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Design and synthesis of fluorescently labeled pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Fredrik Almqvist)
Department of Molecular Microbiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Pilicides and curlicides block formation of the E. coli virulence factors pili and curli. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved using a strategy where key pilicide and curlicide substituents were replaced by fluorophores having similar physicochemical properties. The resulting fluorescent compounds had improved anti-virulence activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing both coumarin and 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide scaffold. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated to the bacteria and seem to discriminate between different bacteria in a population.

National Category
Infectious Medicine Organic Chemistry Organic Chemistry Medicinal Chemistry Organic Chemistry
Research subject
Biorganic Chemistry; Infectious Diseases; läkemedelskemi; Organic Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-37170OAI: oai:DiVA.org:umu-37170DiVA: diva2:358223
Available from: 2010-10-21 Created: 2010-10-21 Last updated: 2011-02-18Bibliographically approved
In thesis
1. Pilicides and Curlicides: Design, synthesis, and evaluation of novel antibacterial agents targeting bacterial virulence
Open this publication in new window or tab >>Pilicides and Curlicides: Design, synthesis, and evaluation of novel antibacterial agents targeting bacterial virulence
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

New strategies are needed to counter the growing problem of bacterial resistance to antibiotics. One such strategy is to design compounds that target bacterial virulence, which could work separately or in concert with conventional bacteriostatic or bactericidal antibiotics. Pilicides are a class of compounds based on a ring-fused 2-pyridone scaffold that target bacterial virulence by blocking the chaperone/usher pathway in E. coli and thereby inhibit the assembly of pili. This thesis describes the design, synthesis, and biological evaluation of compounds based on the pilicide scaffold with the goal of improving the pilicides and expanding their utility. Synthetic pathways have been developed to enable the introduction of substituents at the C-2 position of the pilicide scaffold. Biological evaluation of these compounds demonstrated that some C-2 substituents give rise to significant increases in potency. X-ray crystallography was used to elucidate the structural basis of this improved biological activity. Furthermore, improved methods for the preparation of oxygen-analogues and C-7 substituted derivatives of the pilicide scaffold have been developed. These new methods were used in combination with existing strategies to decorate the pilicide scaffold as part of a multivariate design approach to improve the pilicides and generate structure activity relationships (SARs).

Fluorescent pilicides were prepared using a strategy where selected substituents were replaced with fluorophores having similar physicochemical properties as the original substituents. Many of the synthesized fluorescent compounds displayed potent pilicide activities and can thus be used to study the complex interactions between pilicide and bacteria. For example, when E. coli was treated with fluorescent pilicides, it was found that the compounds were not uniformly distributed throughout the bacterial population, suggesting that the compounds are primarily associated to bacteria with specific properties.

Finally, by studying compounds designed to inhibit the aggregation of Aβ, it was found that some compounds based on the pilicide scaffold inhibit the formation of the functional bacterial amyloid fibers known as curli; these compounds are referred to as 'curlicides'. Some of the curlicides also prevent the formation of pili and thus exhibit dual pilicide-curlicide activity. The potential utility of such 'dual-action' compounds was highlighted by a study of one of the more potent dual pilicide-curlicides in a murine UTI model were the compound was found to significantly attenuate virulence in vivo.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, Kemiska Institutionen, 2010. 83 p.
Keyword
pilicide, curlicide, anti-virulence, chaperone/usher pathway, antibacterial, pili, curli, Escherichia coli, biofilm inhibitor, 2-pyridone, peptidomimetic
National Category
Organic Chemistry Organic Chemistry Medicinal Chemistry Organic Chemistry Infectious Medicine
Research subject
Biorganic Chemistry; Infectious Diseases; läkemedelskemi; Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-37161 (URN)978-91-7459-095-1 (ISBN)
Public defence
2010-11-19, KBC-huset, KB3B1, Umeå Universitet, kemiska institutionen, SE-90187, Umeå, 10:33 (English)
Opponent
Supervisors
Available from: 2010-10-29 Created: 2010-10-21 Last updated: 2011-05-16Bibliographically approved
2. Optical characterization of potential drugs and drug delivery systems
Open this publication in new window or tab >>Optical characterization of potential drugs and drug delivery systems
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This Thesis is a characterization study on substances having potency as drugs as well as on a lipid based drug-delivery matrix. The optical properties of newly synthesized molecules with proven pilicide properties have been characterized with several spectroscopic methods. These methods include optical absorption and fluorescence as well as time-resolved fluorescence. Upon covalently linking compounds with high quantum yields of fluorescence to specific parts of the pilicide, the biological impact was found to increase for some of the derivatives. Furthermore, by expanding the aromatic part of the pilicide molecule, a significant increase in the inherent fluorescence was obtained. The S0-S1 absorption band for these molecules was found to originate from an impure electronic transition, vibronically promoted by intensity borrowing from higher electronic states.

Included in this Thesis is the measurement of how deeply some in this class of newly synthesized molecules become situated when placed inside ganglioside GM1 micelles, and how the molecules’ reorientation is affected. By means of radiation-less energy transfer, it was shown that the molecules place themselves close to the hydrophobic-hydrophilic interface inside the GM1 micelles. As a consequence they are exposed to a densely packed environment, which inhibits the free tumbling of the molecule. This restricted tumbling could be measured by means of time-resolved depolarization experiments.

The release of drug-like fluorescent molecules is investigated from a lipid mixture, which upon equilibrium with water forms a mixture of inverted hexagonal and cubic phases. The lipid matrix displayed an extended release over the course of weeks, in vitro, for molecules having a large variation in hydrophobicity.

Place, publisher, year, edition, pages
Umeå: Kemiska institutionen, Umeå universitet, 2011. 35 p.
Keyword
Pilicide, Ganglioside GM1 micelles, Drug Delivery, Elecronic Energy Transfer, UV-Vis Absorption, Fluorescence Spectroscopy
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-40177 (URN)978-91-7459-157-6 (ISBN)
Public defence
2011-03-11, KBC-huset, KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2011-02-18 Created: 2011-02-16 Last updated: 2011-02-18Bibliographically approved

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