title: Dissecting the Multiple Roles of PqsE in Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds.
abstract: Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Zender
authors.affiliation: Helmholtz Centre for Infection Research , Department Structure and Function of Proteins, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Witzgall
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Münster , Corrensstrasse 3, 48149 Münster, Germany.
authors.forename: Steffen L
authors.initials: SL
authors.lastname: Drees
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Weidel
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.
authors.forename: Christine K
authors.initials: CK
authors.lastname: Maurer
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Münster , Corrensstrasse 3, 48149 Münster, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fetzner
authors.affiliation: Helmholtz Centre for Infection Research , Department Structure and Function of Proteins, Inhoffenstrasse 7, 38124 Braunschweig, Germany.|Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig , Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Wulf
authors.initials: W
authors.lastname: Blankenfeldt
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Empting
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.|Pharmaceutical and Medicinal Chemistry, Department of Pharmacy, Saarland University , Campus E8.1, 66123 Saarbrücken, Germany.
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Hartmann
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D001565:Benzoates / Q000494:pharmacology; D002264:Carboxylic Acids / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D005470:Fluorometry; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011710:Pyocyanine / Q000096:biosynthesis; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015363:Quinolones / Q000378:metabolism; D053038:Quorum Sensing; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors*; D013876:Thiophenes / Q000494:pharmacology; D014774:Virulence / Q000187:drug effects; D037521:Virulence Factors / Q000096:biosynthesis
chemical_list: C000622585:AC30341; D001426:Bacterial Proteins; D001565:Benzoates; C000622584:CC34301; C000622583:CC41801; D002264:Carboxylic Acids; D011725:Pyridines; D011758:Pyrroles; D015363:Quinolones; D013876:Thiophenes; D037521:Virulence Factors; D011710:Pyocyanine; D013869:Thiolester Hydrolases