• 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.
    • Cinnamide Derivatives of d -Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

      Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Wagner, Stefanie; Audfray, Aymeric; Prestel, Andreas; Möller, Heiko M.; Imberty, Anne; Titz, Alexander; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany.; et al. (2015-12)
    • Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging.

      Wagner, Stefanie; Hauck, Dirk; Hoffmann, Michael; Sommer, Roman; Joachim, Ines; Müller, Rolf; Imberty, Anne; Varrot, Annabelle; Titz, Alexander; HIPS, Helmholtz-Institut für pharmazeutische Forchung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-09-28)
      Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing "pathoblockers" preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.
    • Development of a competitive binding assay for the Burkholderia cenocepacia lectin BC2L-A and structure activity relationship of natural and synthetic inhibitors

      Beshr, Ghamdan; Sommer, Roman; Hauck, Dirk; Siebert, David Chan Bodin; Hofmann, Anna; Imberty, Anne; Titz, Alexander (2016)
    • Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas aeruginosa .

      Meiers, Joscha; Zahorska, Eva; Röhrig, Teresa; Hauck, Dirk; Wagner, Stefanie; Titz, Alexander; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (ACS, 2020-10-02)
      Chronic infections by Pseudomonas aeruginosa are characterized by biofilm formation, which effectively enhances resistance toward antibiotics. Biofilm-specific antibiotic delivery could locally increase drug concentration to break antimicrobial resistance and reduce the drug's peripheral side effects. Two extracellular P. aeruginosa lectins, LecA and LecB, are essential structural components for biofilm formation and thus render a possible anchor for biofilm-targeted drug delivery. The standard-of-care drug ciprofloxacin suffers from severe systemic side effects and was therefore chosen for this approach. We synthesized several ciprofloxacin-carbohydrate conjugates and established a structure-activity relationship. Conjugation of ciprofloxacin to lectin probes enabled biofilm accumulation in vitro, reduced the antibiotic's cytotoxicity, but also reduced its antibiotic activity against planktonic cells due to a reduced cell permeability and on target activity. This work defines the starting point for new biofilm/lectin-targeted drugs to modulate antibiotic properties and ultimately break antimicrobial resistance.
    • Efficient Two Step β-Glycoside Synthesis from -Acetyl -Glucosamine: Scope and Limitations of Copper(II) Triflate-Catalyzed Glycosylation

      Sommer, Roman; Hauck, Dirk; Titz, Alexander; HIPS, Helmholtz-Institute für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.
      β‐Linked glycosides of N‐acetyl glucosamine are widespread in nature. Their direct synthesis is hampered by the low reactivity of GlcNAc as a glycosyl donor. We report a selective and rapid copper(II) triflate‐catalyzed two‐step synthesis of β‐glycosides of GlcNAc from cheap GlcNAc as starting material without purification of intermediates. α‐Specific glycosylation can be achieved by increasing the amount of catalyst and extending reaction times.
    • Methylated glycans as conserved targets of animal and fungal innate defense.

      Wohlschlager, Therese; Butschi, Alex; Grassi, Paola; Sutov, Grigorij; Gauss, Robert; Hauck, Dirk; Schmieder, Stefanie S; Knobel, Martin; Titz, Alexander; Dell, Anne; et al. (2014-07-08)
      Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.
    • O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

      Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016)
      Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.
    • Photorhabdus luminescens lectin A (PllA) - a new probe for detecting α-galactoside-terminating glycoconjugates.

      Beshr, Ghamdan; Sikandar, Asfandyar; Jemiller, Eva-Maria; Klymiuk, Nikolai; Hauck, Dirk; Wagner, Stefanie; Wolf, Eckhard; Koehnke, Jesko; Titz, Alexander; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Universitycampus E8.1, 66123 Saarbrücken, Germany. (2017-09-28)
      Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096 coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for alpha-galactoside-terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90 degree twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting alpha-galactosides. The alpha-Gal epitope is present on wild-type pig cells and the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllAs high specificity for alpha-galactoside-containing ligands, and we show that PllA can be used to visualize alpha-Gal epitope on porcine tissues.
    • Synthesis of mannoheptose derivatives and their evaluation as inhibitors of the lectin LecB from the opportunistic pathogen Pseudomonas aeruginosa.

      Hofmann, Anna; Sommer, Roman; Hauck, Dirk; Stifel, Julia; Göttker-Schnetmann, Inigo; Titz, Alexander; hemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany. (2015-05-05)
      Biofilm formation and chronic infections with Pseudomonas aeruginosa depend on lectins produced by the bacterium. The bacterial C-type lectin LecB binds to the two monosaccharides l-fucose and d-mannose and conjugates thereof. Previously, d-mannose derivatives with amide and sulfonamide substituents at C6 were reported as potent inhibitors of the bacterial lectin LecB and LecB-mediated bacterial surface adhesion. Because d-mannose establishes a hydrogen bond via its 6-OH group with Ser23 of LecB in the crystal structure and may be beneficial for binding affinity, we extended d-mannose and synthesized mannoheptoses bearing the free 6-OH group as well as amido and sulfonamido-substituents at C7. Two series of diastereomeric mannoheptoses were synthesized and the stereochemistry was determined by X-ray crystallography. The potency of the mannoheptoses as LecB inhibitors was assessed in a competitive binding assay. The data reveal a diastereoselectivity of LecB for (6S)-mannoheptose derivatives with increased activity over methyl α-d-mannoside.