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Towards novel antibacterials: Synthesis and identification of natural product inspired inhibitors of Chlamydia trachomatis and development of chemical probes targeting virulence of Pseudomonas aeruginosa
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Mikael Elofsson's group)
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 [en]
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: urn:nbn:se:umu:diva-150970ISBN: 978-91-7601-917-7 (print)OAI: oai:DiVA.org:umu-150970DiVA, id: diva2:1240446
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, SB12-0022Available from: 2018-08-24 Created: 2018-08-21 Last updated: 2018-08-21Bibliographically approved
List of papers
1. Natural product inspired library synthesis – Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis
Open this publication in new window or tab >>Natural product inspired library synthesis – Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 143, p. 1077-1089Article in journal (Refereed) Published
Abstract [en]

A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial activity in a panel of test systems including phenotypic, biochemical and image-based screening assays. We identified several potent inhibitors that block intracellular replication of pathogenic Chlamydia trachomatis with IC50 ≤ 3 μM. These new C. trachomatis inhibitors can serve as starting points for the development of specific treatments that reduces the global burden of C. trachomatis infections.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
2, 3-diaryl-2, 3-dihydrobenzofuran, 2, 3-diaryl-benzofuran, Antibacterial, Benzofuran, Chlamydia
National Category
Organic Chemistry Biological Sciences
Identifiers
urn:nbn:se:umu:diva-143062 (URN)10.1016/j.ejmech.2017.11.099 (DOI)000428216700089 ()29232584 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SB12-0022Swedish Research Council, 621-2014-4670
Available from: 2017-12-18 Created: 2017-12-18 Last updated: 2018-08-24Bibliographically approved
2. Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity
Open this publication in new window or tab >>Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity
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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
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.

Keywords
ADP-ribosylation, bacterial toxins, drug discovery, enzyme inhibitors, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-124333 (URN)10.1177/1087057116629923 (DOI)000379694900007 ()26850638 (PubMedID)
Available from: 2016-09-22 Created: 2016-08-04 Last updated: 2018-08-24Bibliographically approved
3. Structure–activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity
Open this publication in new window or tab >>Structure–activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 143, p. 568-576Article in journal (Refereed) Published
Abstract [en]

During infection, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa employs its type III secretion system to translocate the toxin exoenzyme S (ExoS) into the eukaryotic host cell cytoplasm. ExoS is an essential in vivo virulence factor that enables P. aeruginosa to avoid phagocytosis and eventually kill the host cell. ExoS elicits its pathogenicity mainly via ADP-ribosyltransferase (ADPRT) activity. We recently identified a new class of ExoS ADPRT inhibitors with in vitro IC50 of around 20 μM in an enzymatic assay using a recombinant ExoS ADPRT domain. Herein, we report structure-activity relationships of this compound class by comparing a total of 51 compounds based on a thieno [2,3-d]pyrimidin-4(3H)-one and 4-oxo-3,4-dihydroquinazoline scaffolds. Improved inhibitors with in vitro IC50 values of 6 μM were identified. Importantly, we demonstrated that the most potent inhibitors block ADPRT activity of native full-length ExoS secreted by viable P. aeruginosa with an IC50 value of 1.3 μM in an enzymatic assay. This compound class holds promise as starting point for development of novel antibacterial agents.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
2-Aminobenzamide, ADP-Ribosyltransferase, Bacterial exotoxins, ExoS, Pseudomonas aeruginosa, Quinazolines, Type III secretion
National Category
Organic Chemistry Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-143429 (URN)10.1016/j.ejmech.2017.11.036 (DOI)000428216700046 ()29207339 (PubMedID)
Available from: 2017-12-23 Created: 2017-12-23 Last updated: 2018-08-24Bibliographically approved
4. Identification of small molecules blocking the Pseudomonas aeruginosa type III secretion system protein PcrV
Open this publication in new window or tab >>Identification of small molecules blocking the Pseudomonas aeruginosa type III secretion system protein PcrV
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs the type III secretion system (T3SS) during acute phase of infection to translocate cytotoxins into the host cell cytoplasm. PcrV, known as the V-antigen, is a T3S protein that facilitates the integration of the translocators into the eukaryotic cell membrane. In this study, we report surface plasmon resonance screening to identify small molecule binders of PcrV. A follow up primary structure-activity relationship analysis resulted in anti-virulence PcrV binders that protect macrophages in a  P. aeruginosa cell-based infection assay.

Keywords
Pseudomonas aeruginosa, type III secretion, PcrV, surface plasmon resonance, screening, small molecules, macrophages
National Category
Natural Sciences
Research subject
Organic Chemistry; biology
Identifiers
urn:nbn:se:umu:diva-150969 (URN)
Funder
Swedish Foundation for Strategic Research , SB12-0022
Available from: 2018-08-21 Created: 2018-08-21 Last updated: 2018-08-21

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