Small Molecules and Their Conjugates with Peptides: Chemical Strategies to Tackle Resistant Bacteria

Abstract

Antimicrobial resistance is an increasingly important issue as it renders currently used antibiotics ineffective. There are different approaches how to slow down emergence of resistance, including better stewardship of the current treatments, release of drugs in a more selective way, or discovery of new compounds and bacterial targets. This thesis will cover two projects that have tried to address the latter two strategies. The first part focuses on a bacterium with robust resistance mechanisms, Pseudomonas aeruginosa, and how it could be targeted with two different cleavable peptide–drug conjugates. These conjugates aim to simultaneously release two independently-acting antimicrobial agents, selectively in a bacterial environment. To prepare these conjugates, a small-molecule antibacterial compound was attached to an antimicrobial peptide using a cleavable construct that would get cleaved by enzymes produced by the bacteria. The positively-charged antimicrobial peptide should target the negatively-charged bacterial membrane and facilitate an additional level of selectivity. The attachment of the rest of drug–linker construct to the peptide was mediated by stapling of cysteine residues using a divinylheteroarene staple. While the two conjugates shared the same antimicrobial peptide, they differed in the other components. One of the conjugates used bacterial lipase to cleave off a lipidic chain, which would trigger self-immolation of the linker to release a quorum sensing inhibitor. This small molecule was chosen for its known ability to reduce bacterial biofilms, formation of which makes bacteria more resistant. The target peptide–drug conjugate was successfully synthesised together with control compounds. Unfortunately, upon testing of the quorum sensing inhibitor controls, the desired anti-biofilm effects were not observed, which justified the lack of observed activity of the target conjugate. The other conjugate used a β-lactamase cleavable linker to join the peptide to a known antibiotic, ciprofloxacin. The linker ensured that the components of the conjugate were released selectively by bacteria expressing β-lactamase, therefore selectively targeting resistant bacteria while having a prospect of lowering systemic toxicity. Following on previous work from our lab, the most promising and synthetically tractable conjugate was prepared and its activity measured through its minimal inhibitory concentrations (MIC). Further optimisations were attempted to understand factors affecting the MIC. The results lead to a conjugate with proven cleavage by β-lactamase and an improved MIC value compared to the non-cleavable control. The second part of this thesis deals with establishing of a structure-activity relationship of a compound identified in a phenotypic screen of a library of compounds made by diversity-oriented synthesis. The focus of this part is a different bacterium, Mycobacterium abscessus. The importance of targeting this bacterium is high as the strain is commonly found in the environment, presents a variety of resistance mechanisms and disproportionately affects people with underlying medical conditions, such patients with cystic fibrosis or lowered immune response. A series of biochemical assays narrowed over 1500 compounds down to four potential hits. Different structural features of one of the hit compounds were interrogated and their effect on minimal inhibitory concentration against the bacterium was assessed. Three series of compounds have also managed to identify a compound with single-digit micromolar minimal inhibitory concentration in two different bacterial growth media and provided a starting point for identification of the target of the hit compound.