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Peptidomimetics based on ring-fused 2-pyridones: probing pilicide function in uropathogenic E. coli and identification of Aβ-peptide aggregation inhibitors
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the synthesis and biological evaluation of highly substituted, ring-fused 2-pyridones. The utility of the bicyclic 2-pyridones to gain fundamental insights into the disease processes of bacterial infections and Alzheimer’s disease has been investigated.

The 2-pyridones have mainly been studied as a new class of anti-infective agents termed pilicides. The function of the pilicides has been explored using uropathogenic E. coli (UPEC) as a prototype pathogen and urinary tract infection as a model disease. The pilicides target the infectious ability of UPEC by inhibiting key proteins (chaperones) in the so-called chaperone-usher pathway, thus preventing the assembly of bacterial surface organelles (pili/fimbriae).

Synthetic pathways to aminomethylate the 2-pyridones have been developed in order to increase their aqueous solubility while retaining biological activity. Also, the importance of a carboxylic acid has been demonstrated in studies with various carboxylate derivatives and by bioisosteric replacement. Moreover, synthetic procedures to extend the backbone of the rigid, dipeptide-mimicking 2-pyridones have been established. This rendered peptidomimetic building blocks and structures that alongside their potential use as pilicides are of more general interest in peptidomimetic-related research.

The potential pilicides have been screened for chaperone affinity using relaxation-edited 1H-NMR spectroscopy. In addition, their ability to inhibit pilus biogenesis in E. coli has been demonstrated by assays of hemagglutination, biofilm formation and attachment to bladder cells, as well as with electron and atomic force microscopy. Moreover, it has been confirmed that pilicides regulate the expression of pili without affecting the biofunctional properties of the pilus rod. This was verified by measurements of individual P pili, on living bacteria, using force measuring optical tweezers. The pilicide binding site was investigated using NMR spectroscopy and X-ray crystallography of a pilicide-chaperone complex. Based on the results obtained, a mechanism whereby the pilicides may inhibit pilus assembly was proposed, which was subsequently experimentally supported by surface plasmon resonance assays and genetic analysis.

Finally, based on the generic 2-pyridone scaffold, a new collection of substituted compounds has been synthesized and validated as inhibitors of Amyloid β (Aβ)-peptide aggregation, which has been suggested to be involved in Alzheimer’s disease.

Place, publisher, year, edition, pages
Umeå: Kemi , 2006. , 101 p.
Keyword [en]
2-pyridone, peptidomimetic, antibacterial, pili, Escherichia coli, virulence, amyloid, Alzheimer’s.
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-909ISBN: 91-7264-157-6 (print)OAI: oai:DiVA.org:umu-909DiVA: diva2:145001
Public defence
2006-11-24, KB3B1, KBC, Linneaus väg 7, 901 87 Umeå, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2006-11-02 Created: 2006-11-02 Last updated: 2011-04-21Bibliographically approved
List of papers
1. Microwave-assisted synthesis of highly substituted aminomethylated 2-pyridones
Open this publication in new window or tab >>Microwave-assisted synthesis of highly substituted aminomethylated 2-pyridones
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2004 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 69, no 23, 7830-7835 p.Article in journal (Refereed) Published
Abstract [en]

By employing microwave-assisted organic synthesis (MAOS) efficient conditions to introduce aminomethylene substituents in highly substituted bicyclic 2-pyridones have been established. Primary amino methylene substituents were introduced via a cyanodehalogenation followed by a borane dimethyl sulfide reduction of the afforded nitrile. In both of these transformations, microwave irradiation proved to be superior to traditional conditions and the primary amines were obtained in good overall yields (55-58% over three steps). To incorporate tertiary aminomethylene substituents in the 2-pyridone framework, a microwave-assisted Mannich reaction using preformed iminium salts proved to be effective. Thus highly substituted 2-pyridones were obtained in 48-93% yields.

