• Flexible Fragment Growing Boosts Potency of Quorum Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

      Zender, Michael; Witzgall, Florian; Kiefer, Alexander Felix; Kirsch, Benjamin; Maurer, Christine K; Kany, Andreas M; Xu, Ningna; Schmelz, Stefan; Börger, Carsten; Blankenfeldt, Wulf; et al. (Wiley-VCH, 2019-11-11)
      Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-amino pyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa . We pursue an innovative treatment strategy by interfering with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.
    • A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms

      Schütz, Christian; Ho, Duy‐Khiet; Hamed, Mostafa Mohamed; Abdelsamie, Ahmed Saad; Röhrig, Teresa; Herr, Christian; Kany, Andreas Martin; Rox, Katharina; Schmelz, Stefan; Siebenbürger, Lorenz; et al. (Wiley and Sons Inc., 2021-03-18)
      Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development.
    • The N‐terminal peptide of the transglutaminase‐activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross‐linking sites

      Juettner, Norbert E.; Schmelz, Stefan; Anderl, Anita; Colin, Felix; Classen, Moritz; Pfeifer, Felicitas; Scrima, Andrea; Fuchsbauer, Hans‐Lothar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2019-08-29)
      Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).
    • Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.

      Schubert, Maren; Nimtz, Manfred; Bertoglio, Federico; Schmelz, Stefan; Lukat, Peer; van den Heuvel, Joop; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2021-05-21)
      he expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
    • Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

      Scheithauer, Lina; Thiem, Stefanie; Schmelz, Stefan; Dellmann, Ansgar; Büssow, Konrad; Brouwer, René M H J; Ünal, Can M; Blankenfeldt, Wulf; Steinert, Michael; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2021-01-24)
      ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.