title: Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity.
abstract: The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N(6)-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β-stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development.
authors: Department of Medicinal Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors: Ana Filipa
authors: AF
authors: Pinto
authors: Department of Medicinal Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors: Mahsa
authors: M
authors: Ebrahimi
authors: Department of Chemistry, Umeå University, Umeå, Sweden.
authors: Michael
authors: M
authors: Saleeb
authors: Department of Molecular Biology, Umeå University, Umeå, Sweden.
authors: Åke
authors: Å
authors: Forsberg
authors: Department of Chemistry, Umeå University, Umeå, Sweden.
authors: Mikael
authors: M
authors: Elofsson
authors: Department of Medicinal Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden herwig.schuler@ki.se.
authors: Herwig
authors: H
authors: Schüler
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics; D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics; D055808:Drug Discovery; D024881:Drug Resistance, Bacterial; D005098:Exotoxins / Q000037:antagonists & inhibitors / Q000235:genetics; D054884:Host-Pathogen Interactions / Q000235:genetics*; D006801:Humans; D011552:Pseudomonas Infections / Q000188:drug therapy* / Q000235:genetics / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000472:pathogenicity; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013379:Substrate Specificity; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; C107953:RAC1 protein, human; D054852:Small Molecule Libraries; C482982:YWHAB protein, human; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D020830:rac1 GTP-Binding Protein
keywords: ADP-ribosylation; Pseudomonas aeruginosa; bacterial toxins; drug discovery; enzyme inhibitors