 

Place, publisher, year, edition, pages
Easton, Pa.: American Chemical Society, 2004
Identifiers
urn:nbn:se:umu:diva-5469 (URN)10.1021/jo048554y (DOI)
Available from: 2006-11-02 Created: 2006-11-02 Last updated: 2017-12-14Bibliographically approved
2. C-Terminal properties are important for ring-fused 2-pyridones that interfere with the chaperone function in uropathogenic E-coli
Open this publication in new window or tab >>C-Terminal properties are important for ring-fused 2-pyridones that interfere with the chaperone function in uropathogenic E-coli
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2005 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 3, no 21, 3886-3892 p.Article in journal (Refereed) Published
Abstract [en]

Virulence-associated organelles, termed pili or fimbriae, are assembled via the highly conserved chaperone–usher pathway in a vast number of pathogenic bacteria. Substituted bicyclic 2-pyridones, pilicides, inhibit pilus formation, possibly by interfering with the active site residues Arg8 and Lys112 of chaperones in uropathogenic E. coli. In this article we describe the synthesis and evaluation of nine analogues of a biologically active pilicide. Derivatization was performed with respect to its C-terminal features and the affinities for the chaperone PapD were studied with 1H relaxation-edited NMR spectroscopy. It could be concluded that the carboxylic acid functionality and also its spatial location was important for binding. In all cases, binding was significantly reduced or even abolished when the carboxylic acid was replaced by other substituents. In addition, in vivoresults from a hemagglutination assay are presented where the derivatives have been evaluated for their ability to inhibit pilus formation in uropathogenic E. coli.

Place, publisher, year, edition, pages
London: Royal Society of Chemistry, 2005
Keyword
target pilus biogenesis, solid-phase synthesis, pathogenic bacteria, nmr-spectroscopy, structural basis, subunit recognition, papd chaperone, adhesin, protein, cycloaddition
Identifiers
urn:nbn:se:umu:diva-13340 (URN)10.1039/b509376g (DOI)
Available from: 2007-05-08 Created: 2007-05-08 Last updated: 2017-12-14Bibliographically approved
3. Design, synthesis and evaluation of peptidomimetics based on substituted bicyclic 2-pyridones-targeting virulence of uropathogenic E. coli
Open this publication in new window or tab >>Design, synthesis and evaluation of peptidomimetics based on substituted bicyclic 2-pyridones-targeting virulence of uropathogenic E. coli
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2006 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 14, no 22, 7563-7581 p.Article in journal (Refereed) Published
Abstract [en]

Substituted bicyclic 2-pyridones, termed pilicides, are dipeptide mimetics that prevent pilus assembly in uropathogenic Escherichia coli. Here, we apply rational design to produce four classes of extended peptidomimetics based on two bioactive 2-pyridones. The key intermediate in the synthesis was an amino-functionalised 2-pyridone scaffold, which could be obtained via a mild and selective nitration and subsequent reduction. Procedures were then developed to further derivatize this amino-substituted core and a total of 24 extended peptidomimetics were synthesised and evaluated for chaperone affinity and in vivo antivirulence activity in P pili producing E. coli. Enhanced affinities for the target protein were observed within the generated set of compounds, while the ability to prevent pilus assembly in vivo was significantly decreased compared to the parent lead compounds. The results suggest that the limited in vivo potencies of the analogues are either uptake/distribution related or due to loss in binding specificity.

Place, publisher, year, edition, pages
Oxford: Pergamon Press, 2006
Keyword
Bicyclo Compounds; Heterocyclic/*chemical synthesis/chemistry/*pharmacology, Biomimetics, Crystallography; X-Ray, Drug Design, Escherichia coli/*drug effects/*pathogenicity, Magnetic Resonance Spectroscopy, Molecular Chaperones/chemistry/metabolism, Molecular Structure, Peptides/*chemistry, Pyridones/*chemistry, Structure-Activity Relationship, Urinary Tract/microbiology
Identifiers
urn:nbn:se:umu:diva-12343 (URN)10.1016/j.bmc.2006.07.017 (DOI)16904898 (PubMedID)
Available from: 2008-04-10 Created: 2008-04-10 Last updated: 2017-12-14Bibliographically approved
4. Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria
Open this publication in new window or tab >>Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 47, 17897-17902 p.Article in journal (Refereed) Published
Abstract [en]

A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone–usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by 90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone–subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.

