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Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2016 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 21, no 6, 590-595 p.Article in journal (Refereed) PublishedText
Abstract [en]

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 14-3-3-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-y l)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.

Place, publisher, year, edition, pages
2016. Vol. 21, no 6, 590-595 p.
Keyword [en]
ADP-ribosylation, bacterial toxins, drug discovery, enzyme inhibitors, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology Medicinal Chemistry
URN: urn:nbn:se:umu:diva-124333DOI: 10.1177/1087057116629923ISI: 000379694900007PubMedID: 26850638OAI: diva2:973804
Available from: 2016-09-22 Created: 2016-08-04 Last updated: 2016-09-22Bibliographically approved

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Saleeb, MichaelForsberg, ÅkeElofsson, Mikael
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