• 7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

      Xiao, Zhangping; Chen, Deng; Song, Shanshan; van der Vlag, Ramon; van der Wouden, Petra E; van Merkerk, Ronald; Cool, Robbert H; Hirsch, Anna K H; Melgert, Barbro N; Quax, Wim J; et al. (American Chemical Society, 2020-10-13)
      Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.
    • -Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases.

      Konstantinović, Jelena; Yahiaoui, Samir; Alhayek, Alaa; Haupenthal, Jörg; Schönauer, Esther; Andreas, Anastasia; Kany, Andreas M; Müller, Rolf; Koehnke, Jesko; Berger, Fabian K; et al. (ACS, 2020-06-17)
      In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.
    • 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.
    • Discovery and Structure-Based Optimization of 2-Ureidothiophene-3-carboxylic Acids as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors.

      Elgaher, Walid A M; Sharma, Kamal K; Haupenthal, Jörg; Saladini, Francesco; Pires, Manuel; Real, Eleonore; Mély, Yves; Hartmann, Rolf W; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-07-07)
      We are concerned with the development of novel anti-infectives with dual antibacterial and antiretroviral activities for MRSA/HIV-1 co-infection. To achieve this goal, we exploited for the first time the mechanistic function similarity between the bacterial RNA polymerase (RNAP) "switch region" and the viral non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Starting from our previously discovered RNAP inhibitors, we managed to develop potent RT inhibitors effective against several resistant HIV-1 strains with maintained or enhanced RNAP inhibitory properties following a structure-based design approach. A quantitative structure-activity relationship (QSAR) analysis revealed distinct molecular features necessary for RT inhibition. Furthermore, mode of action (MoA) studies revealed that these compounds inhibit RT noncompetitively, through a new mechanism via closing of the RT clamp. In addition, the novel RNAP/RT inhibitors are characterized by a potent antibacterial activity against S. aureus and in cellulo antiretroviral activity against NNRTI-resistant strains. In HeLa and HEK 293 cells, the compounds showed only marginal cytotoxicity.
    • Novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from Lipopolysaccharide-Induced Cytotoxicity.

      Guo, Hao; Verhoek, Iris C; Prins, Gerian G H; van der Vlag, Ramon; van der Wouden, Petra E; van Merkerk, Ronald; Quax, Wim J; Olinga, Peter; Hirsch, Anna K H; Dekker, Frank J; et al. (ACS, 2019-04-19)
      Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.