Place, publisher, year, edition, pages
Washtington: National Academy of Sciences, 2006
Keyword
antimicrobials, chaperone–usher pathway, pilicide, urinary tract infection
Identifiers
urn:nbn:se:umu:diva-12796 (URN)doi:10.1073/pnas.0606795103 (DOI)
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2017-12-14Bibliographically approved
5. Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod
Open this publication in new window or tab >>Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod
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2007 (English)In: Molecular BioSystems, ISSN 1742-206X, Vol. 3, 214-218 p.Article in journal (Refereed) Published
Abstract [en]

The infectious ability of uropathogenic Escherichia coli relies on adhesive fibers, termed pili or fimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that are formed via a highly preserved assembly and secretion system called the chaperone-usher pathway. We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt both type 1 and P pilus biogenesis in E. coli. In this study, we show that the pilicides do not affect the structure, dynamics or function of the pilus rod. This was demonstrated by first suppressing the expression of P pili in E. coli by pilicide treatment and, next, measuring the biophysical properties of the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic force microscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers were determined by optical tweezers force measurements on individual pili and were found to be intact. The presented results establish a potential use of pilicides as chemical tools to study important biological processes e.g. adhesion, pilus biogenesis and the role of pili in infections and biofilm formation.

Place, publisher, year, edition, pages
Cambridge: Royal Society of Chemistry, 2007
Identifiers
urn:nbn:se:umu:diva-12792 (URN)10.1039/B613441F (DOI)
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2011-03-23Bibliographically approved
6. Carboxylic acid isosteres improve the activity of ring-fused 2-pyridones that inhibit pilus biogenesis in E. coli
Open this publication in new window or tab >>Carboxylic acid isosteres improve the activity of ring-fused 2-pyridones that inhibit pilus biogenesis in E. coli
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5474 (URN)
Available from: 2006-11-02 Created: 2006-11-02 Last updated: 2010-01-13Bibliographically approved
7. Microwave-assisted decarboxylation of bicyclic 2-pyridone scaffolds and identification of A beta-peptide aggregation inhibitors
Open this publication in new window or tab >>Microwave-assisted decarboxylation of bicyclic 2-pyridone scaffolds and identification of A beta-peptide aggregation inhibitors
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2005 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 3, no 15, 2817-2823 p.Article in journal (Refereed) Published
Abstract [en]

A reagent-free microwave-assisted decarboxylation procedure for carboxylic acid functionalized bicyclic 2-pyridones has been developed. This new method, based on microwave heating at 220 degrees C for 600 seconds in N-methyl pyrrolidone (NMP), proved to be practical and very efficient, resulting in decarboxylated 2-pyridones in near-quantitative yields. The decarboxylated products and the intermediate 2-pyridones in the form of carboxylic acid methyl esters and carboxylic acids were screened for their effect on A beta-peptide aggregation. Two out of the 21 2-pyridones described in this study inhibited amyloid formation of the Alzheimer A beta(1-40) peptide. The effect was seen even at a 4 : 1 ratio of 2-pyridone and monomeric A beta-peptide.

Place, publisher, year, edition, pages
London, U.K.: Royal Society of Chemistry, 2005
Keyword
RING-FUSED 2-PYRIDINONES, ALZHEIMERS-DISEASE, ELECTRON-TRANSFER, OLIGOMERS, ACIDS, NEUROTOXICITY, DERIVATIVES, MECHANISM, RADICALS, ESTERS
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:umu:diva-13466 (URN)10.1039/b503294f (DOI)16032359 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2017-12-14Bibliographically approved

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  • modern-language-association-8th-edition
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