Kinetic and Thermodynamic Characterization of the Bacterial Lectin FimH

One fundamental aim of drug discovery is the development of new molecular entities that have a considerably advantage over already existing therapies. Urinary tract infections (UTIs) urgently require an alternative to the conventional antibiotic therapy as resistance rates for antibiotics are increasing. The development of an anti-adhesive UTI treatment strategy with the bacterial lectin FimH as target is a promising approach to remedy such alarming tendencies. FimH is presented by uropathogenic E. coli (UPEC) strains on the tip of type 1 pili and mediates adhesion to mannosylated residues on the urothelium. This interaction prevents the clearance of UPECs during micturition and enables internalization of the pathogens by urothelial cells. Mannoside-derived FimH antagonists are under development and are considered as promising treatment option for UTIs. In contrast to antibiotics, FimH antagonists do not necessarily exert resistance mechanisms against drugs because they block the adhesion of bacteria to the urothelium without killing them or inhibiting their growth.
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In the present thesis, FimH and its interaction with mannose-based antagonists were biophysically characterized. Additionally, new methodical approaches are introduced, which are relevant not only for a strategic development of FimH antagonists but also for drugs of other therapeutic areas. The following aspects were investigated:
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Publication 2: The publication “KinITC – One method supports both thermo-dynamic and kinetic SARs” (Chemistry, 2018,24(49), 13049-13057) comments on kinITC-ETC, a new method based on ITC data to reveal the kinetic fingerprint of a drug–target interaction. In this study, kinITC-ETC was independently validated for the first time. Moreover, structural properties of FimH antagonists could be correlated with kinetic parameters of FimH–antagonist interactions.
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Manuscript 1: The development of an off-rate screening approach is presented in the study “Off-rate screening by surface plasmon resonance – The search for promising lead structures targeting low-affinity FimH”. The method is subsequently applied to screen a mannose-based compound library against full-length FimH. The assay allows classification of structurally diverse FimH antagonist in order to spot chemical classes exhibiting long dissociative half-lives.
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Publication 3: The lectin domain is conformationally rigid and needs the pilin domain for allosteric propagation. However, the crosstalk between allosteric sites within the lectin domain takes also place in the absence of the pilin domain as demonstrated in the publication “Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain – Engineering a minimalistic allosteric system” (J. Biol. Chem., 2018, 293(5), 1835-1849). Mutants of the isolated lectin domain, FimHLD R60P and V27C/L34C, exhibited a low-affinity state and mimic full-length FimH regarding its conformational transition upon mannoside binding.
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Publication 4: The publication “Target-directed dynamic combinatorial chemistry: A study on potentials and pitfalls as exemplified on a bacterial target” (Chemistry, 2017, 23, 11570-11577) illustrates a target-directed dynamic combinatorial chemistry (tdDCC) approach employing reversible acylhydrazone formation with FimH full-length as target. Optimal sample preparation and data procession are discussed in detail. Finally, the results of the tdDCC assay were subsequently compared with the affinity of library constituents by SPR.
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Publication 5: In the publication “Comparison of affinity ranking by target-directed dynamic combinatorial chemistry and surface plasmon resonance” larger FimH antagonist libraries were screened using the tdDCC method established in publication 3. The comparison of amplification rates of library substituents with respective binding affinities determined by SPR revealed a linear association. Furthermore, the hazardous acylhydrazone moiety could be replaced by various bioisosteres without changing the affinity of the parent compound.
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Manuscript 2: The hydrogen bond network formed between mannose derivates and the CRD of FimH is extensively elucidated in the manuscript ”High-affinity carbohydrate–lectin interaction: How nature makes it possible”. Computational methods and structural prediction in combination with binding data revealed that the hydrogen bond network forms a unified whole. The removal of only a single hydroxyl group leads to a disruption of the cooperative interplay within the network and consequently results in a dramatic loss in binding affinity.
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Manuscript 3: In the study “The tyrosine gate of the bacterial adhesion FimH – An evolutionary remnant paves the way for drug discovery”, ITC measurements demonstrated the influence of the tyrosine gate on binding affinity between FimH and natural ligands. While the tyrosine gate is exploited to form optimal hydrophobic interactions with aryl aglycones of synthetic FimH antagonists in order to increase their binding affinity, the tyrosine gate has only a marginal impact on the KD of natural ligands. In contrast to wild-type FimH, mutants that partially or completely lack the tyrosine gate exhibited a comparable binding affinity to dimannoside.
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Publication 6: The publication “Improvement of aglycone π-stacking yields nanomolar to sub-nanomolar FimH antagonists” displays that fluorination of biphenyl mannosides further improved π-π stacking with the tyrosine gate, reaching nanomolar affinities with FimHFL and even picomolar affinities with FimHLD. It also could be shown that ligand binding to FimHFL occurs with a highly favorable enthalpic and a considerably unfavorable entropic contribution.
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Publication 7: In the publication “Enhancing the enthalpic contribution of hydrogen bonds by solvent shielding” microcalorimetric studies of FimH could reveal that conformational adaptions of the binding site can establish a solvent-free cavity. Shielding the solvent results in a lower dielectric environment, in which the formation of hydrogen bonds has a considerable enthalpic contribution to the binding free energy. In the case of FimH approximately -13 kJ mol-1 for mannoside binding.