Society, 2019-10-24)
      Biofilm formation is a key mechanism of antimicrobial resistance. We have recently reported two classes of orally bioavailable C-glycosidic inhibitors of the Pseudomonas aeruginosa lectin LecB with antibiofilm activity. They proved efficient in target binding, were metabolically stable, nontoxic, selective, and potent in inhibiting formation of bacterial biofilm. Here, we designed and synthesized six new carboxamides and 24 new sulfonamides for a detailed structure-activity relationship for two clinically representative LecB variants. Sulfonamides generally showed higher inhibition compared to carboxamides, which was rationalized based on crystal structure analyses. Substitutions at the thiophenesulfonamide increased binding through extensive contacts with a lipophilic protein patch. These metabolically stable compounds showed a further increase in potency toward the target and in biofilm inhibition assays. In general, we established the structure-activity relationship for these promising antibiofilm agents and showed that modification of the sulfonamide residue bears future optimization potential.

    • Antiviral drug discovery: broad-spectrum drugs from nature.

      Martinez, J P; Sasse, F; Brönstrup, M; Diez, J; Meyerhans, A; Helmholtz Centre for infection research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-01)
      Covering: up to April 2014. The development of drugs with broad-spectrum antiviral activities is a long pursued goal in drug discovery. It has been shown that blocking co-opted host-factors abrogates the replication of many viruses, yet the development of such host-targeting drugs has been met with scepticism mainly due to toxicity issues and poor translation to in vivo models. With the advent of new and more powerful screening assays and prediction tools, the idea of a drug that can efficiently treat a wide range of viral infections by blocking specific host functions has re-bloomed. Here we critically review the state-of-the-art in broad-spectrum antiviral drug discovery. We discuss putative targets and treatment strategies, with particular focus on natural products as promising starting points for antiviral lead development.
    • Archazolid A-15-O-β-D-glucopyranoside and iso-archazolid B: potent V-ATPase inhibitory polyketides from the myxobacteria Cystobacter violaceus and Archangium gephyra.

      Horstmann, Nicole; Essig, Sebastian; Bockelmann, Svenja; Wieczorek, Helmut; Huss, Markus; Sasse, Florenz; Menche, Dirk; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2011-05-27)
      Two structurally novel analogues of the macrolides archazolids A and B, archazolid A-15-O-β-D-glucopyranoside (archazolid E, 5) and iso-archazolid B (archazolid F, 6), were isolated from the myxobacterium Cystobacter violaceus and Archangium gephyra, respectively. Macrolactone 5 represents the first 15-O-glycoside of the archazolids. iso-Archazolid B (6) incorporates a C-3 alkene and presents the first constitutional isomer reported for this natural product class. The structures of these polyketides were determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations and by chemical degradation. iso-Archazolid B (6) demonstrated extremely high antiproliferative and V-ATPase inhibitory effects, with IC(50) values in the picomolar range, while only moderate activity was observed for glycoside 5. iso-Archazolid B presents the most potent archazolid known.
    • Archazolid and apicularen: novel specific V-ATPase inhibitors.

      Huss, Markus; Sasse, Florenz; Kunze, Brigitte; Jansen, Rolf; Steinmetz, Heinrich; Ingenhorst, Gudrun; Zeeck, Axel; Wieczorek, Helmut; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2005-08-04)
      V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.
    • An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

      Overwin, Heike; González, Myriam; Méndez, Valentina; Seeger, Michael; Wray, Victor; Hofer, Bernd; Hel,holtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-09)
      The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.
    • Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.

      Spadaccini, Roberta; Reidt, Ulrich; Dybkov, Olexandr; Will, Cindy; Frank, Ronald; Stier, Gunter; Corsini, Lorenzo; Wahl, Markus C; Lührmann, Reinhard; Sattler, Michael; et al. (2006-03)
      The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.
    • Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.

      Lukat, Peer; Katsuyama, Yohei; Wenzel, Silke; Binz, Tina; König, Claudia; Blankenfeldt, Wulf; Brönstrup, Mark; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-11-01)
      Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
    • Characteristics, chemical compositions and biological activities of propolis from Al-Bahah, Saudi Arabia.

      Elnakady, Yasser A; Rushdi, Ahmed I; Franke, Raimo; Abutaha, Nael; Ebaid, Hossam; Baabbad, Mohannad; Omar, Mohamed O M; Al Ghamdi, Ahmad A; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-06)
      Propolis has been used to treat several diseases since ancient times, and is an important source of bioactive natural compounds and drug derivatives. These properties have kept the interest of investigators around the world, leading to the investigation of the chemical and biological properties and application of propolis. In this report, the chemical constituents that are responsible for the anticancer activities of propolis were analyzed. The propolis was sourced from Al-Baha in the southern part of the Kingdom of Saudi Arabia. Standard protocols for chemical fractionation and bioactivity-guided chemical analysis were used to identify the bio-active ethyl acetate fraction. The extraction was performed in methanol and then analyzed by gas chromatography-mass spectrometry (GC-MS). The major compounds are triterpenoids, with a relative concentration of 74.0%; steroids, with a relative concentration of 9.8%; and diterpenoids, with a relative concentration of 7.9%. The biological activity was characterized using different approaches and cell-based assays. Propolis was found to inhibit the proliferation of cancer cells in a concentration-dependent manner through apoptosis. Immunofluorescence staining with anti-α-tubulin antibodies and cell cycle analysis indicated that tubulin and/or microtubules are the cellular targets of the L-acetate fraction. This study demonstrates the importance of Saudi propolis as anti-cancer drug candidates.

