title: Development and optimization of a competitive binding assay for the galactophilic low affinity lectin LecA from Pseudomonas aeruginosa.
abstract: Infections with the Gram-negative bacterium Pseudomonas aeruginosa result in a high mortality among immunocompromised patients and those with cystic fibrosis. The pathogen can switch from planktonic life to biofilms, and thereby shields itself against antibiotic treatment and host immune defense to establish chronic infections. The bacterial protein LecA, a C-type lectin, is a virulence factor and an integral component for biofilm formation. Inhibition of LecA with its carbohydrate ligands results in reduced biofilm mass, a potential Achilles heel for treatment. Here, we report the development and optimization of a fluorescence polarization-based competitive binding assay with LecA for application in screening of potential inhibitors. As a consequence of the low affinity of d-galactose for LecA, the fluorescent ligand was optimized to reduce protein consumption in the assay. The assay was validated using a set of known inhibitors of LecA and IC50 values in good agreement with the known Kd values were obtained. Finally, we employed the optimized assay to screen sets of synthetic thio-galactosides and natural blood group antigens and report their structure-activity relationship. In addition, we evaluated a multivalent fluorescent assay probe for LecA and report its applicability in an inhibition assay.
authors.affiliation: Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany. alexander.titz@helmholtz-hzi.de and Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover, Braunschweig, Germany and Department of Chemistry and Graduate School Chemical Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Joachim
authors.affiliation: Department of Chemistry and Graduate School Chemical Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rikker
authors.affiliation: Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany. alexander.titz@helmholtz-hzi.de and Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover, Braunschweig, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hauck
authors.affiliation: Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Research Campus Golm, 14424 Potsdam, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ponader
authors.affiliation: Heinrich-Heine-University Duesseldorf, Institute of Organic Chemistry and Macromolecular Chemistry, D-40225 Düsseldorf, Germany.
authors.forename: Sophia
authors.initials: S
authors.lastname: Boden
authors.affiliation: Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany. alexander.titz@helmholtz-hzi.de and Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover, Braunschweig, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Sommer
authors.affiliation: Heinrich-Heine-University Duesseldorf, Institute of Organic Chemistry and Macromolecular Chemistry, D-40225 Düsseldorf, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Hartmann
authors.affiliation: Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66123 Saarbrücken, Germany. alexander.titz@helmholtz-hzi.de and Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover, Braunschweig, Germany and Department of Chemistry and Graduate School Chemical Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Titz
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D001789:Blood Group Antigens / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology*; D005690:Galactose / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D018829:Adhesins, Bacterial; D001789:Blood Group Antigens; D005456:Fluorescent Dyes; C102929:LecA protein, bacteria; D008024:Ligands; D005690:Galactose