Table of Contents:
The rapid development of antimicrobial resistance is one of the major challenges facing the global healthcare system. Today, many bacterial species already develop pronounced resistance to a variety of antibiotics. Due to neglected antibiotic research, the development pipeline of novel antibacterial agents has not yielded any innovative lead structures so far. An alternative class of substances are antimicrobial peptides, in particular those with intracellular targets and specific effects. A molecule cluster within this substance class is represented by proline-rich antimicrobial peptides, so-called PrAMPs. Due to their specific intracellular effects such as the inhibition of protein biosynthesis, this group moved into the focus of research.
A new proline-rich antimicrobial peptide in salivary excretion of the medicinal leech was identified and characterized. Initially, the sequence obtained by de novo sequencing was synthesized by solid-phase peptide synthesis and its antimicrobial activity was verified. Both gram-groups showed significant inhibitions against reference organisms and up to 50 % growth inhibition against multi-resistant Klebsiella isolates including carbapenem resistance genes and ESBL. The proteolytic activity of the lead structure has also been investigated. In particular, a high stability in human serum and simulated gastric fluid could be demonstrated. Preliminary in vivo data for HP01 were generated in an infection model with Galleria mellonella. The median survival rate of 80 % after seven days post infectum at a drug dose of 10.3 nM was shown, whereas after the same incubation period a lethality of 100 % occurred in the untreated control group.
Further work packages generated a large number of derivatives in order to increase the antimicrobial activity of the lead structure. The modified sequences were selected using rational design methods based on already published data from known PrAMPs and defined design rules. Thereby, the main focus was on physicochemical parameters such as net charge and amphiphilicity. After completing the studies on the structure-activity relationships of the derivatives, both the dose-activity profile and the parameters IC50 and MIC, which are important for further development, were significantly improved. Following modification, a MIC of 3.82 nM was reported. Furthermore, investigations were carried out on the cytotoxic potential of the lead structure and its derivatives. The first cell damaging effects for the lead structure could be detected at 14 times the IC50 value in in vitro cell culture models with HeLa cells. For the optimized derivatives, an increase in tolerability could be achieved. Cytotoxic effects only occurred after 50 times the effective concentration.
Finally, studies on the mode of action and target identification of the lead structure and its derivatives were carried out. The cellular uptake was studied by confocal laser scanning microscopy. Surface plasmon resonance was used investigating the interaction and binding of the lead structure and its derivatives with the 70S ribosome of gram-negative bacteria. After covalent immobilization of the ribosomes on the sensor chip surface, specific binding constants in the low nM range could be detected. Thereby, the 70S ribosome of gram-negative pathogens could be identified as a possible intracellular target for the identified proline-rich lead structure of salivary excretion of the medicinal leech.