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group leader: Prof. Brönstrup

Recent Submissions

  • SAR Studies of the Leupyrrins: Design and Total Synthesis of Highly Potent Simplified Leupylogs.

    Wosniok, Paul R; Knopf, Christopher; Dreisigacker, Sandra; Orozco-Rodriguez, J Manuel; Hinkelmann, Bettina; Mueller, Peter P; Brönstrup, Mark; Menche, Dirk; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-11-11)
    Invited for the cover of this issue is the group of Dirk Menche at the University of Bonn. The image depicts the natural product leupyrrin A1 and a synthetic leupylog in balance on an IC50 weighing scale. Read the full text of the article at 10.1002/chem.202002622.
  • The Peptide Chain Release Factor Methyltransferase PrmC Influences the PA14 Endo- and Exometabolome.

    Depke, Tobias; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-18)
    Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain.
  • Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

    Rahim, Muhammad Imran; Winkel, Andreas; Lienenklaus, Stefan; Stumpp, Nico S; Szafrański, Szymon P; Kommerein, Nadine; Willbold, Elmar; Reifenrath, Janin; Mueller, Peter P; Eisenburger, Michael; et al. (MDPI, 2020-10-21)
    The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-β) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-β expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-β expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.
  • 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.
  • Host-induced spermidine production in motile triggers phagocytic uptake.

    Felgner, Sebastian; Preusse, Matthias; Beutling, Ulrike; Stahnke, Stephanie; Pawar, Vinay; Rohde, Manfred; Brönstrup, Mark; Stradal, Theresia; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (elifeSciences, 2020-09-22)
    Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.
  • Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

    Tegtmeyer, Nicole; Neddermann, Matthias; Lind, Judith; Pachathundikandi, Suneesh Kumar; Sharafutdinov, Irshad; Gutiérrez-Escobar, Andrés Julián; Brönstrup, Mark; Tegge, Werner; Hong, Minsun; Rohde, Manfred; et al. (Cell Press, 2020-11-15)
    Helicobacter pylori (Hp) is an important human pathogen associated with gastric inflammation and neoplasia. It is commonly believed that this bacterium avoids major immune recognition by Toll-like receptors (TLRs) because of low intrinsic activity of its flagellin and lipopolysaccharides (LPS). In particular, TLR5 specifically detects flagellins in various bacterial pathogens, while Hp evolved mutations in flagellin to evade detection through TLR5. Cancerogenic Hp strains encode a type IV secretion system (T4SS). The T4SS core component and pilus-associated protein CagY, a large VirB10 ortholog, drives effector molecule translocation. Here, we identify CagY as a flagellin-independent TLR5 agonist. We detect five TLR5 interaction sites, promoting binding of CagY-positive Hp to TLR5-expressing cells, TLR5 stimulation, and intracellular signal transduction. Consequently, CagY constitutes a remarkable VirB10 member detected by TLR5, driving crucial innate immune responses by this human pathogen.
  • Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa

    Depke, Tobias; Thöming, Janne Gesine; Kordes, Adrian; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-13)
    Pseudomonas aeruginosa is a facultative pathogen that can cause, inter alia, acute or chronic pneumonia in predisposed individuals. The gram-negative bacterium displays considerable genomic and phenotypic diversity that is also shaped by small molecule secondary metabolites. The discrimination of virulence phenotypes is highly relevant to the diagnosis and prognosis of P. aeruginosa infections. In order to discover small molecule metabolites that distinguish different virulence phenotypes of P. aeruginosa, 35 clinical strains were cultivated under standard conditions, characterized in terms of virulence and biofilm phenotype, and their metabolomes were investigated by untargeted liquid chromatography-mass spectrometry. The data was both mined for individual candidate markers as well as used to construct statistical models to infer the virulence phenotype from metabolomics data. We found that clinical strains that differed in their virulence and biofilm phenotype also had pronounced divergence in their metabolomes, as underlined by 332 features that were significantly differentially abundant with fold changes greater than 1.5 in both directions. Important virulence-associated secondary metabolites like rhamnolipids, alkyl quinolones or phenazines were found to be strongly upregulated in virulent strains. In contrast, we observed little change in primary metabolism. A hitherto novel cationic metabolite with a sum formula of C12H15N2 could be identified as a candidate biomarker. A random forest model was able to classify strains according to their virulence and biofilm phenotype with an area under the Receiver Operation Characteristics curve of 0.84. These findings demonstrate that untargeted metabolomics is a valuable tool to characterize P. aeruginosa virulence, and to explore interrelations between clinically important phenotypic traits and the bacterial metabolome.
  • Filovirus antiviral activity of cationic amphiphilic drugs is associated with lipophilicity and ability to induce phospholipidosis.

