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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, p. 590-595Article in journal (Refereed) Published
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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, p. 590-595
Keywords [en]
ADP-ribosylation, bacterial toxins, drug discovery, enzyme inhibitors, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology Medicinal Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-124333DOI: 10.1177/1087057116629923ISI: 000379694900007PubMedID: 26850638OAI: oai:DiVA.org:umu-124333DiVA, id: diva2:973804
Available from: 2016-09-22 Created: 2016-08-04 Last updated: 2018-08-24Bibliographically approved
In thesis
1. Towards novel antibacterials: Synthesis and identification of natural product inspired inhibitors of Chlamydia trachomatis and development of chemical probes targeting virulence of Pseudomonas aeruginosa
Open this publication in new window or tab >>Towards novel antibacterials: Synthesis and identification of natural product inspired inhibitors of Chlamydia trachomatis and development of chemical probes targeting virulence of Pseudomonas aeruginosa
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotic resistance has evolved significantly to become one of the serious threats to public health today. Yet, the pipeline of new antibiotics is drying up and is lagging behind the challenging needs. As a contribution to this recurrent need for novel antibacterials, we applied multidisciplinary strategies to identify small-molecule antibacterials against Chlamydia trachomatis and antivirulence agents against Pseudomonas aeruginosa infections. These strategies included:

1. Synthesis of a focused compounds library inspired by natural product scaffolds followed by phenotypic screening against Chlamydia trachomatis. (Paper I)

(-)-Hopeaphenol is a polyphenol natural product that was identified as an antivirulence agent against Y. pseudotuberculosis and P. aeruginosa. Hopeaphenol core scaffold, 2,3-diaryl-2,3-dihydrobenzofuran, is ubiquitous in polyphenolic phytochemicals. In this thesis, a focused library of forty-eight compounds was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3- dihydrobenzofuran. The library was then explored for antibacterial properties in a number of screening assays and resulted in five novel antichlamydial compounds with inhibition potency down to sub-micromolar. The identified molecules also inhibited the growth of different clinical presentations of C. trachomatis, one of the most common sexually transmitted disease worldwide.

2. Target-based screening against the P. aeruginosa virulence factor using enzymatic and biophysical assays. (Paper II-IV)

P. aeruginosa is a Gram-negative opportunistic pathogen with remarkable antibiotic resistance that is associated with a wide range of clinical infections. An alternative strategy to develop novel and selective antibacterials is to target the bacterial virulence factors, i.e. the ability of the bacteria to promote disease, thus ‘disarming’ the pathogens instead of killing them. P. aeruginosa employs its virulence factor, the type III secretion system (T3SS), to inject toxins (e.g. ExoS) into the eukaryotic cytosol. In one part of this thesis, we utilized enzymatic assay and identified inhibitors against the P. aeruginosa T3S toxin (ExoS). A follow up structure-activity relationship analysis was established and resulted in five (low micromolar) inhibitors of ExoS ADP-ribosylation enzymatic activity. In another part, we used surface plasmon resonance biophysical assay and identified small molecule binders of T3S translocation protein (PcrV). The primary SAR analysis was established and showed the antivirulence properties of these molecules and the potential to expand them further as novel antibacterials.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 95
Keywords
Antibacterials, antibiotics, small molecules, natural products, benzofuran, dihydrobenzofuran, the type III secretion system, Pseudomonas aeruginosa, Chlamydia trachomatis, phenotypic screening, high-throughput screening, surface plasmon resonance, drug discovery, bacterial toxins, enzyme inhibitors
National Category
Natural Sciences Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-150970 (URN)978-91-7601-917-7 (ISBN)
Public defence
2018-09-14, KB.E3.03 (Stora hörsalen), KBC-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research , SSF, SB12-0022
Available from: 2018-08-24 Created: 2018-08-21 Last updated: 2018-08-21Bibliographically approved

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Saleeb, MichaelForsberg, ÅkeElofsson, Mikael

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