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Biological characterization of the lectins DC-SIGN and FimH : putative targets for novel anti-infectives

Scharenberg, Meike. Biological characterization of the lectins DC-SIGN and FimH : putative targets for novel anti-infectives. 2013, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10698

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Abstract

Urinary tract infection (UTI) is one of the most common infections, with millions of people affected every year. Besides women, who bear a risk of 40 - 50% to experience at least one symptomatic UTI episode during a life-time, patients with diabetes, spinal cord injuries, and suppressed immune system are particularly at risk. Without treatment UTI may lead to bladder infection (cystitis) and, in a later infection state, to kidney infection (pyelonephritis). The initial and most fundamental step in the pathogenesis of UTIs is the type 1 pili-dependent adhesion of uropathogenic Escherichia coli (UPEC) to ?-mannoside-containing glycoprotein receptors on the surface of uroepithelial cells, such as uroplakin Ia (UPIa). The bacterial adhesion is mediated by the lectin FimH, localized at the tip of type 1 pili, which recognizes mono- and oligomannosides. The adhesion triggers the bacterial cell invasion, resulting in the development of an infection. FimH antagonists such as alpha-D-mannopyranosides have been shown to interfere with the attachment of UPEC to their host cells, thus providing a novel therapeutic opportunity for the treatment and prevention of UTIs as an alternative to antibiotic treatment.
A potent FimH antagonist has to fulfill several requirements to also achieve a high in vivo efficacy. Besides a high affinity for FimH (KD), slow off-rates and irreversibility of the antagonist-FimH interaction are beneficial for the in vivo efficacy, as prolonged occupancy of the target by the drug results in an extended duration of the pharmacological effect. Furthermore, target selectivity of FimH antagonists is a pivotal concern, since the reported FimH antagonists are alpha-D-mannopyranosides and therefore are potential ligands for mannose receptors of the human host system. Non-selective interactions of FimH antagonists with the various mannose receptors would have a profound impact on physiological processes and could cause severe side effects.
This thesis adresses some major issues in the development and biological evaluation of FimH antagonists:
-Development of a cell-based competition assay for the determination of IC50 values of FimH antagonists using flow cytometry.
-Determination of kinetic properties and KD values of FimH antagonists by surface plasmon resonance.
- Investigation of the selectivity of FimH antagonists towards human mannose binding receptors.
Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a pathogen recognition receptor (PRR) and abundantly expressed on immature dendritic cells (DCs). The binding of pathogens via PRRs mediates phagocytosis, DC maturation and migration from peripheral tissues to draining lymph nodes. After lysosomal degradation, the processed antigen particles are presented to naïve T-cells, resulting in the stimulation of adaptive immune responses. However, a variety of pathogens including HIV-1 use the interaction with DC-SIGN on DCs as initial entry port to their host. These pathogens are able to circumvent the intracellular degradation process and impair DC maturation. DC-SIGN recognizes mannose-containing glycoconjugates and fucose-containing blood-group antigens, such as Lewisx (Lex), Lewisa (Lea), and Lewisy (Ley), in the envelope of viruses and the membranes of parasites. DC-SIGN is therefore considered as a potential drug target for the treatment and prevention of a number of infectious diseases. Consequently, considerable efforts are made to develop DC-SIGN antagonists. These new anti-infectives would inhibit DC-SIGN-pathogen interaction and block the initial step of an infection, as well as the pathogen dissemination.
This thesis addresses the issue of improving the recombinant expression of the carbohydrate recognition domain of DC-SIGN by investigating the effect of different signal peptides on the expression of recombinant protein in CHO-K1 cells. The usage of the luciferase signal peptide of the copepod Gaussia princeps led to a drastic improvement of the protein yield compared to the standard interleukin-2 signal peptide. The recombinant protein was used for the evaluation of binding potentials of novel FimH antagonists. A target-based binding assay was developed and a series of antagonists were measured, with the focus on Lewis type structures. Combination of binding assays, mutational analysis, STD NMR studies, and computational modeling revealed a new binding mode with improved affinity for phenyl group-substituted Lea compounds. Therefore these compounds display a novel class of potential anti-infectives.
Advisors:Ernst, Beat
Committee Members:Glockshuber, Rudolf
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molekulare Pharmazie (Ernst)
Item Type:Thesis
Thesis no:10698
Bibsysno:Link to catalogue
Number of Pages:159 p.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:55
Deposited On:02 May 2014 09:32

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