    • The CLU-files: disentanglement of a mystery.

      Rohne, Philipp; Prochnow, Hans; Koch-Brandt, Claudia; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunshweig, Germany. (2016-02)
      The multifaceted protein clusterin (CLU) has been challenging researchers for more than 35 years. The characterization of CLU as a molecular chaperone was one of the major breakthroughs in CLU research. Today, secretory clusterin (sCLU), also known as apolipoprotein J (apoJ), is considered one of the most important extracellular chaperones ever found. It is involved in a broad range of physiological and pathophysiological functions, where it exerts a cytoprotective role. Descriptions of various forms of intracellular CLU have led to further and even contradictory functions. To untangle the current state of knowledge of CLU, this review will combine old views in the field, with new discoveries to highlight the nature and function of this fascinating protein(s). In this review, we further describe the expression and subcellular location of various CLU forms. Moreover, we discuss recent insights into the structure of CLU and assess how structural properties as well as the redox environment determine the chaperone activity of CLU. Eventually, the review connects the biochemistry and molecular cell biology of CLU with medical aspects, to formulate a hypothesis of a CLU function in health and disease.
    • Coprinuslactone protects the edible mushroom Coprinus comatus against biofilm infections by blocking both quorum-sensing and MurA.

      de Carvalho, Maira P; Gulotta, Giuseppe; do Amaral, Matheus W; Lünsdorf, Heinrich; Sasse, Florenz; Abraham, Wolf-Rainer; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-10-03)
      Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared to planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria on their sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3,4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin and rhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications. This article is protected by copyright. All rights reserved.


    • Cytotoxic and antivascular 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5-(halophenyl)-imidazoles.

      Biersack, Bernhard; Muthukumar, Yazh; Schobert, Rainer; Sasse, Florenz (2011-11-01)
      A series of 1-methyl-4,5-diphenylimidazoles 6 with various patterns of m-halogen substitution at the 5-phenyl ring were tested for cytotoxicity in cancer and nonmalignant cell lines and for their capacity to prevent tube formation in HUVEC cultures. Unlike the monofluoro and difluoro derivatives 6a and 6e, the monobromo and diiodo analogs 6c and 6h were strongly cytotoxic and inhibited the polymerization of tubulin and the tube formation by HUVEC. The dibromo derivative 6g displayed a unique selectivity for KB-3-1 cervix and PC-3 prostate cancer cells. It also inhibited the tube formation by HUVEC and the polymerization of tubulin which is indicative of its potential antiangiogenic activity in solid tumors.
    • Detection and Investigation of Eagle Effect Resistance to Vancomycin in With an ATP-Bioluminescence Assay.

      Jarrad, Angie M; Blaskovich, Mark A T; Prasetyoputri, Anggia; Karoli, Tomislav; Hansford, Karl A; Cooper, Matthew A; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Vancomycin was bactericidal against Clostridium difficile at eightfold the minimum inhibitory concentration (MIC) using a traditional minimum bactericidal concentration (MBC) assay. However, at higher concentrations up to 64 × MIC, vancomycin displayed a paradoxical “more-drug-kills-less” Eagle effect against C. difficile. To overcome challenges associated with performing the labor-intensive agar-based MBC method under anaerobic growth conditions, we investigated an alternative more convenient ATP-bioluminescence assay to assess the Eagle effect in C. difficile. The commercial BacTiter-GloTM assay is a homogenous method to determine bacterial viability based on quantification of bacterial ATP as a marker for metabolic activity. The ATP-bioluminescence assay was advantageous over the traditional MBC-type assay in detecting the Eagle effect because it reduced assay time and was simple to perform; measurement of viability could be performed in less than 10 min outside of the anaerobic chamber. Using this method, we found C. difficile survived clinically relevant, high concentrations of vancomycin (up to 2048 μg/mL). In contrast, C. difficile did not survive high concentrations of metronidazole or fidaxomicin. The Eagle effect was also detected for telavancin, but not for teicoplanin, dalbavancin, oritavancin, or ramoplanin. All four pathogenic strains of C. difficile tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in C. difficile could be more prevalent than previously appreciated, with potential clinical implications. The ATP-Bioluminescence assay can thus be used as an alternative to the agar-based MBC assay to characterize the Eagle effect against a variety of antibiotics, at a wide-range of concentrations, with much greater throughput. This may facilitate improved understanding of Eagle effect resistance and promote further research to understand potential clinical relevance.