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Two complementary strategies to tackle the growing health crisis of antibiotic resistance were pursued in this thesis. A modular concept of caged electrophiles that are selectively liberated in bacteria by enzymatic activation was established. This strategy has the potential to deliver antibiotics with reduced side-effects and resistance formation. For the development of novel narrow-spectrum antibiotics, the covalent ligandability of cysteines in the pathogen Moraxella catarrhalis was profiled and covalent binding was validated for three essential proteins., In dieser Arbeit wurden zwei komplementäre Strategien zur Bekämpfung der Antibiotikakrise verfolgt. Zur Vermeidung von Nebenwirkungen und Resistenzbildung wurde ein modulares Konzept geschützter Elektrophile erarbeitet, welches das Potenzial hat, Antibiotika selektiv in Bakterien durch enzymatische Aktivierung freizusetzen. Zur Entwicklung neuer pathogenspezifischer Antibiotika wurde die kovalente Adressierbarkeit von Cysteinen im Krankheitserreger Moraxella catarrhalis untersucht und die kovalente Bindung dreier essenzieller Proteine validiert.

The herein developed chemoproteomic isoDTB-ABPP platform streamlines the identification of binding sites of covalent ligands across bacterial proteomes, providing starting points for the development of novel antibiotics. Moreover, the described advances in the profiling of nucleophilic amino acids other than cysteine will help to expand the target space of covalent ligands and thus foster the development of antibiotics with new modes-of-action., Die hier entwickelte chemoproteomische isoDTB-ABPP-Plattform ermöglicht die proteomweite Identifizierung von Bindestellen kovalenter Liganden und liefert so Ansatzpunkte für die Entwicklung neuartiger Antibiotika. Zudem werden die beschriebenen Fortschritte in der Untersuchung anderer nukleophiler Aminosäuren als Cystein dazu beitragen, die Anzahl potentieller Zielstrukturen kovalenter Liganden zu erhöhen und so die Entwicklung von Antibiotika mit neuartigen Wirkmechanismen fördern.

Covalently acting compounds experience a strong interest within chemical biology both as molecular probes in studies of fundamental biological mechanisms and/or as novel drug candidates. In this context, the identification of new classes of reactive groups is particularly important as these can expose novel reactivity modes and, consequently, expand the ligandable proteome. Here, we investigated the electrophilic reactivity of the 3-acyl-5-hydroxy-1,5-dihydro-2H-pyrrole-2-one (AHPO) scaffold, a heterocyclic motif that is e.g. present in various bioactive natural products. Our investigations were focused on the compound MT-21 - a simplified structural analogue of the natural product epolactaene - which is known to have both neurotrophic activity and ability to trigger apoptotic cell death. We found that the central N-acyl hemiaminal group of MT-21 can function as an electrophilic centre enabling divergent reactivity with both amine- and thiol-based nucleophiles, which furthermore translated to reactivity with proteins in both cell lysates and live cells. We found that in live cells MT-21 strongly engaged the lipid transport protein fatty acid-binding protein 5 (FABP5) by direct binding to a cysteine residue in the bottom of the ligand binding pocket. Through preparation of a series of MT-21 derivatives, we probed the specificity of this interaction which was found to be strongly dependent on subtle structural changes. Our study suggests that MT-21 may be employed as a tool compound in future studies of the biology of FABP5, which remains incompletely understood. Furthermore, our study has also made clear that other natural products containing the AHPO-motif may likewise possess covalent reactivity and that this property may underlie their biological activity.

The herein developed chemoproteomic isoDTB-ABPP platform streamlines the identification of binding sites of covalent ligands across bacterial proteomes, providing starting points for the development of novel antibiotics. Moreover, the described advances in the profiling of nucleophilic amino acids other than cysteine will help to expand the target space of covalent ligands and thus foster the development of antibiotics with new modes-of-action., Die hier entwickelte chemoproteomische isoDTB-ABPP-Plattform ermöglicht die proteomweite Identifizierung von Bindestellen kovalenter Liganden und liefert so Ansatzpunkte für die Entwicklung neuartiger Antibiotika. Zudem werden die beschriebenen Fortschritte in der Untersuchung anderer nukleophiler Aminosäuren als Cystein dazu beitragen, die Anzahl potentieller Zielstrukturen kovalenter Liganden zu erhöhen und so die Entwicklung von Antibiotika mit neuartigen Wirkmechanismen fördern.