    Gunesch, Antonia P; Zapatero-Belinchon, Francisco J; Pinkert, Lukas; Steinmann, Eike; Manns, Michael P; Schneider, Gisbert; Pietschmann, Thomas; Brönstrup, Mark; von Hahn, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2020-06-08)
    Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood.We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition.We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analysed correlation of antiviral activity with four chemical properties: pKa, ClogP, molecular weight and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structure (analogues) to strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall 11 out of 45 (24 %) CADs inhibited MARVpp by 40 % or more. The strongest antiviral compounds were dronedarone, triparanol and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP>4), and DIPL. Moreover, pKa and intra-molecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity, but to a lesser extent. We also showed that in contrast to analogues, derivatives had similar antiviral activity as the seed compound dronedarone. Overall, one quarter of CADs inhibits MARVpp entry in vitro and antiviral activity of CADs mostly relies on their hydrophobicity, yet is promoted by the individual structure.
  • Expansion of functional personalized cells with specific transgene combinations.

    Lipps, Christoph; Klein, Franziska; Wahlicht, Tom; Seiffert, Virginia; Butueva, Milada; Zauers, Jeannette; Truschel, Theresa; Luckner, Martin; Köster, Mario; MacLeod, Roderick; et al. (Springer Nature, 2018-03-08)
    Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
  • Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.

    Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (AAAS, 2020-03-20)
    The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
  • Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

    Blockus, Sebastian; Sake, Svenja M; Wetzke, Martin; Grethe, Christina; Graalmann, Theresa; Pils, Marina; Le Goffic, Ronan; Galloux, Marie; Prochnow, Hans; Rox, Katharina; et al. (Elsevier, 2020-03-18)
    Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
  • 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.
  • 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.
  • T4SS-dependent TLR5 activation by Helicobacter pylori infection.

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Arnold, Isabelle Catherine; Lind, Judith; Neddermann, Matthias; Falkeis-Veits, Christina; Chattopadhyay, Sujay; Brönstrup, Mark; Tegge, Werner; Hong, Minsun; et al. (Nature publishing group, 2019-12-16)
    Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.
  • 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.
  • The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates.

    Klahn, Philipp; Fetz, Verena; Ritter, Antje; Collisi, Wera; Hinkelmann, Bettina; Arnold, Tatjana; Tegge, Werner; Rox, Katharina; Hüttel, Stephan; Mohr, Kathrin I; et al. (Royal Society of Chemistry, 2019-05-28)
    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 (e.g. 16R-aminoratjadone 16 with IC50 = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC50 = 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 FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.
  • Evidence for inoculum size and gas interfaces as critical factors in bacterial biofilm formation on magnesium implants in an animal model.

    Rahim, Muhammad Imran; Szafrański, Szymon P; Ingendoh-Tsakmakidis, Alexandra; Stiesch, Meike; Mueller, Peter P; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-11-28)
    Infections of medical implants caused by bacterial biofilms are a major clinical problem. Bacterial colonization is predicted to be prevented by alkaline magnesium surfaces. However, in experimental animal studies, magnesium implants prolonged infections. The reason for this peculiarity likely lies within the ‒still largely hypothetical‒ mechanism by which infection arises. Investigating subcutaneous magnesium implants infected with bioluminescent Pseudomonas aeruginosa via in vivo imaging, we found that the rate of implant infections was critically dependent on a surprisingly high quantity of injected bacteria. At high inocula, bacteria were antibiotic-refractory immediately after infection. High cell densities are known to limit nutrient availability, restricting proliferation and trigger quorum sensing which could both contribute to the rapid initial resistance. We propose that gas bubbles such as those formed during magnesium corrosion, can then act as interfaces that support biofilm formation and permit long-term survival. This model could provide an explanation for the apparent ineffectiveness of innovative contact-dependent bactericidal implant surfaces in patients. In addition, the model points toward air bubbles in tissue, either by inclusion during surgery or by spontaneous gas bubble formation later on, could constitute a key risk factor for clinical implant infections
  • Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

    Prochnow, Hans; Rox, Katharina; Birudukota, N V Suryanarayana; Weichert, Loreen; Hotop, Sven-Kevin; Klahn, Philipp; Mohr, Kathrin; Franz, Sergej; Banda, Dominic H; Blockus, Sebastian; et al. (ASM, 2019-10-30)
    To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.
  • 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.
  • Hydantoin analogs inhibit the fully assembled ClpXP protease without affecting the individual peptidase and chaperone domains.

    Fetzer, Christian; Korotkov, Vadim S; Sieber, Stephan A; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Royal Society of Chemistry, 2019-08-14)
    Proteolysis mediated by ClpXP is a crucial cellular process linked to bacterial pathogenesis. The development of specific inhibitors has largely focused on ClpP. However, this focus was challenged by a recent finding showing that conformational control by ClpX leads to a rejection of ClpP binders. Thus, we here follow up on a hit molecule from a high throughput screen performed against the whole ClpXP complex and demonstrate that stable inhibition with high potency is possible. Further investigations revealed that the small molecule binds to ClpP without affecting its activity. Likewise, the molecule does not inhibit ClpX and retains the overall oligomeric state of ClpXP upon binding. Structure activity relationship studies confirmed structural constraints in all three parts of the molecule suggesting binding into a defined stereospecific pocket. Overall, the inhibition of ClpXP without affecting the individual components represents a novel mechanism with perspectives for further optimization for in situ applications.

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